Ball, G.H., Department of Biology, University of California, Los Angeles, .... Rens, D., Institut für Genetik der Universität zu Köln, Köln, Federal Republic of .... Université de Montréal, for their support in the organization of the conference and ...... (Rehacek, 1958, a,b.) .... cing masses, filling the whole cytoplasm (Rehacek et a/.c.
M a i n S p e a k e r s a n d S e s s i o n C h a i r m e n o f t h e F o u r t h I n t e r n a t i o n a l C o n f e r e n c e on I n v e r t e b r a t e T i s s u e C u l t u r e . Standing,
I.
to r.:
A.
D ü b e n d o r f e r ( S w i t z e r l a n d ) , D.
Peters
( N e t h e r l a n d s ) , K.A.
Harrap (Great
Britain), J . J . Lipa
( P o l a n d ) , P. F a u l k n e r ( C a n a d a ) , J . M i t s u h a s h i ( J a p a n ) , S.S. Sohi ( C a n a d a ) , J . R e h a c e k ( C z e c h o s l o v a k i a ) , J . C . L a n d u r e a u ( F r a n c e ) ; seated ( U . S . A . ) . (CP.A.
Photo,
I. to r.:
S. B u c k l e y ( U . S . A . ) ,
Montreal)
K. M a r a m o r o s c h ( U . S . A . ) ,
E. K u r s t a k ( C a n a d a ) , C M . W i l l i a m s
INVERTEBRATE TISSUE CULTURE APPLICATIONS IN MEDICINE, BIOLOGY, AND AGRICULTURE Edited
by
Edouard Kurstak Groupe de Recherche en Virologie Comparée Faculté de Médecine Université de Montréal Montréal, P.Q., Canada
Karl Maramorosch Waksman New
A c a d e m i c Press
Institute of Microbiology Rutgers University Brunswick, New Jersey, U.S.A.
New York
San Francisco
London
A Subsidiary o f H a r c o u r t Brace J o v a n o v i c h , Publishers
1976
ACADEMIC PRESS RAPID MANUSCRIPT
C o p y r i g h t
a ll r i no p a t r a n s or m e i n f o r p e r m i
©
1976,
b y A c a d e m i c
Press,
I n c .
g hst r e s e r v .e d tr o f t h si p u b l i c a tni om a y b e r e p r o d u dc e o r m i t dt ei n a n y f o rm o r b y a n y m e a n, s e l e c t r ocn i c h a n i c, a li n c l u d gi n p h o t o c o ,p yr e c o r d i ,n go r a n y m a t ni o s t o r ae g a n d r e t r i e vl a s y s t e , m w i t h ot u p u b l i s h. e r s s ni o i n w r i t i gn f r om t h e
A C A D E M I C PRESS, I N C . I l l F i f t h A v e n u e , N e w Y o r k , N e w Y o r k 10003
United Kingdom Edition published by A C A D E M I C PRESS, I N C . ( L O N D O N ) L T D . 24/28 O v a l R o a d , L o n d o n N W 1
L i b r a r y o f C o n g r e s s C a t a l o g i n g in P u b l i c a t i o n D a t a International Conference on Invertebrate Tissue Culture, 4th, Mont Gabriel, Que., 1975. I n v e r t e b r a t e tissue c u l t u r e . Sponsored by the Université de Montréal and others. Bibliography: p. Includes index. 1. Tissue culture-Congresses. Cultures and culture media-Congresses. Edouard. II. Maramorosch, Karl. de Montréal. IV. Title. QH585.I54 1975 ISBN 0 - 1 2 - 4 2 9 7 4 0 - 4
PRINTED
IN THE UNITED
592'.08'21
STATES OF
AMERICA
2. I.
InvertebratesKurstak, III. Université 76-14911
REPRODUCTION
Contributors
Aitcken,
T.H.G.,
Yale U n i v e r s i t y
School o f M e d i c i n e , Y a l e A r b o v i r u s
Research
U n i t , D e p t . of E p i d e m i o l o g y & P u b l i c H e a l t h , New H a v e n , C o n n e c t i c u t , U . S . A . B a l l , G . H . , D e p a r t m e n t of B i o l o g y , U n i v e r s i t y of C a l i f o r n i a , Los A n g e l e s , C a l i f o r nia, U.S.A. Bergtrom, G . , The B i o l o g i c a l C o n n e c t i c u t , U.S.A.
Sciences Group, U n i v e r s i t y
of C o n n e c t i c u t , S t o r r s ,
B i r d , F.T., C a n a d i a n Forestry S e r v i c e , I n s e c t Pathology Research I n s t i t u t e , Sault Ste-Marie, Ontario, Canada. Brooks, M . A . , D e p a r t m e n t of E n t o m o l o g y , F i s h e r i e s & W i l d l i f e , U n i v e r s i t y of M i n n e s o t a , S t - P a u l , M i n n e s o t a , U.S.A. Brown, D 0T . , I n s t i t u t für G e n e t i c der U n i v e r s i t ä t zu K ö l n , K ö l n , Federal R e p u b l i c of Germany. Brown, Μ β, D e p a r t m e n t of M i c r o b i o l o g y and I m m u n o l o g y , Q u e e n ' s U n i v e r s i t y , K i n g s ton, Ontario, Canada. B u c k l e y , S . M . , Yale U n i v e r s i t y School of M e d i c i n e , Y a l e A r b o v i r u s Research U n i t , D e p t . of
Epidemiology
C a s a l s , J 0 , Yale U n i v e r s i t y Dept.
of E p i d e m i o l o g y
& Public School
H e a l t h , New
Haven, Connecticut,
of M e d i c i n e , Y a l e A r b o v i r u s
& Public
Health, New
U.S.A.
Research
Haven, Connecticut,
Unit, U.S.A.
C e c i l , J . T . , O s b o r n L a b o r a t o r i e s of M a r i n e S c i e n c e s , New York A q u a r i u m , N e w York Z o o l o g i c a l S o c i e t y , B r o o k l y n , New York, U . S . A . C h a o , J . , Department of B i o l o g y , U n i v e r s i t y of C a l i f o r n i a , Los A n g e l e s , C a l i f o r n i a , U.S.A. C h e n , T . T . , D e p a r t m e n t of B i o l o g y , Q u e e n ' s U n i v e r s i t y , K i n g s t o n , O n t a r i o , C a n a d a . C o u b l e , P., D e p a r t m e n t of B i o l o g y , Q u e e n ' s U n i v e r s i t y , K i n g s t o n , O n t a r i o , C a n a d a . D i C a p u a , R.A., School of Pharmacy, U n i v e r s i t y of C o n n e c t i c u t , S t o r r s , C o n n e c t i c u t , U.S.A. Dübendorfer, Α . , Zoological Zürich, Switzerland.
I n s t i t u t e of t h e U n i v e r s i t y of Z u r i c h , K u n s t l e r g a s s e ,
E c h a l i e r , G . , S e r v i c e de B i o l o g i e A n i m a l e , U n i v e r s i t é de Paris V I , P a r i s , F r a n c e . F a u l k n e r , P., D e p a r t m e n t of M i c r o b i o l o g y Kingston, Ontario, Canada. Filtzer, D.f
and I m m u n o l o g y ,
Queen's
University,
I n s t i t u t für G e n e t i k der U n i v e r s i t ä t zu K ö l n , K ö l n , Federal R e p u b l i c of
Germany. Fowler, G . L . , D e p a r t m e n t of B i o l o g y , U n i v e r s i t y of O r e g o n , Eugene, O r e g o n , U . S . A .
ix
CONTRIBUTORS Gardiner,
G.R.,
Shell Research L i m i t e d , Woodstock L a b o r a t o r y ,
S i t t i n g b o u r n e Re-
search C e n t r e , S i t t i n g b o u r n e , Kent, E n g l a n d . G i l b e r t , L . I . , Department of B i o l o g i c a l S c i e n c e s , N o r t h w e s t e r n U n i v e r s i t y , Evanst o n , I l l i n o i s , U.S.A. G l i e d m a n , J . B . , I n s t i t u t f ü r G e n e t i k der U n i v e r s i t ä t zu K ö l n , K ö l n , Federal R e p u b l i c of
Germany.
G r a n a d o s , R.R., Boyce Thompson I n s t i t u t e , Y o n k e r s , New York, U . S . A . Hansen,
E.L.,
Clinical
Pharmacology
Research
Institute,
Berkeley,
California,
U.S.A. H a r r a p , K.A., NERC U n i t of I n v e r t e b r a t e V i r o l o g y , South Parks Road, O x f o r d , England H a y e s , C . G . , Yale U n i v e r s i t y School of M e d i c i n e , Yale A r b o v i r u s Research U n i t , D e p t . of E p i d e m i o l o g y
& Public
H e a l t h , New H a v e n , C o n n e c t i c u t ,
U.S.A.
H i n k , W.F., Department of Entomology, The O h i o State U n i v e r s i t y , C o l u m b u s , O h i o , U.S.A. H i r u m i , H e, Boyce Thompson I n s t i t u t e , Y o n k e r s , New York, U.S.A. H i r u m i , K.,
Boyce Thompson I n s t i t u t e , Y o n k e r s , New York, U.S.A.
I g a r a s h i , Α . , Department of M i c r o b i o l o g y , C M D N J - R u t g e r s M e d i c a l S c h o o l , P i s c a t a way, New Jersey, U.S.A. J o h a n n i s s o n , R.,
I n s t i t u t e for General B i o l o g y , U n i v e r s i t y of D ü s s e l d o r f ,
Düssel-
dorf, Federal Republic of Germany. J o n a t h a n , J . F . , I n s t i t u t für G e n e t i k der U n i v e r s i t ä t zu K ö l n , K ö l n , Federal Republic of Germany. Kimura, M.,
Medical
Research
I n s t i t u t e , Wakayama
Medical
College,
Wakayama,
Japan. Kiss,
L,
Knudson,
Institute D.L.,
of
Genetics,
NERC U n i t
of
Biological
Invertebrate
Research Virology,
Center,
Szeged,
South Parks
Hungary.
Road, O x f o r d ,
England. Koeppe, J . ,
Department of Z o o l o g y ,
University
of
North C a r o l i n a ,
Chapel H i l l ,
North C a r o l i n a , U.S.A. Koo, R„, Department of M i c r o b i o l o g y , C M D N J - R u t g e r s M e d i c a l S c h o o l , P i s c a t a w a y , New J e r s e y , U . S . A . K u r t t i , T . J . , Department of E n t o m o l o g y , F i s h e r i e s and W i l d l i f e , U n i v e r s i t y of M i n n e sota, St-Paul, Minnesota, U.S.A. L a n d u r e a u , J . C . , S e r v i c e de B i o l o g i e A n i m a l e , F a c u l t é des S c i e n c e s , U n i v e r s i t é de Paris V I , P a r i s , France. L a u f e r , H . , The B i o l o g i c a l S c i e n c e s Group, U n i v e r s i t y of C o n n e c t i c u t , S t o r r s , C o n n e c t i c u t , U.S.A.
χ
CONTRIBUTORS L e l o u p , A,M», U n i t é de M o r p h o l o g i e A n i m a l e , U n i v e r s i t é C a t h o l i q u e de L o u v a i n , Louvain, Belgique. L i c h t e r , W., D e p a r t m e n t of M i c r o b i o l o g y , U n i v e r s i t y of M i a m i , School of M e d i c i n e , M i a m i , F l o r i d a , U.S.A. Lipman,
M., Y a l e U n i v e r s i t y
Dept.
of E p i d e m i o l o g y
School of M e d i c i n e , Yale A r b o v i r u s & Public
Research
Health, New Haven, Connecticut,
Unit, U.S.A.
L o p e z , D . M . , D e p a r t m e n t of M i c r o b i o l o g y , U n i v e r s i t y of M i a m i , School of M e d i c i n e , M i a m i , F l o r i d a , U.S.A. M a l o n e y , J . M . , Yale U n i v e r s i t y School of M e d i c i n e , Yale A r b o v i r u s Research U n i t , Dept. of E p i d e m i o l o g y & P u b l i c H e a l t h , N e w H a v e n , C o n n e c t i c u t , U.S.A. M a r k s , E.P., A g r i c u l t u r a l Research S e r v i c e , U.S. Department of A g r i c u l t u r e , Metabol i s m & R a d i a t i o n Research L a b o r a t o r y , Fargo, N o r t h D a k o t a , U . S . A . M a z z o n e , H . M . , Forest I n s e c t and D i s e a s e L a b o r a t o r y , Forest S e r v i c e , U.S. D e p a r t ment of A g r i c u l t u r e , H a m d e n , C o n n e c t i c u t , U . S . A . M c i n t o s h , A . H . , Waksman I n s t i t u t e of M i c r o b i o l o g y , Rutgers U n i v e r s i t y , N e w Brunswick,
New J e r s e y , U . S . A .
M i t s u h a s h i , J . , D i v i s i o n of E n t o m o l o g y , N a t i o n a l I n s t i t u t e of A g r i c u l t u r a l S c i e n c e s , Tokyo, Japan. M i y a k e , T., M i t s u b i s h i - K a s e i
I n s t i t u t e of L i f e S c i e n c e s , M i n a m i o o y a , M a c h i d a s h i ,
Tokyo,Japan. N a k a j i m a , S., M i t s u b i s h i - K a s e i I n s t i t u t e of L i f e S c i e n c e s , M i n a m i o o y a , M a c h i d a s h i , Tokyo, Japan. Naughton,
M., Boyce Thompson I n s t i t u t e , Y o n k e r s , N e w York, U.S.A.
N i g r e l l i , R.F., Osborn L a b o r a t o r i e s of M a r i n e S c i e n c e s , New York A q u a r i u m , New York Z o o l o g i c a l S o c i e t y , B r o o k l y n , N e w York, U . S . A . N o r t o n , P.W., School of Pharmacy, U n i v e r s i t y
of C o n n e c t i c u t , S t o r r s , C o n n e c t i c u t ,
U.S.A. N o w o c k , J . , D e p a r t m e n t of B i o l o g i c a l S c i e n c e s , N o r t h w e s t e r n U n i v e r s i t y , E v a n s t o n , I l l i n o i s , U.S.A. O b e r l a n d e r , H . , A g r i c u l t u r a l Research S e r v i c e , U S D A , G a i n e s v i l l e , F l o r i d a , U . S . A . Ofengand, Padhi,
J . , Roche I n s t i t u t e of M o l e c u l a r B i o l o g y , N u t l e y , New J e r s e y , U . S . A .
S.B., Waksman
I n s t i t u t e of M i c r o b i o l o g y ,
Rutgers U n i v e r s i t y ,
N e w Bruns-
w i c k , N e w J e r s e y , U.S.A. Potter,
K.M.,
Department
of
Microbiology
and
Immunology,
Queen's
University,
Kingston, Ontario, Canada. Ramoska, W . A . , Department of E n t o m o l o g y , The O h i o S t a t e U n i v e r s i t y , O h i o , U.S.A.
xi
Columbus,
CONTRIBUTORS Rehäcek,
J.,
Institute
of
Virology,
Slovak
Academy
of
Sciences,
Bratislava,
Czechoslovakia. Rens, D.,
I n s t i t u t f ü r G e n e t i k der U n i v e r s i t ä t zu K ö l n , K ö l n , Federal R e p u b l i c of
Germany. R i d d i f o r d , L . M . , Department of Z o o l o g y , U n i v e r s i t y of W a s h i n g t o n , S e a t l e , W a s h i n g ton,
U.S.A.
R i e d e l , Β., I n s t i t u t f ü r G e n e t i k der U n i v e r s i t ä t zu K ö l n , K ö l n , Federal Republic of Germany. Rogers, R., The B i o l o g i c a l S c i e n c e s G r o u p , U n i v e r s i t y of C o n n e c t i c u t , S t o r r s , C o n n e c t i c u t , U.S.A. Rosales-Sharp, M.C., Department of B i o l o g y , U n i v e r s i t y of New M e x i c o , A l b u q u e r q u e , New M e x i c o , U.S.A. R u g g i e r i , G . D . , Osborn L a b o r a t o r i e s of Marine S c i e n c e s , New York A q u a r i u m , N e w York Z o o l o g i c a l S o c i e t y , B r o o k l y n , New York, U.S.A. Schlesinger,
R.W.,
Department
of
Microbiology,
CMDNJ-Rutgers
Medical
School,
P i s c a t a w a y , New J e r s e y , U.S.A. Shenk, T.E., D e p a r t m e n t of B i o c h e m i s t r y , Stanford U n i v e r s i t y , School of M e d i c i n e , S t a n f o r d , C a l i f o r n i a , U.S.A. S i g e l , M.M., Department of M i c r o b i o l o g y , U n i v e r s i t y of M i a m i , School of M e d i c i n e , M i a m i , F l o r i d a , U.S.A. S i l v e r m a n , P.H.,
Department of B i o l o g y , U n i v e r s i t y of New M e x i c o , A l b u q u e r q u e ,
New M e x i c o , U.S.A. S m i t h , J . B . , I n s t i t u t f ü r G e n e t i k der U n i v e r s i t ä t zu K ö l n , K ö l n , Federal Republic of Germany. S o h i , S.S., C a n a d i a n Forestry S e r v i c e , I n s e c t Pathology Research I n s t i t u t e , Sault Ste-Marie, O n t a r i o , C a n a d a . Speyer, G., Boyce Thompson I n s t i t u t e , Y o n k e r s , New York, U.S.A. S t o l l a r , V . , Department of M i c r o b i o l o g y , C M D N J - R u t g e r s M e d i c a l S c h o o l , P i s c a t a w a y , New J e r s e y , U S . A . S t r a u s s , E., Department of E n t o m o l o g y , O h i o State U n i v e r s i t y , U.S.A.
Columbus, Ohio,
S t o c k d a l e , H . , Shell Research L i m i t e d , Woodstock search C e n t r e , S i t t i n g b o u r n e , Kent, England.
S i t t i n g b o u r n e Re-
Summers, M . D . ,
Laboratory,
C e l l Research I n s t i t u t e and D e p a r t m e n t of B o t a n y ,
U n i v e r s i t y of
T e x a s , A u s t i n , T e x a s , U.S.A. V o l k m a n , L.E., C e l l
Research
I n s t i t u t e and Department of B o t a n y , U n i v e r s i t y
Texas, A u s t i n , Texas, U.S.A.
xii
of
CONTRIBUTORS.
W e l l h a m , L . L . , D e p a r t m e n t of M i c r o b i o l o g y , U n i v e r s i t y of M i a m i , School of M e d i c i n e , M i a m i , F l o r i d a , U.S.A. Williams,
CM.,
Biological
Laboratories, Harvard U n i v e r s i t y ,
Cambridge,
Massa-
c h u s e t t s , U.S.A. Wyatt, G.R., D e p a r t m e n t of B i o l o g y , Q u e e n ' s U n i v e r s i t y , K i n g s t o n , O n t a r i o , C a n a d a . Wyatt, S.S., D e p a r t m e n t of B i o l o g y , Q u e e n ' s U n i v e r s i t y , K i n g s t o n , O n t a r i o , C a n a d a .
xiii
Preface The p r e s e n t v o l u m e c o m p r i s e s t h e P r o c e e d i n g s of t h e IV I n t e r n a t i o n a l C o n f e r e n c e on I n v e r t e b r a t e T i s s u e C u l t u r e , h e l d J u n e 5-8, 1975 at Mont G a b r i e l , Q u e b e c , C a n a d a . The s c h e d u l i n g of t h i s c o n f e r e n c e , as w e l l as t h e c h o i c e of l o c a t i o n , w e r e c o o r d i n a t e d so as to f o l l o w t h e A n n u a l M e e t i n g of t h e T i s s u e C u l t u r e A s s o c i a t i o n h e l d at t h e U n i v e r s i t é de M o n t r é a l . Since t h e P r o c e e d i n g s of t h e e a r l i e r c o n f e r e n c e s w e r e not w i d e l y d i s t r i b u t e d , i t seems a p p r o p r i a t e t o p r e s e n t here a b r i e f h i s t o r y of t h i s series. The F i r s t I n t e r n a t i o n a l C o n f e r e n c e on I n v e r t e b r a t e T i s s u e C u l t u r e w a s h e l d O c t o ber 2 2 - 2 4 , 1962 at M o n t p e l l i e r , France. It w a s o r g a n i z e d under t h e a u s p i e c e s of t h e C e n t r e N a t i o n a l de la Recherche S c i e n t i f i q u e , t h e I n s t i t u t N a t i o n a l de la Recherche A g r o n o m i q u e of France, t h e I n s t i t u t Pasteur, and t h e U n i v e r s i t é de M o n t p e l l i e r . The papers p r e s e n t e d at t h i s m e e t i n g appeared as s p e c i a l v o l u m e of t h e A n n a l e s d e s E p i p h y t i e s , P a r i s , 1963. The Second C o n f e r e n c e w a s h e l d at V i l l a C a r l o t t a , T r e m e z z o , C o m o , I t a l y , September 9 - 1 0 , 1 9 6 7 , under the s p o n s o r s h i p of t h e I t a l i a n A c a d e m y of S c i e n c e s a n d t h e I n s t i t u t e Lombardo ( B a s e l I i F o u n d a t i o n ) . The P r o c e e d i n g s of t h i s c o n f e r e n c e w e r e p u b l i s h e d in a s e p a r a t e , s o f t - c o v e r v o l u m e in M i l a n o in 1968. The T h i r d C o n f e r e n c e w a s h e l d in S m o l e n i c e near B r a t i s l a v a , C z e c h o s l o v a k i a , J u n e 2 2 - 2 5 , 1 9 7 1 , under t h e a u s p i e c e s of t h e S l o v a k A c a d e m y of S c i e n c e s and t h e I n s t i t u t e of V i r o l o g y at B r a t i s l a v a . The P r o c e e d i n g s appeared in 1973 as a s e p a r a t e v o l u m e , p r o d u c e d by t h e P u b l i s h i n g H o u s e of t h e S l o v a k A c a d e m y of S c i e n c e s . The s c o p e of t h e Fourth I n t e r n a t i o n a l C o n f e r e n c e w a s to d i s c u s s i n v e r t e b r a t e org a n , t i s s u e , and c e l l c u l t u r e , i t s l i m i t a t i o n s , p i t f a l l s , p r e s e n t and p o t e n t i a l a p p l i c a t i o n s in m e d i c i n e , b i o l o g y , and a g r i c u l t u r e , in s t u d i e s of m o r p h o g e n e s i s , d i f f e r e n t i a t i o n , v i r u s e s , s y m b i o n t s , p a r a s i t e s , and n e u r o p h y s i o l o g y . T h i s t i m e , t h e s p o n s o r s h i p i n c l u d e d the U n i v e r s i t é de M o n t r é a l , t h e N a t i o n a l I n s t i t u t e s of H e a l t h in B e t h e s d a , M a r y l a n d , U . S . A . , t h e N a t i o n a l Research C o u n c i l of C a n a d a , O t t a w a , t h e M e d i c a l Research C o u n c i l of C a n a d a , O t t a w a , and t h e F a c u l t y of M e d i c i n e of t h e U n i v e r s i t é de M o n t r é a l . The program w a s arranged by Prof. E. Kurstak of t h e U n i v e r s i t é de M o n t r é a l and Prof. Κ. Maramorosch of Rutgers - t h e State U n i v e r s i t y of New J e r s e y , w h o a c t e d as c o n f e r e n c e c o - c h a i r m e n . They w e r e a s s i s t e d by an I n t e r n a t i o n a l A d v i s o r y C o m m i t t e e , c o n s i s t i n g of Prof. C. B a r i g o z z i ( I t a l y ) , Dr. A . D ü b e n d o r f e r ( S w i t z e r l a n d ) , Prof. G. E c h a l i e r ( F r a n c e ) , Dr. P. F a u l k n e r ( C a n a d a ) , Dr. T . D . C . Grace ( A u s t r a l i a ) , Prof. E. Hadorn ( S w i t z e r l a n d ) , Dr. J . M i t s u h a s h i ( J a p a n ) , Dr. J . Peleg ( I s r a e l ) , Dr. D. Peters ( N e t h e r l a n d s ) , Dr. J . Rehàcek ( C z e c h o s l o v a k i a ) , Dr. K.A. Harrap ( U . K . ) , and Dr. I.V. T a r a s e v i c h ( U . S . S . R . ) . Papers w e r e p r e s e n t e d by 62 s c i e n t i s t s from B e l g i u m , C a n a d a , C z e c h o s l o v a k i a , Federal R e p u b l i c of Germany, F r a n c e , H u n g a r y , I n d i a , J a p a n , N e t h e r l a n d s , P o l a n d , S w i t z e r l a n d , U n i t e d K i n g d o m , U . S . A . , and U.S.S.R.
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PREFACE New a p p l i c a t i o n s of i n v e r t e b r a t e t i s s u e c u l t u r e in m e d i c i n e and in b i o l o g y , t h e l a t e s t d e v e l o p m e n t s in m o l l u s c t i s s u e c u l t u r e , c e l l c l o n i n g , and t h e i r a p p l i c a t i o n s t o t h e study of v i r u s e s and m i c r o s p i r i d i a n s w e r e among t h e h i g h l i g h t s of t h e c o n f e r e n c e . The m e e t i n g brought t o g e t h e r t h e o u t s t a n d i n g and most a c t i v e research l e a d e r s in i n v e r t e b r a t e c e l l , t i s s u e and organ c u l t u r e and p r o v i d e d a d i r e c t c o n t a c t of t h e s e e x p e r t s w i t h younger w o r k e r s and a t t e n d i n g graduate s t u d e n t s . New a v e n u e s of research w e r e e x p l o r e d and s u g g e s t i o n s made f o r n o v e l a p p r o a c h e s t o a d v a n c e t h e f i e l d . Throughout the c o n f e r e n c e d i s c u s s i o n s w e r e i n t e n s e and i l l u m i n a t i n g , l a s t i n g u n t i l l a t e at n i g h t , and r a i s i n g new q u e s t i o n s and c h a l l e n g e s . In order t o make t h i s i n f o r m a t i o n a v a i l a b l e to t h e w i d e s t p o s s i b l e s c i e n t i f i c c o m m u n i t y , i t w a s d e c i d e d t o p u b l i s h the i n v i t e d and c o n t r i b u t e d p a p e r s , in an e x p a n d e d , e d i t e d f o r m , as a h a r d - c o v e r b o o k , rather than as c o n f e r e n c e p r o c e e d i n g s . A v o l u m e d e v o t e d t o the b a s i c a s p e c t s of i n v e r t e b r a t e t i s s u e c u l t u r e and c o m p l e m e n t i n g t h e p r e s e n t o n e , i s b e i n g p u b l i s h e d at the same t i m e by A c a d e m i c Press under t h e t i t l e : Invertebrate Tissue Culture in Basic Research. The e d i t o r s f e l t t h a t i t w a s of u t m o s t i m p o r t a n c e t o p u b l i s h t h e o r i g i n a l p a p e r s , often in a much longer v e r s i o n , as w e l l as t h e c o m p r e h e n s i v e and i n c i s i v e r e v i e w s of i n v e r t e b r a t e t i s s u e c u l t u r e a p p l i c a t i o n s w i t h no d e l a y , and at a r e a s o n a b l e p r i c e . These g o a l s w e r e a c h i e v e d , t h a n k s t o t h e generous support of t h e o r i g i n a l s p o n s o r s of t h e C o n f e r e n c e , and t h e e f f i c i e n c y of the P u b l i s h e r . The e d i t o r s hope t h a t t h i s book w i l l be u s e f u l and s t i m u l a t i n g , and w i l l p r o v i d e in a s i n g l e v o l u m e t h e l a t e s t r e s u l t s o b t a i n e d in the d i v e r s e areas of r e s e a r c h p u r s u e d by the l e a d i n g e x p o n e n t s of i n v e r t e b r a t e t i s s u e c u l t u r e from A m e r i c a , Europe, A s i a , and A u s t r a l i a . The p r e s e n t a t i o n of t h e l a t e s t t e c h n i q u e s u s e d in l a b o r a t o r i e s around t h e w o r l d w i l l a l s o be of i m m e d i a t e v a l u e in f u r t h e r i n g s t u d i e s in i n f e c t i o u s d i s e a s e s and p o s s i b l y lead t o the d e v e l o p m e n t of new methods of disease control. The v o l u m e p r o v i d e s the most recent i n f o r m a t i o n on s o p h i s t i c a t e d l a b o r a t o r y met h o d s and on numerous u t i l i z a t i o n s of i n v e r t e b r a t e c e l l c u l t u r e t e c h n i q u e s . A p p l i c a t i o n s to t h e study of a r b o v i r u s e s , m a l a r i a l p a r a s i t e b i o l o g y , t h e use of s n a i l c e l l l i n e s in S c h i s t o s o m a w o r k , a p p l i c a t i o n s of t h e s e t e c h n i q u e s t o e m b r y o l o g y , genet i c s , e n d o c r i n o l o g y and p h y s i o l o g y are o n l y a f e w of t h e f a s c i n a t i n g areas i n c l u d e d . T h i s book w i l l be of i n t e r e s t to many, i n c l u d i n g r e s e a r c h e r s and s t u d e n t s in m e d i cal and b i o m e d i c a l s c i e n c e s , such as v i r o l o g y , i m m u n o l o g y , p a t h o l o g y , p a r a s i t o l o gy, e n d o c r i n o l o g y , d e v e l o p m e n t a l b i o l o g y , m i c r o b i o l o g y , e n t o m o l o g y , p l a n t p a t h o l o g y , and b i o l o g i c a l c o n t r o l of v e c t o r s . The p r e s e n t a t i o n s of c u r r e n t s and of t h e m o s t r e c e n t r e s u l t s of t h e l e a d i n g invert e b r a t e t i s s u e c u l t u r e e x p e r t s , t h e i r own i n t e r p r e t a t i o n s and o r i g i n a l c o n c l u s i o n s , as w e l l as t h e i n c l u s i o n of numerous i l l u s t r a t i o n s make t h i s book a t i m e l y s o u r c e of i n f o r m a t i o n and b r i n g i n t o sharp f o c u s t h e r a p i d l y m o v i n g f r o n t i e r and new d i r e c t i o n s of i n v e r t e b r a t e t i s s u e c u l t u r e . The c h a i r m e n of the Fourth I n t e r n a t i o n a l C o n f e r e n c e on I n v e r t e b r a t e T i s s u e C u l t u r e and e d i t o r s of t h i s book w i s h to e x p r e s s t h e i r g r a t i t u d e t o a l l c o n t r i b u t o r s f o r the e f f o r t and c a r e , as w e l l as p r o m p t n e s s w i t h w h i c h t h e y have prepared t h e i r c h a p t e r s ; to t h e F a c u l t y of M e d i c i n e of t h e U n i v e r s i t é de M o n t r é a l , t o t h e U n i v e r s i t é de M o n t r é a l , to t h e I n s t i t u t e of A l l e r g y and I n f e c t i o u s D i s e a s e s , N a t i o n a l I n s t i t u t e s of H e a l t h , Bethesda, M a r y l a n d , to t h e N a t i o n a l Research C o u n c i l of Canada and t o t h e M e d i c a l Research C o u n c i l of C a n a d a , f o r t h e i r f i n a n c i a l s u p p o r t ; t o Dr. M a u r i c e L ' A b b é , V i c e - R e c t e u r f o r Research of t h e U n i v e r s i t é de M o n t r é a l , t o
xv i
PREFACE Dr. Pierre B o i s , Dean of t h e F a c u l t y of M e d i c i n e , t o D r . G a s t o n de L a m i r a n d e , V i c e Dean f o r Research of t h e F a c u l t y of M e d i c i n e of t h e U n i v e r s i t é de M o n t r é a l , a n d t o Dr. Sorin Sonea, D i r e c t o r of t h e D e p a r t m e n t of M i c r o b i o l o g y and Immunology of t h e U n i v e r s i t é de M o n t r é a l , for t h e i r s u p p o r t in t h e o r g a n i z a t i o n of t h e c o n f e r e n c e a n d e d i t i n g m a t t e r s . Our t h a n k s are e x t e n d e d t o M i s s G h i s l a i n e Montagne f o r her p a r t in e d i t i n g t h i s v o l u m e and l a s t , b u t not l e a s t , t o t h e s t a f f of A c a d e m i c Press f o r t h e i r p a r t in t h e p r o d u c t i o n o f t h e v o l u m e .
Prof. Edouard Kurstak Prof. Karl Maramorosch
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Chapter 1
ARBOVIRUS STUDIES IN INVERTEBRATE CELL LINES
S.M. B u c k l e y , C . G . H a y e s , Ü.M. M a l o n e y , M. Lipman, T . H . G . A i t c k e n , and J . C a s a l s
I.
Introduction
3
II.
Kinetic aspects of arbovirus multiplication
4
III.
Persistence
IV.
Diagnostic
5 10
tools
12
V.
Contamination
VI.
Conclusions
14
VII.
References
17
I.
...
Introduction
By d e f i n i t i o n , " a r b o v i r u s e s are v i r u s e s w h i c h are m a i n t a i n e d in n a t u r e p r i n c i p a l l y or t o an important e x t e n t , through b i o l o g i c a l t r a n s m i s s i o n b e t w e e n s u s c e p t i b l e v e r t e b r a t e h o s t s by hematophagous a r t h r o p o d s " (WHO S c i e n t i f i c G r o u p , 1 9 6 7 ) 0 A t t h e end of 1 9 7 4 , 350 a r b o v i r u s e s w e r e r e c o g n i z e d . These h e t e r o g e n e o u s v i r u s e s , i n c o r p o r a t e d i n t o a general s y s t e m of v i r u s c l a s s i f i c a t i o n ( C a s a l s , 1 9 7 1 ) , h a v e been arranged f o r the most p a r t i n t o groups of r e l a t e d , but d i s t i n c t a g e n t s . In a s much as c h a r a c t e r i z e d by s e r o l o g y , m o r p h o l o g y , m o r p h o g e n e s i s , b i o c h e m i s t r y and b i o p h y s i c s , arboviruses belong to six taxons: alphavirus, bunyavirus, f l a v i v i r u s , i r i d o v i r u s , O r b i v i r u s , and r h a b d o v i r u s . The range of n a t u r a l v e c t o r s e x t e n d s from m o s q u i t o e s t o t i c k s , p h l e b o t o m i n e s and Culicoides (Casa\s„ 1 9 7 1 ) . Since t h e d o u b l e b r e a k - t h r o u g h w i t h regard t o h a e m o l y m p h - f r e e medium ( M i t s u h a s h i - M a r a m o r o s c h , 1964) and e s t a b l i s h m e n t of s t a b l e c e l l l i n e s f r o m Aedes m o s q u i t o e s ( G r a c e , 1966; S i n g h , 1967; P e l e g , 1 9 6 8 ) , in vitro s t u d i e s of v i r u s e s in i n v e r t e b r a t e t i s s u e c u l t u r e s y s t e m s have i n c r e a s e d in p a r a l l e l w i t h t h e p r o d u c t i o n of new i n v e r t e b r a t e c e l l l i n e s . A c c o r d i n g t o H i n k ( 1 9 7 2 ) , D i p t e r a c e l l l i n e s are r e p r e s e n t e d by 14 s p e c i e s w i t h primary e x p i a n t s d e r i v e d from e m b r y o s , l a r v a e , i m a g i n a i d i s c s , a d u l t o v a r i e s , and Aedes pseudoor a d u l t s p e c i e s . Two i n v e r t e b r a t e s p e c i e s , Aedes malayensis scutellaris, both b e l o n g i n g t o the scute/fan's subgroup of the s u b g e n u s Stegomyia, h a v e been used r e c e n t l y f o r t h e e s t a b l i s h m e n t of t w o new Aedes c e l l l i n e s by Varma et ai. ( 1 9 7 4 ) . A real b r e a k - t h r o u g h , h o w e v e r , is t h e e s t a b l i s h m e n t of t h r e e c e l l l i n e s from t h e t i c k Rhipicephalus appendiculatus by Varma et al.(1974). Thus, a r b o v i r u s s t u d i e s c a n be c a r r i e d out in p a r a l l e l in c e l l l i n e s d e r i v e d from t w o i m p o r t a n t v e c t o r s , i . e . , m o s q u i t o and t i c k . Recent and c o m p r e h e n s i v e r e v i e w s have c o v e r e d a r t h r o p o d c e l l c u l t u r e s and t h e i r v a l u e in a r b o v i r u s in vitro s t u d i e s ( S i n g h , 1 9 7 1 , 1 9 7 2 ; Yunker, 1 9 7 1 ; R e h a c e k , 1972; D a l g a r n o and D a v e y , 1973; B u c k l e y , 1 9 7 6 ) . In t h i s p r e s e n t a t i o n , a d e t a i l e d survey of the l i t e r a t u r e i s , t h e r e f o r e , not a t t e m p t e d . Examples i n d i c a t i v e of t h e p r e v a l e n t d i r e c t i o n of r e s e a r c h are p r e s e n t e d .
3
S. M. BUCKLEY et al. II.
Kinetics
of a r b o v i r u s
multiplication
Studies of q u a n t i t a t i v e a s p e c t s of a r b o v i r u s m u l t i p l i c a t i o n in i n v e r t e b r a t e c e l l l i n e s l o g i c a l l y depend on p r e l i m i n a r y i n v e s t i g a t i o n s w i t h regard t o growth or n o n g r o w t h of an agent in a p a r t i c u l a r i n v e r t e b r a t e c e l l System» The s u s c e p t i b i l i t y of D i p t e r a c e l l l i n e s t o members of the d i f f e r e n t t a x o n s of a r b o v i r u s e s has been r e v i e w e d r e c e n t l y ( B u c k l e y , 1976)„ B r i e f l y , t h e f o l l o w i n g d e t e r m i n a n t s w e r e f o u n d t o be i m p o r t a n t : 1) innate d i f f e r e n c e s between i n v e r t e b r a t e c e l l l i n e s , and 2) h e t e r o g e n e i t y of v i r u s e s w i t h regard t o a) v e c t o r and b) p r e s e n c e or a b s e n c e of a v i rion envelope,, Enveloped a r b o v i r u s e s are s e n s i t i v e t o t h e a c t i o n of s o d i u m d e o x y c h o l a t e or r e l a t i v e l y r e s i s t a n t ( T h e i l e r , 1957; C a s a l s , 1 9 7 1 ; Borden et al., 1 9 7 1 ) ; u n e n v e l o p e d a r b o v i r u s e s ( o r b i v i r u s e s ) are r e l a t i v e l y r e s i s t a n t (Borden et al., 1 9 7 1 ) , T h u s , in as much as r e p o r t e d , u n e n v e l o p e d o r b i v i r u s e s m u l t i p l y in S i n g h ' s Aedes albopictus c e l l s ( S i n g h , 1 9 6 7 ) , r e g a r d l e s s of v e c t o r ; m o s q u i t o - o r Culicoides-bome e n v e l o p e d a l p h a , b u n y a - , f l a v i - a n d r h a b d o v i r u s e s reproduce a l s o 0 H o w e v e r , e n v e loped t i c k - b o r n e f l a v i v i r u s e s or e n v e l o p e d t i c k - o r p h l e b o t o m i n e - b o r n e b u n y a v i r u s e s cell f a i l to m u l t i p l y „ T a b l e 1 summarizes r e s u l t s o b t a i n e d in t h e Aedes albopictus l i n e . In i n f e c t i o n e x p e r i m e n t s w i t h t h e t i c k c e l l l i n e T T C - 2 4 3 , Varma et al. (1974) r e p o r t e d r e p l i c a t i o n of t h e f l a v i v i r u s e s West N i l e , L o u p i n g i l l , Langat as w e l l a s of the s t i l l u n c l a s s i f i e d t i c k - b o r n e v i r u s Q u a r a n f i L W h i l e West N i l e v i r u s has been i s o l a t e d from d i f f e r e n t m o s q u i t o e s , T a y l o r et aL, (1956) s u c c e e d e d in i n f e c t i n g savigni w i t h West N i l e v i r u s and on one o c c a s i o n were a b l e t o o b t a i n Ornithodorus t r a n s m i s s i o n of the v i r u s f o l l o w i n g e x p e r i m e n t a l f e e d i n g of t h e i n f e c t e d t i c k s on s u s c e p t i b l e v e r t e b r a t e s . It w o u l d be of great i n t e r e s t t o i n v e s t i g a t e growth or n o n g r o w t h of A f r i c a n s w i n e f e v e r v i r u s , a t i c k - b o r n e D N A v i r u s of t h e i r i d o v i r u s t a x o n , in a t i c k c e l l l i n e . TABLE 1 Growth
Taxon
No.
of arboviruses
of
arboviruses
in Singh's
AEDES ALBOPICTUS
cell
Proven
suspected
to SDC
or
Sensitive
cultures
or
Growth
tested
vector*
alphavirus
8
mosquito
sensitive
yes
( 8/8
)
bunyavirus
3
mosquito
sensitive
yes
( 3/3
)
f lavivirus
relatively
resistant
11
mosquito
Orbivirus
3
mosquito
rhabdovirus
4
mosquito
sensitive
sensitive relatively
resistant
yes
(11/11)
yes
(
yes
( 4/4 )
3/
3)
bunyavirus
3
tick
sensitive
no
( 0/3
f lavivirus
3
tick
sensitive
no
( 0/3
)
Orbivirus
5
tick
(
5/5
)
not
5
tick
( 0/5
)
bunyavirus
3
phlebotomine
Orbivirus
9
phlebotomine
classified
rhabdovi rus
1
Culicoides
Orbivirus
1
Culicoides
* In a d d i t i o n t o h a v i n g been i s o l a t e d from m o s q u i t o e s , viruses (VSV subgroup)
have also
been
relatively
resistant
sensitive sensitive relatively
resistant
sensitive relatively bunyavirus
resistant
4
no no
( 0/3
)
yes
( 9/9
)
yes
( 1/1 )
yes
( 1/1 )
Ganjam and some
isolated from t i c k s and phlebotomine
tively.
yes
)
flies
rhabdorespec-
ARBOVIRUS STUDIES IN INVERTEBRATE CELL LINES A r e v i e w p e r t a i n i n g t o k i n e t i c s , b i o c h e m i s t r y , a n d u l t r a s t r u c t u r a l s t u d i e s in i n v e r t e b r a t e and i n v e r t e b r a t e c e l l c u l t u r e s w i t h s p e c i a l r e f e r e n c e t o a l p h a v i r u s e s such as S e m l i k i F o r e s t , S i n d b i s , eastern e q u i n e e n c e p h a l i t i s , w e s t e r n e q u i n e e n c e p h a l i t i s , V e n e z u e l a n e q u i n e e n c e p h a l i t i s and t o a f l a v i v i r u s , K u n j i n , has been p u b l i s h e d by D a l g a r n o and Davey ( 1 9 7 3 ) . O p t i m a l t o g a v i r u s r e p l i c a t i o n in i n v e r t e b r a t e and v e r t e b r a t e c e l l s y s t e m s w a s , a p p a r e n t l y , r e l a t e d t o o p t i m u m t e m p e r a t u r e of c e l l g r o w t h , h o w e v e r , i n v e r t e b r a t e c e l l l i n e s m a i n t a i n e d by i n d i v i d u a l i n v e s t i g a t o r s as w e l l as h e t e r o g e n e i t y of a r b o v i r u s e s , v i r a l s t r a i n , v i r a l p a s s a g e h i s t o r y , m u l t i p l i c i t y of i n f e c t i o n are f a c t o r s d e t e r m i n i n g t h e s p e c i f i c s of k i n e t i c s o f v i r a l g r o w t h in vitro in a d d i t i o n t o t e m p e r a t u r e of i n c u b a t i o n . E x a m p l e s w i t h regard t o a r b o v i r u s e s b e l o n g i n g to f i v e d i f f e r e n t t a x o n s h a v e been d i s c u s s e d in d e t a i l by Buckley (1975). III.
Persistence
S p e c u l a t i n g on o r i g i n and e v o l u t i o n of a r b o v i r u s e s , A n d r e w s (1973) s t a t e s : " i t seems a l m o s t c e r t a i n t h a t one is d e a l i n g e i t h e r w i t h i n s e c t - p a r a s i t e s w h i c h have b e c o m e s e c o n d a r i l y a d a p t e d t o l i v i n g in v e r t e b r a t e s of the other w a y r o u n d . The f i r s t a l t e r n a t i v e seems p r e f e r a b l e " . In a n a l o g y t o t h e in vivo s i t u a t i o n , inapparent p e r s i s t e n t i n f e c t i o n of m o s q u i t o c e l l c u l t u r e s may be i n d u c e d w i t h any a r b o v i r u s alboc a p a b l e of r e p l i c a t i o n in a g i v e n c e l l s y s t e m ( B u c k l e y , 1976) s u c h as Aedes pictus and Aedes aegypti ( m o n o l a y e r ; h o l l o w v e s i c l e s ) ( S i n g h , 1 9 6 7 ) , Aedes aegypti ( P e l e g , 1 9 6 8 ) , and Aedes w-a/bus (Singh and Bhat, 1 9 7 1 ) . 1 . A l p h a v i r u s . Production of c a r r i e r c u l t u r e s of S i n g h ' s Aedes albopictus cell line i n f e c t e d w i t h d i f f e r e n t a r b o v i r u s e s has been d e s c r i b e d f i r s t by Banerjee and Singh ( 1 9 6 8 ) . The same i n v e s t i g a t o r s , s u b s e q u e n t l y , r e p o r t e d l o s s of mouse v i r u l e n c e in Chikungunya v i r u s , an a l p h a v i r u s from t h e c a r r i e r c u l t u r e of Aedes albopictus cell l i n e (Banerjee and S i n g h , 1 9 6 9 ) . We h a v e been a b l e t o c o n f i r m t h e i r r e s u l t s , u s i n g t h e same C h i k u n g u n y a v i r u s ( s t r a i n I 6 3 4 0 2 9 ; 4th mouse p a s s a g e ) and an Aedes albopictus s u b l i n e brought p e r s o n n a l y t o t h e Yale A r b o v i r u s Research U n i t b y monolayer c u l Singh in Spring 1 9 7 0 . B r i e f l y , primary i n f e c t i o n of Aedes albopictus t u r e s w a s i n i t i a t e d w i t h a p p r o x i m a t e l y 20 p l a q u e f o r m i n g u n i t s (PFU) of the O r i e n t a l s t r a i n . C u l t u r e s w e r e i n c u b a t e d at 3 0 ° C f o r 4 days and t h e r e a f t e r at room t e m p e r a t u r e ( 2 0 - 2 5 ° C ) . I n f e c t e d c a r r i e r c u l t u r e s as w e l l as u n i n o c u l a t e d c o n t r o l c u l t u r e s w e r e t r a n s f e r r e d at w e e k l y i n t e r v a l s by a 1:8 s p l i t r a t i o . C e l l s in p e r s i s t e n t l y i n f e c t e d c u l t u r e s w e r e i n d i s t i n g u i s h a b l e from t h e u n i n o c u l a t e d c u l t u r e s w i t h regard t o morphology and growth p o t e n t i a l . A t each t r a n s f e r l e v e l , f l u i d p h a s e s of t w o c u l t u r e s w e r e p o o l e d , c e n t r i f u g e d (2000 RPM f o r 10 m i n u t e s ) and a s s a y e d f o r t h e p r e s e n c e of v i r u s in 2 - d a y - o l d m i c e , in BHK-21 c e l l c u l t u r e s (Karabatsos and Buckl e y , 1967) and in V e r o c e l l c u l t u r e s ( K a r a b a t s o s , 1969). The i n f e c t i v i t y t i t e r s e x p r e s s e d as " d e x " ( d e c i m a l e x p o n e n t ) ( H a l d a n e , 1960) per ml are l i s t e d in T a b l e 2 . T i t e r s in mice s t a r t e d t o d e c r e a s e as of t h e 5th t r a n s f e r ; f l u i d s of t h e 9th and 10th t r a n s f e r s i n d u c e d an o c c a s i o n a l death in m i c e w h i c h w e r e o b s e r v e d f o r 14 d a y s p o s t - i n o c u l a t i o n . No s i g n i f i c a n t p l a q u e f o r m i n g u n i t (PFU) or t i s s u e c y t o p a t h i c d o s e (TCD)50 t i t e r d i f f e r e n c e s w e r e n o t i c e d in t h e v e r t e b r a t e c e l l l i n e s w i t h one e x c e p t i o n (3rd t r a n s f e r ; BHK-21) in w h i c h poor q u a l i t y c u l t u r e s w e r e u s e d . In t h e V e r o plaque a s s a y s , p l a q u e s i z e s w e r e u n i f o r m up to the 3rd t r a n s f e r in w h i c h a s m a l l plaque (SP) v a r i a n t w a s o b s e r v e d , m e a s u r i n g 3 χ 3 mm in d i a m e t e r . The numbers of SP i n c r e a s e d d u r i n g s u b s e q u e n t t r a n s f e r s w i t h 6 / 1 0 p l a q u e s m e a s u r i n g 3 χ 3 mm and 4 / 1 0 p l a q u e s r e p r e s e n t i n g large p l a q u e s (LP), m e a s u r i n g 5 χ 5 mm and 6 χ 6 mm, at t h e 8th t r a n s f e r l e v e l (10"5 d i l u t i o n ) . In a p r e v i o u s s t u d y ( B u c k l e y , 1973a) w i t h the high mouse b r a i n p a s s a g e C h i k u n g u n y a v i r u s (Ross s t r a i n ; A f r i c a n 5
S. M . B U C K L E Y et al.
TABLE
Infectivity
titers
I 634029-infected
of Chikungunya fluid
phases
virus,
Material
chik.
(I 6 3 4 0 2 9 )
strain
of transfer
I 634029,
levels
L D 5 *0 (stock)
2
8.5
No.
mouse
brain
tissue
stock,
1-10 AEDES ALBOPICTUS dex per ml
cultures.
T C D 50
PFU 9.0
9.3
1st
transfer
6.2
7.5
6.5
2nd
transfer
4.9
7.4
6.2
3rd
transfer
5.7
6.4
4.5
4th
transfer
5.0
5.2
5.3
5th
transfer
3.4
6.5
5.5
6th
transfer
2.6
7.2
6.3
7th
transfer
2.5
5.5
5.7
8th
transfer
2.2
6.2
6.5
9th
transfer
trace
6.9
5.7
10th
transfer
trace
6.9
6.3
* determined in 2-day-old
and of
carrier
mice
o r i g i n ) , it w a s shown t h a t c o n t i n u e d s u b c u l t u r i n g of Aedes albopictus carrier c u l t u r e v i r u s f o r 800 d a y s in vitro r e s u l t e d in a change of v i r u l e n c e f o r 3 - d a y - o l d m i c e . With t h e A f r i c a n s t r a i n in c o n t r a s t t o t h e I n d i a n s t r a i n , t h e e x t e n t t o w h i c h v i r u l e n c e w a s l o s t depended upon the t r a n s f e r l e v e l and s p e c i f i c a l l y upon p l a q u e s i z e as a n a l y z e d in V e r o c e l l s . Two in vitro m a r k e r s , SP and LP v a r i a n t s of Chikungunya v i r u s were c h a r a c t e r i z e d , , Progenies of t h e LP v a r i a n t w e r e c o m p a r a b l e in mouse p a t h o g e n i c i t y t o the parent s t r a i n , w h e r e a s p r o g e n i e s of t h e SP v a r i a n t f a i l e d t o i n d u c e i l l n e s s in newborn m i c e . In a more r e c e n t s t u d y ( B u c k l e y et al, 1976K t h e LP v a r i a n t t e n t e d to m o d i f y t o w a r d the SP v a r i a n t . On a c o m p a r a t i v e b a s i s , t h e Indian s t r a i n of Chikungunya (I 634029) has m o d i f i e d much more e a s i l y w i t h regard t o p a t h o g e n i c i t y for newborn m i c e than t h e A f r i c a n s t r a i n (Ross). S e r o l o g i c a l l y , i t has been shown by C a s a l s (1961) t h a t O r i e n t a l s t r a i n s of Chikungunya d i f f e r s l i g h t l y from A f r i c a n s t r a i n s . A t t e n u a t i o n d u r i n g p e r s i s t e n c e in m o s q u i t o c e l l c u l t u r e s has been reported a l s o f o r S e m l i k i Forest v i r u s by Peleg (1971 ) and by D a v e y and D a l g a r n o ( 1 9 7 4 ) . 2 . R h a b d o v i r u s . A r t s o b and Spence (1974a) e s t a b l i s h e d a p e r s i s t e n t i n f e c t i o n w i t h v e s i c u l a r s t o m a t i t i s v i r u s ( V S V ) , r h a b d o v i r u s t a x o n , in S i n g h ' s Aedes aegypti a n d Aedes albopictus c e l l l i n e s . In t h e r a b i e s serogroup (Shope et aL„ 1970; Shope, 1974; Shope et al.f 1975), a subgroup w i t h i n t h e r h a b d o v i r u s t a x o n , O b o d h i a n g v i uniformis) and k o t o n k a n v i r u s ( i s o l a t e d from Culicoirus ( i s o l a t e d from Mansonia des) (Kemp et al., 1973) m u l t i p l y in S i n g h ' s Aedes albopictus c e l l l i n e , but not in Aedes aegypti c e l l s ( B u c k l e y , 1973b). A t h i r d member, Mokola v i r u s ( i s o l a t e d f r o m Croc/dura s p . ) (Kemp et al., 1972) b e h a v e s s i m i l a r l y ( B u c k l e y , 1976); a t p r e s e n t , t h e r e is no other e v i d e n c e i n d i c a t i v e o f an arthropod c y c l e in t h e m a i n t e n a n c e of M o k o l a v i r u s . A c c o r d i n g t o Murphy ( 1 9 7 4 ) , the in vivo s i t e of b u d d i n g f o r t h e t w o p r e s u m p t i v e a r b o v i r u s e s is t h e plasma membrane, w h e r e a s Mokola v i r u s b u d s
6
A R B O V I R U S S T U D I E S IN I N V E R T E B R A T E C E L L LINES
Fig. 2 . I n f e c t i o n o f V e r o c e l l s w i t h c l o n e d O b o d h i a n g v i r u s . V i r a l a n t i g e n l o c a l i z e d s p e c i f i c a l l y in t h e c y t o p l a s m by i m m u n o f l u o r e s c e n c e .
t h r o u g h and a c q u i r e s i t s outer c o a t both from p l a s m a membrane as w e l l as f r o m t h e e n d o p l a s m i c r e t i c u l u m . N e g r i - l i k e b o d i e s w e r e f o u n d w i t h M o k o l a v i r u s in i n f e c t e d monkey brain (Percy et ah, 1971). In i n f e c t e d V e r o c e l l c u l t u r e s , c y t o p l a s m i c i n c l u s i o n b o d i e s w e r e f o u n d by i m m u n o f l u o r e s c e n c e as w e l l as by a t i n c t o r i a l method ( G i e m s a ) w i t h both k o t o n k a n and O b o d h i a n g v i r u s e s . F i g u r e s 1 and 2 i l l u s t r a t e r e s u l t s o b t a i n e d w i t h O b o d h i a n g v i r u s . The methods used here w e r e t h e same as d e s c r i b e d in d e t a i l e l s e w h e r e ( B u c k l e y and C l a r k e , 1970; B u c k l e y and cells with cloned kotonC a s a l s , 1 9 7 0 ) . P e r s i s t e n t i n f e c t i o n of Aedes albopictus kan, Obodhiang and M o k o l a v i r u s e s ( B u c k l e y and T i g n o r , 1975) w a s e s t a b l i s h e d as f o l l o w s : m o s q u i t o c e l l s w e r e i n f e c t e d w i t h c l o n e d k o t o n k a n and O b o d h i a n g v i r u s e s a t a m u l t i p l i c i t y of i n f e c t i o n (MOI) of one PFU per 1,000 c e l l s . Primary i n f e c t i o n w a s s u c c e s s f u l as a s s a y e d both by p l a q u e a s s a y in V e r o c e l l s and mouse i n o c u l a t i o n w i t h c e n t r i f u g e d f l u i d phase of i n f e c t e d c u l t u r e s . These c u l t u r e s w e r e k e p t at room t e m p e r a t u r e ( 2 0 - 2 5 ° C ) f o r 2 4 months p r i o r t o i n i t i a t i o n of t r a n s f e r s .
7
S. M. B U C K L E Y et al. Primary i n f e c t i o n of m o s q u i t o c e l l s w i t h c l o n e d Mokola v i r u s w a s e s t a b l i s h e d at a MOI of one PFU per 20 c e l l s . T r a n s f e r s of t h e i n f e c t e d c e l l s w a s c a r r i e d o u t a p p r o x i m a t e l y t h r e e months f o l l o w i n g primary i n f e c t i o n . S u b s e q u e n t l y , a l l t h r e e c a r r i e r c u l t u r e s w e r e t r a n s f e r r e d r e g u l a r l y at 2 - w e e k - i n t e r v a l s by a l o w s p l i t of 1:2. U n i n f e c t e d c o n t r o l c e l l s w e r e t r a n s f e r r e d in p a r a l l e l . Two t e m p e r a t u r e s of i n c u b a t i o n were r o u t i n e l y c o m b i n e d : 3 0 ° C for 4 days f o l l o w e d by room temperature ( 2 0 - 2 5 ° C ) f o r ten d a y s ; c a r r i e r c u l t u r e s were f e d o n c e b e t w e e n t r a n s f e r s . A s d e t e r m i n e d by i n f e c t i o u s c e n t e r a s s a y in V e r o c e l l s by methods d e s c r i b e d p r e v i o u s l y ( p e l e g , 1969; L i k i k o v a and B u c k l e y , 1971) o n l y a s m a l l p e r c e n t a g e of c e l l s w a s cells i n f e c t e d as documented in Table 3. Figures 3, 4 , and 5 show Aedes albopictus p e r s i s t e n t l y i n f e c t e d w i t h c l o n e d k o t o n k a n , O b o d h i a n g and Mokola v i r u s and s t a i n e d by the i n d i r e c t method (Weiler and C o o n s , 1954) of t h e f l u o r e s c e n t a n t i b o d y t e c h n i q u e . While s p e c i f i c s t a i n i n g of k o t o n k a n - and O b o d h i a n g - i n f e c t e d c e l l s w a s d i f f u s e , b r i g h t c y t o p l a s m i c b o d i e s as w e l l as d i f f u s e i m m u n o f l u o r e s c e n c e w a s o b s e r v e d in M o k o l a - i n f e c t e d c e l l s . Mokola c a r r i e r c u l t u r e s r e m a i n e d p a t h o g e n i c for 2-day-old m i c e , whereas kotonkan-and Obodhiang carrier cultures lost pathogen i c i t y for i n f a n t m i c e in as much as t e s t e d ; moreover, O b o d h i a n g - a n d k o t o n k a n c e l l s f a i l e d t o p r o d u c e a n t i b o d i e s in m i c e p e r s i s t e n t l y i n f e c t e d Aedes albopictus aegypti f o l l o w i n g one i n t r a c e r e b r a l i n o c u l a t i o n . A l l c a r r i e r c u l t u r e s i n f e c t e d Aedes m o s q u i t o e s in one e x p e r i m e n t in w h i c h k o t o n k a n v i r u s w a s r e c o v e r e d both by p l a que a s s a y in V e r o c e l l s as w e l l as by mouse i n o c u l a t i o n 12 days p o s t - i n o c u l a t i o n , O b o d h i a n g 9 days and Mokola 11 d a y s a f t e r the i n o c u l a t i o n of m o s q u i t o e s , r e s p e c t i v e l y . A i t k e n ( u n p u b l i s h e d o b s e r v a t i o n ) has shown t h a t both kotonkan and O b o d h i a n g v i r u s e s i n f e c t Aedes aegypti by i n t r a t h o r a c i c i n o c u l a t i o n and m u l t i p l i e d in s a l i v a r y g l a n d s . A t t h e t i m e of the w r i t i n g of t h i s c o m m u n i c a t i o n , Mokola v i r u s
TABLE 3 Percentage of AEDES ALBOPICTUS Mokola virus Virus
Strain
cells
Transfer level of infected eel Is
containing
C e l l s per ml o f tested cell suspension 6 (Χ 1 0 )
infective
kotonkan,
PFU* per ml o f tested cell s u s p e n s3i o n (Χ 1 0 )
Obodhiang,
Ratio o f infective to non i n f e c t iive cells
and
Percentc of 1 infecti cells
kotonkan
lb A n 23380
6
7.50
2.5
1/3000
0.03
kotonkan
l b A n 23380
10
10.0
1/184
kotonkan
lb A n 23380
11
1.84 5.80
39.0
1/149
0.55 0.77
6
7.60
27.5
1/276
0.36
CO
9.40
10.0
1/940
0.11
10
1.14
37.5
1/30
3.33
Obodhiang
Sud A r 1154-61
Obodhiang
Sud A r 1154-61
Obodhiang
Sud A r 1154-61
Mokola Mokola
lb A n 27377
2 5
8.60 9.00
190.0 425.0
1/45 1/21
2.22
lb A n 27377
Mokola
l b A n 27377
8
19.60
940.0
1/20
5.00
* PFU = p l a q u e f o r m i n g u n i t s ( p l a q u e a s s a y i n V e r o c e l l s )
8
4.76
ARBOVIRUS STUDIES IN INVERTEBRATE CELL LINES has p r o p a g a t e d p e r s i s t e n t l y in the Aedes albopictus f o r 15 m o n t h s ; a l t h o u g h an a r t h r o p o d v e c t o r has not been f o u n d , in vitro e v i d e n c e p r e s e n t e d here p o i n t s t o t h e f a c t t h a t Mokola v i r u s in a l l p r o b a b i l i t y i s an a r b o v i r u s , r e l a t e d t o r a b i e s (Shope et al., 1970) and known t o be p a t h o g e n i c f o r homo s a p i e n s ( F a m i l u s i and Moore, 1972).
Fig. 3. I m m u n o f l u o r e s c e n c e p a t t e r n o f Aedes cloned kotonkan virus; transfer # 1 8 .
albopictus
cells persistently infected with
Fig. 4. I m m u n o f l u o r e s c e n c e p a t t e r n o f Aedes cloned Obodhiang virus; transfer # 1 8 .
albopictus
cells persistently infected with
9
S. M. B U C K L E Y et al.
Fig. 5. I m m u n o f l u o r e s c e n c e p a t t e r n o f Aedes cloned Mokola virus; transfer # 1 2 .
IV.
albopictus
cells persistently
infected with
Diagnostic tools
M o s q u i t o c e l l s have been f o u n d r e f r a c t o r y t o i n f e c t i o n w i t h v i r u s e s not b e l o n g i n g t o a r b o v i r u s t a x o n s ( S i n g h , 1 9 7 2 ) ; t h u s , i n v e r t e b r a t e c e l l s y s t e m s can be used a d v a n t a g e o u s l y in a s s i s t i n g in the i d e n t i f i c a t i o n of unknown v i r u s s t r a i n s i s o l a t e d i n the f i e l d or g e n e r a l l y in the c h a r a c t e r i z a t i o n of v i r u s e s . D u r i n g L a s s a v i r u s s t u d i e s ( B u c k l e y and C a s a l s , 1 9 7 0 ) , the f a c t t h a t the a g e n t f a i l e d t o m u l t i p l y in Aedes c e l l s made i t l i k e l y t h a t the a g e n t w a s n o t an a r b o v i r u s . L i k e w i s e , t h e f a c t t h a t Mokola v i r u s , r a b i e s serogroup (Shope et al., 1 9 7 0 ) , p r o p a g a t e d p e r s i s t e n t l y in m o s q u i t o c e l l s f o r 15 months f a v o r s t h e h y p o t h e s i s of an a r b o v i r u s a l though an arthropod v e c t o r may never be f o u n d . S i n g h ' s Aedes c e l l l i n e s have been an a i d t o us r e c e n t l y in t h e c h a r a c t e r i z a t i o n of an unknown v i r u s s t r a i n ( U . V . Str.) w h i c h w a s k i n d l y sent t o the Yale A r b o v i r u s Research U n i t (YARU) by t h e M i c r o b i o l o g i c a l Research E s t a b l i s h m e n t , Porton D o w n , S a l i s b u r y , E n g l a n d . The U . V . Str. had been i s o l a t e d from " a pool of 100 Mansonia uniformis c o l l e c t e d in Kampong T i j i r a k , Sarawak on 15 J u l y , 1 9 6 9 " ( S i m p s o n , personal communication). Apparently, adaptation to mice was a l i t t l e troublesome. A t YARU, a t e n t a t i v e d i a g n o s i s of a r h a b d o v i r u s w a s q u i c k l y s u r m i s e d based on t h e f a c t t h a t the U . V . Str. m u l t i p l i e d b e s t in Aedes aegypti c e l l s . It has been shown by Buckley ( 1 9 6 9 ) , Singh (1971) and A r t s o b and Spence (1974b) t h a t in the rhabdov i r u s t a x o n , v i r u s e s b e l o n g i n g t o the v e s i c u l a r s t o m a t i t i s subgroup m u l t i p l y in both Aedes c e l l l i n e s w i t h t h e Aedes aegypti c e l l l i n e a p p a r e n t l y b e i n g more s e n s i t i v e . B r i e f l y , Aedes c e l l s as w e l l as v e r t e b r a t e BHK-21 and V e r o c e l l s grown in large L e i g h t o n t u b e c u l t u r e s w e r e i n o c u l a t e d w i t h a 1:1000 d i l u t i o n of the i n f e c t e d mouse b r a i n s u s p e n s i o n . Two d a y s p o s t - i n o c u l a t i o n , t h e i n f e c t e d f l u i d phases w e r e a s s a y e d f o r v i r u s in 2 - d a y - o l d m i c e . The i n f e c t e d c e l l s w e r e f i x e d w i t h a c e t o n e and s t a i n e d by the i n d i r e c t method (Weiler and C o o n s , 1954) of i m m u n o f l u o r e s c e n c e . The procedures used in t h i s l a b o r a t o r y h a v e been d e s c r i b e d in d e t a i l e l s e w h e r e ( B u c k l e y and C l a r k e , 1 9 7 1 ) . S p e c i f i c s t a i n i n g w a s most b r i l l i a n t in the c y t o p l a s m
10
A R B O V I R U S STUDIES IN I N V E R T E B R A T E C E L L LINES of i n f e c t e d Aedes aegypti c e l l s as d e m o n s t r a t e d in Figure 6 and a l s o s h o w n in T a b l e 4 . Moreover, u n d i l u t e d i n f e c t i v e f l u i d phase of s u c h Aedes aegypti c u l t u r e s k i l l e d a l l m i c e w i t h an average s u r v i v a l t i m e of 1„9 d a y s . E x a m i n a t i o n of s e c t i o n e d c e l l s in t h e e l e c t r o n m i c r o s c o p e r e v e a l e d i n f e c t e d BHK-21 and Aedes aegypti r h a b d o v i r u s - l i k e s t r u c t u r e s . M u l t i p l i c a t i o n of the U . V . Str. in Aedes aegypti c e l l s w a s n o t i n h i b i t e d by the i n c o r p o r a t i o n of 5 - b r o m o d e o x y u r i d i n e in t h e medium (Webb et ahg 1 9 6 7 ) ; hence t h e v i r u s p r o b a b l y c o n t a i n s r i b o n u c l e i c a c i d . A p e r s i s t e n t c e l l s in a d d i t i o n i n f e c t i o n w a s e s t a b l i s h e d w i t h t h e U . V . Str. in Aedes albopictus t o Aedes aegypti c e l l s ; t h u s , it is u n l i k e l y t h a t t h e a g e n t b e l o n g s t o t h e r a b i e s s e r o g r o u p , subgroup of the r h a b d o v i r u s t a x o n , in as much as it has been shown t h a t k o t o n k a n , O b o d h i a n g a n d Mokola v i r u s i n f e c t Aedes albopictus c e l l s only (Buckley, 1976), but d o n ' t m u l t i p l y in Aedes aegypti c e l l s . The f a c t t h a t the U . V . Str. m u l t i p l i e d in t h e m o s q u i t o c e l l s s t r o n g l y s u g g e s t s t h a t i t is a t r u e a r b o v i r u s . The a g e n t may be a new r h a b d o v i r u s , as i t d i d not r e a c t by c o m p l e m e n t - f i x a t i o n w i t h any of t h e immune r e a g e n t s a v a i l a b l e in the l a b o r a t o r y . P r e s e n t a t i o n here of some of t h e c h a r a c t e r i s t i c s of t h e U . V . Str. is not in any w a y i n t e n d e d t o s u p e r c e d e the d e f i n i t i v e d e s c r i p t i o n and i d e n t i f i c a t i o n of the v i r u s w h i c h w i l l be done by the M i c r o b i o l o g i c a l Research E s t a b l i s h m e n t , Porton D o w n , S a l i s b u r y , E n g l a n d . TABLE 4 Mouse pathogenicity and immunofluorescence systems with U.V.Str. Cells inoculated with infected mouse brain suspension
observed
in invertebrate
Mouse Pathogenicity Mortality Average Survival time 2-day-old-mice (days)
16/16
1.9
12/16
1.9
BHK-21
5/16
3.4
Vero
1/16
3.0
Aedes Aedes
aegypti albopictus
* I m m u n o f l u o r e s c e n c e graded o n a s c a l e of
-Γ++,
and vertebrate
cell
Immunofluorescence* (cytoplasmic)
-H+ -H-
+ +
Ή - , and +
Pavri and Ghose (1969) s h o w e d t h a t u n t r e a t e d f l u i d p h a s e s o b t a i n e d from Aedes albopictus c e l l c u l t u r e s i n o c u l a t e d w i t h a r b o v i r u s e s can be u s e d s a t i s f a c t o r i l y as a n t i g e n s f o r v i r a l i d e n t i f i c a t i o n in t h e c o m p l e m e n t - f i x a t i o n (CF) t e s t . S u b s e q u e n t l y , Singh and Paul (1969) and C a s a l s and B u c k l e y (1973) used t h i s t e c h n i q u e s u c c e s s f u l l y to i d e n t i f y dengue v i r u s e s , t y p e s I t o I V , i s o l a t e d d i r e c t l y in m o s q u i t o c e l l s from f i e l d m a t e r i a l . R e c e n t l y , A j e l l o et al. (1975) have e x t e n d e d t h i s t e c h n i q u e t o West N i l e v i r u s . B r i e f l y , CF a n t i g e n w i t h t i t e r s up t o 1:128 w a s d e m o n s t r a t e d in Aedes albopictus c e l l c u l t u r e s i n o c u l a t e d w i t h the Egypt 101 s t r a i n , 11th mouse b r a i n p a s s a g e . The CF a n t i g e n w a s s p e c i f i c . There w e r e no c r o s s r e a c t i o n s w i t h t i c k - b o r n e e n c e p h a l i t i s mouse s e r u m . The s e n s i t i v i t y of t h e s y s t e m w a s d o c u m e n t e d by d e m o n s t r a t i n g t h a t West N i l e v i r u s d i l u t e d t o 8 dex ( H a l d a n e , 1960) m u l t i p l i e d in the m o s q u i t o c e l l s as a s c e r t a i n e d b o t h by V e r o p l a q u e a s s a y as w e l l as by CF r e a c t i o n . A s a t i s f a c t o r y CF a n t i g e n has r e c e n t l y been o b t a i n e d in t h i s l a b o r a t o r y a l s o w i t h f l u i d phases of Aedes albopictus c e l l c u l t u r e s i n o c u l a t e d w i t h an O r i e n t a l s t r a i n of Chikungunya v i r u s (I 6 3 4 0 2 9 ) . In our h a n d s , such a n t i g e n s have not been a n t i - c o m p l e m e n t a r y at any t i m e .
11
S. M. B U C K L E Y et al.
Fig.
V.
6.
I m m u n o f l u o r e s c e n c e p a t t e r n w i t h U . V . S t r . i n Aedes
aegypti
c e l l s ; f i r s t passage»
Contamination
In v e r t e b r a t e c e l l c u l t u r e s , a c y t o p a t h i c e f f e c t (CPE) has been d e s c r i b e d w i t h a r b o v i r u s e s of the s i x t a x o n s . Under f l u i d m e d i u m , CPE c o n s i s t s of moderate t o marked c e l l d e s t r u c t i o n (Scherer and S y v e r t o n , 1954; B u c k l e y , 1964; Karabatsos a n d B u c k l e y , 1 9 6 7 ) , whereas under n u t r i e n t agar o v e r l a y p l a q u e f o r m a t i o n is i n d u c e d ( D u l b e c c o , 1952; S t i m , 1969). A d d i t i o n of s p e c i f i c a n t i b o d y t o c e l l c u l t u r e s a l o n g w i t h t h e v i r a l i n o c u l u m p r e v e n t s t h e d e v e l o p m e n t of CPE or p l a q u e s . With i n v e r t e brate c e l l c u l t u r e s , CPE (Paul et al., 1 9 6 9 ; Suitor and P a u l , 1 9 6 9 ; Varma et al., 1974) or p l a q u e s ( S u i t o r , 1969; Cory and Yunker, 1972; Yunker and C o r y , 1975) c e l l l i n e ( S i n g h , 1967) and have a l s o been d e s c r i b e d in S i n g h ' s Aedes albopictus and Aedes r e c e n t l y in t w o new c e l l l i n e s e s t a b l i s h e d f r o m Aedes ma/ayensis pseudoscutellaris (Varma et a/., 1 9 7 4 ) . What is p u z z l i n g w i t h regard t o c e l l u l a r damage is the f a c t t h a t t h e e x t e n t of t h e CPE seems t o depend on t h e i n d i v i d u a l s u b l i n e s u s e d and a l s o on the t y p e of c o n t a i n e r ( i . e . g l a s s - o r p l a s t i c v e s s e l ) in A. malayensis or A. pseudoscutellaris c e l l s are w h i c h the Aedes albopictus, grown (Suitor and P a u l , 1969; Varma et al., 1 9 7 4 ) . A c c o r d i n g to D a l g a r n o and c e l l s o n l y s h o w CPE " w h e n D a v e y ( 1 9 7 3 ) , a r b o v i r u s - i n f e c t e d Aedes albopictus s t r e s s e d in a p a r t i c u l a r w a y " . T h u s , dengue v i r u s , t y p e 2 , f a i l e d t o p r o d u c e CPE i n the hands of S i n a r a c h a t a n a n t and O l s e n ( 1 9 7 3 ) , w h e r e a s CPE w i t h t h e same v i r u s w a s reported b y Paul et aL (1969) and c o n f i r m e d b y S u i t o r and Paul (1969) as w e l l as by Sweet and U n t h a n k , 1 9 7 1 ) . That the CPE c a u s e d by a s t r e s s s i t u a t i o n m i g h t be dependent on l a t e n t v i r a l c o n t a m i n a t i o n came to l i g h t when t w o i n s t i t u t i o n s . The Boyce Thompson I n s t i t u t e , Yonkers ( H i r u m i , p e r s o n a l c o m m u n i c a t i o n ) and Purdue U n i v e r s i t y , West L a f a y e t t e (Webb, p e r s o n a l c o m m u n i c a t i o n ) r e p o r t e d albopictus s p o n t a n e o u s s y n c y t i a f o r m a t i o n in t w o s u b l i n e s of un i n o c u l a t e d Aedes c e l l s , here d e s i g n a t e d " H i r u m i " and " W e b b " s u b l i n e s . In the m e a n t i m e , i s o l a t i o n of C h i k u n g u n y a v i r u s c o n t a m i n a t i n g the " W e b b " s u b l i n e has been d e s c r i b e d ( C u n ningham et al., 1 9 7 5 ) .
12
A R B O V I R U S S T U D I E S IN I N V E R T E B R A T E C E L L L I N E S
We report here on some f u r t h e r s t u d i e s w i t h regard t o t h e " H i r u m i " s u b l i n e and i t s c o n t a m i n a t i n g a g e n t , i s o l a t e d and i d e n t i f i e d by CF t e s t a n d p l a q u e r e d u c t i o n n e u t r a l i z a t i o n t e s t as C h i k u n g u n y a v i r u s and c h a r a c t e r i z e d as a p a t h o g e n i c f o r n e w born m i c e . A s d e t e r m i n e d b y i n f e c t i o u s c e n t e r a s s a y in V e r o c e l l s b y methods d e s c r i b e d ( P e l e g , 1969; L i b i k o v a and B u c k l e y , 1 9 7 1 ) , a p p r o x i m a t e l y 1.6% of a l l t h e c e l l s were i n f e c t e d w i t h Chikungunya v i r u s w h i c h w a s l o c a l i z e d s p e c i f i c a l l y in t h e c y t o p l a s m as d e t e r m i n e d b y the i n d i r e c t method (Weiler and C o o n s , 1954) of i m m u n o f l u o r e s c e n c e by p r o c e d u r e s d e s c r i b e d in d e t a i l ( B u c k l e y and C l a r k e , 1970) (See Figure 7 ) . The f l u i d phase of the " H i r u m i " c e l l l i n e , t r a n s f e r # 3 , i n d u c e d h a z y p l a q u e s ( F i g u r e 8) in V e r o c e l l s . In a t t e m p t i n g t o r e s t o r e mouse p a t h o g e n i c i t y , t h e Chikungunya i s o l a t e w a s p l a q u e - p u r i f i e d in V e r o c e l l s and p a s s e d for 17 p a s s a g e s in BHK-21 c e l l s . A t t h i s t i m e , large and c l e a r p l a q u e s w e r e o b t a i n e d in V e r o c e l l s (Figure 9 ) ; h o w e v e r , the v i r u s s t r a i n f a i l e d t o i n d u c e i l l n e s s in 2 - d a y - o l d m i c e b y i n t r a c e r e b r a l i n o c u l a t i o n . S u b s e q u e n t l y , b l i n d v i r u s p a s s a g e s in newborn m i c e were c a r r i e d o u t at 3-4 day i n t e r v a l s . Mouse b r a i n s u s p e n s i o n s w e r e p l a q u e a s s a y e d in V e r o c e l l s for p r e s e n c e of v i r u s . Some of t h e i n o c u l a t e d m i c e w e r e observed f o r 24 d a y s a f t e r i n o c u l a t i o n , then s a c r i f i c e d under deep ether a n e s t h e s i a and t h e i r r e s p e c t i v e sera e x a m i n e d f o r d e v e l o p m e n t of n e u t r a l i z i n g a n t i b o d i e s by plaque r e d u c t i o n n e u t r a l i z a t i o n t e s t in V e r o c e l l s . The r e s u l t s are s u m m a r i z e d in Table 5. V i r u s w a s p r e s e n t t h r o u g h s e v e n p a s s a g e s and d i s a p p e a r e d t h e r e a f t e r . N o d e a t h s w e r e o b s e r v e d . The p l a q u e f o r m i n g u n i t (PFU) t i t e r s v a r i e d f r o m 2.2 t o 4.9 dex per m l . Mice of t h e f i r s t t o t h e s i x t h p a s s a g e l e v e l d e v e l o p e d n e u t r a l i z i n g a n t i b o d i e s . I n a c t i v a t e d , u n d i l u t e d sera reduced the p l a q u e c o u n t s f r o m 60 t o 100%; h o w e v e r , as c a n be seen from t h e r e c i p r o c a l of serum t i t e r s o b t a i n e d by the 50% plaque r e d u c t i o n m e t h o d , the v i r u s w a s a poor immunogen. Ten s e r i a l in vivo p a s s a g e s of t h e a v i r u l e n t Chikungunya v i r u s w e r e made t h e r e after in Aedes aegypti. B r i e f l y , f e m a l e m o s q u i t o e s w e r e i n o c u l a t e d i n t r a t h o r a c i c a l l y w i t h a p p r o x i m a t e l y 2 5 , 0 0 0 PFU of the c l o n e d v i r u s . Seven days a f t e r i n o c u l a t i o n , t e n m o s q u i t o e s were r e m o v e d , p o o l e d and t r i t u r a t e d in 2 . 0 ml of d i l u e n t (phosphate b u f f e r e d saline, pH 7 . 2 , containing 0.75% b o v i n e albumin, f r a c t i o n V ) . The undiluted s u p e r n a t a n t of t h i s s u s p e n s i o n w a s i n o c u l a t e d i n t r a t h o r a c i c a l l y into a new batch of m o s q u i t o e s and a l s o p l a q u e - a s s a y e d in V e r o c e l l s . M o s q u i t o e s from each p a s s a g e l e v e l s were a l l o w e d t o f e e d on newborn m i c e ; in a d d i t i o n , m o s q u i t o s u s p e n s i o n s were i n o c u l a t e d i n t r a c e r e b r a l l y into i n f a n t m i c e . The l a t t e r w e r e o b s e r v e d f o r d e v e l o p m e n t of i l l n e s s , t h e n s a c r i f i c e d 21 d a y s after i n o c u l a t i o n as d e s c r i b e d above f o r a s s a y s of n e u t r a l i z i n g a n t i b o d i e s . The r e s u l t s are s u m m a r i z e d in Table 6 . B r i e f l y , t h e v i r u s r e p l i c a t e d w e l l in Aedes aegypti at a l l p a s s a g e l e v e l s w i t h no apprec i a b l e c h a n g e in the maximum PFU t i t e r o b t a i n e d in t h e m o s q u i t o e s t h r o u g h o u t t h e e x p e r i m e n t . Plaques in V e r o c e l l s were a l s o m o n i t o r e d a t each p a s s a g e l e v e l f o r c h a n g e in s i z e , s i n c e the c l o n e d p r e p a r a t i o n used i n i t i a l l y had been s e l e c t e d f o r large p l a q u e s i z e in an a t t e m p t t o r e s t o r e mouse p a t h o g e n i c i t y . H o w e v e r , d u r i n g t h e ten s e r i a l in vivo p a s s a g e s both large and s m a l l p l a q u e s w e r e p r o d u c e d . T h i s c h a r a c t e r i s t i c was c o n s t a n t w i t h o u t u n d e r g o i n g any s e l e c t i o n w h a t s o e v e r d u r i n g t h e s e e x p e r i m e n t s . The v i r u s remained a v i r u l e n t f o r 2 - d a y - o l d m i c e t h r o u g h a l l ten p a s s a ge l e v e l s in m o s q u i t o e s . S p e c i f i c n e u t r a l i z i n g a n t i b o d i e s w e r e p r e s e n t in t h e sera of mice i n o c u l a t e d i n t r a c e r e b r a l l y w i t h l o w d i l u t i o n s of i n f e c t e d m o s q u i t o s u s p e n s i o n s and s a c r i f i c e d t h r e e w e e k s p o s t - i n o c u l a t i o n . P o s i t i v e t r a n s m i s s i o n of v i r u s a l s o o c c u r r e d as e v i d e n c e by t h e d e v e l o p m e n t of n e u t r a l i z i n g a n t i b o d i e s in m i c e exposed t o t h e b i t e s of i n f e c t e d m o s q u i t o e s at v a r i o u s p a s s a g e l e v e l s . Based on t h e s e r e s u l t s i t c a n be s t a t e d t h a t an a p p a r e n t l y s t a b l e mutant of Chikungunya v i r u s has been i s o l a t e d from t h e u n i n o c u l a t e d Aedes albopictus "Hirumi" 13
S. M. B U C K L E Y etat.
Fig. 7. I m m u n o f l u o r e s c e n c e p a t t e r n of Aedes albopictus c e l l s , " H i r u m i " subline, Transfer # 3 , p e r s i s t e n t l y c o n t a m i n a t e d w i t h an a v i r u l e n t C h i k u n g u n y a v i r u s s t r a i n .
s u b l i n e . The main c h a r a c t e r i s t i c s are a p a t h o g e n i c i t y f o r newborn m i c e as w e l l as poor i m m u n o g e n i c i t y . In the l a t t e r r e s p e c t , t h i s v i r u s s t r a i n d i f f e r s from the a t t e n u a t e d C h i k u n g u n y a v i r u s (Ross s t r a i n ) ( B u c k l e y , 1973) and the a t t e n u a t e d S e m l i k i Forest v i r u s ( P e l e g , 1 9 7 1 ) , both c h a r a c t e r i z e d as good i m m u n o g e n s . On a n a t i o n a l and i n t e r n a t i o n a l l e v e l , t h i s l a b o r a t o r y has d i s t r i b u t e d S i n g h ' s Aedes c e l l l i n e s s i n c e 1968. It i s r e m a r k a b l e t h a t t h e m a j o r i t y of i n v e s t i g a t o r s h a v e requested a d d i t i o n a l s h i p m e n t s of c e l l s due to the f a c t t h a t t h e i r c e l l l i n e " h a d become r e f r a c t o r y " t o the p a r t i c u l a r v i r u s t h e y had been s t u d y i n g . T h i s s t a t e of r e f r a c t o r i n e s s may be e x p l a i n e d , p e r h a p s , by homologous i n t e r f e r e n c e . A s p o i n t ed out by Peleg ( 1 9 7 5 ) , c e l l s in S i n d b i s ( a l p h a v i r u s ) p e r s i s t e n t l y i n f e c t e d c u l t u r e s , s u b c u l t u r e d or n o t , are i n d i s t i n g u i s h a b l e from c e l l s in u n i n o c u l a t e d c o n t r o l c u l t u r e s of the same age in r e s p e c t t o growth p o t e n t i a l and m o r p h o l o g y . Yet t h e s e c e l l s are r e s i s t a n t to s u p e r i n f e c t i o n by the homologous v i r u s . These t w o i n v e s t i g a t o r s have f o u n d f u r t h e r t h a t the t r a n s i t i o n to a s t a t e of c e l l u l a r r e s i s t a n c e in c u l t u r e s c o i n c i d e d w i t h t h e appearance in S i n d b i s v i r u s i n f e c t e d Aedes aegypti t h e c u l t u r e of a s m a l l p l a q u e - f o r m i n g mutant d e s i g n a t e d SV-S. The c l o n e d SV-S w a s shown t o i n t e r f e r e w i t h t h e g r o w t h of t h e w i l d s t r a i n of S i n d b i s v i r u s (SV-W) i n a number of i n v e r t e b r a t e and v e r t e b r a t e c e l l c u l t u r e s . T h u s , t h e a c c i d e n t a l , u n i n t e n t i o n a l c o n t a m i n a t i o n of an i n v e r t e b r a t e c e l l l i n e w i t h any a r b o v i r u s s t u d i e d in any laboratory at any t i m e appears to b e t h e g r a v e s t hazard e n c o u n t e r e d in i n v e r t e b r a t e eel I c u l t u r e w o r k . VI.
Conclusions
T w o a s p e c t s appear t o w a r r a n t s p e c i a l c o n s i d e r a t i o n . F i r s t l y , of t h e l a r g e number of r e g i s t e r e d a r b o v i r u s e s (350 as of 1 9 7 4 ) , c o n c l u s i v e e v i d e n c e t h a t t h e y are de facto arthropod-borne v i r u s e s is a v a i l a b l e f o r r e l a t i v e l y f e w . C l a s s i f i c a t i o n of v i r u s e s , g e n e r a l l y , is based e s s e n t i a l l y on t h e p r o p e r t i e s of the v i r i o n and t h e i n t e r a c t i o n of v i r u s and host at t h e c e l l u l a r l e v e l . U s u a l l y , s e r o l o g i c e v i d e n c e i s t h e
14
A R B O V I R U S S T U D I E S IN I N V E R T E B R A T E C E L L LINES
Fig. 8. Plaque m o r p h o l o g y o b s e r v e d i n V e r o c e l l s w i t h t h e f l u i d p h a s e of Aedes c e l l s , " H i r u m i " s u b l i n e , Transfer # 3 .
albopictus
Fig. 9. Plaque m o r p h o l o g y o b s e r v e d in V e r o c e l l s w i t h t h e a v i r u l e n t C h i k u n g u n y a v i r u s s t r a i n f o l l o w i n g c l o n i n g i n V e r o c e l l s a n d 17 a d d i t i o n a l h i g h - d i l u t i o n p a s s a g e s in BHK-21 c e l l s ; apathogenic for newborn mice.
most c o m p l e t e l i n e of c h a r a c t e r i z a t i o n n e x t to morphology and m o r p h o g e n e s i s 0 We h a v e d e m o n s t r a t e d here t h e p r a c t i c a l i t y and u s e f u l n e s s of m o s q u i t o c e l l s as a h o s t s y s t e m in t h e rapid c h a r a c t e r i z a t i o n o f an u n k n o w n v i r u s s t r a i n i s o l a t e d from Mansonia un/'formis. With t h e e s t a b l i s h m e n t of m o s q u i t o - and more r e c e n t l y of t h r e e t i c k - c e l l l i n e s , the a v a i l a b i l i t y of i n v e r t e b r a t e t i s s u e c u l t u r e as a d i a g n o s t i c t o o l i n a d d i t i o n t o m o s q u i t o e s s h o u l d be mandatory in any l a b o r a t o r y engaged in arbov i r u s s t u d i e s . A t t h e p r e s e n t t i m e , t h e s e e s t a b l i s h e d i n v e r t e b r a t e c e l l l i n e s are as
15
S. M. B U C K L E Y et al.
TABLE 5 I nfectivity
titers
production
of complement-fixing
obtained
during
blind (CF)
Number
I nfectivity titers
of
( d e x per ml )
passages
of mou se-avirulent
or neutralizing
antibodies
Chikungunya
as well
as
(NT)
Antibody production
blind
L D 50
PFU
CF
passage
(mouse)
(Vero)
(serum
% plaque
NT
di I.
reduction
1:2)
(serum u n d i l u t e d )
50% reduction titer**
0
4.7
0
90
8
0
4.3
0
100
< 2
3
0
4.8
0
75
< 2
4
0
2.2
0
90
8
5
0
4.9
0
90
8 < 2
CO
1 2
0
3.4
0
60
7
0
3.1
nd
nd
nd
8
0
0
nd
nd
nd
9
0
0
nd
nd
nd
10
0
0
nd
nd
nd
* M i c e b l e d 20 t o 24 d a y s after i n o c u l a t i o n * * Serum t i t e r s e x p r e s s e d as t h e r e c i p r o c a l of t h e h i g h e s t s e r u m d i l u t i o n g i v i n g a 50% r e d u c t i o n in t h e p l a q u e c o u n t
TABLE 6 Infectivity titers obtained during passages as well as transmission status and plaque cells Number of
Infectivity titers
Chikungunya in AEDES passage levels obtained
Plaque s i z e
( d e x per m l ) PFU L D 50
range (mm)
AEGYPTI, in Vero
Transmission* status
mean (mm)
(mouse)
(Vero)
1
0
6.4
1-7
4.3
2
0
5.9
3-8
5.5
CO
mosquito intrathoracic passage number
of mouse-aviru/ent size at individual
0
5.3
3-6
4.5
4
0
5.8
1-7
3.3
+ + + + + +
5
0
5.7
1-6
3.9
6
0
5.5
2-6
3.7
7
0
5.8
1-6
3.0
nd**
8
0
5.9
1-5
2.3
nd
9
0
5.6
2-5
3.5
nd
10
0
5.3
1-5
2.6
nd
* M i c e b l e d 21 d a y s a f t e r b e i n g f e d on by i n f e c t e d m o s q u i t o e s * * nd = not done
16
A R B O V I R U S S T U D I E S IN I N V E R T E B R A T E C E L L L I N E S
e a s y t o h a n d l e as v e r t e b r a t e c e l l
l i n e s . S e c o n d l y , o n e of t h e r e a l l y s e r i o u s h a z a r d s
l i n k e d w i t h t h e u s e of m o s q u i t o - or m a y b e a l s o t i c k - c e l l arbovirus
c a p a b l e of
replication
i n o n e or t h e o t h e r
l i n e s i s t h e f a c t t h a t any
invertebrate
cell
a l s o a b l e t o i n d u c e a p e r s i s t e n t i n f e c t i o n in a n a l o g y t o t h e in vivo
system
is
situation where
arthropods, once i n f e c t e d , remain infected for l i f e . Thus, extraneous
contamination
o f i n v e r t e b r a t e c e l l l i n e s s h o u l d be m o n i t o r e d on a r o u t i n e b a s i s b y e l e c t r o n m i c r o s c o p y , i m m u n o f l u o r e s c e n c e and i n f e c t i o u s c e n t e r a s s a y i n a v e r t e b r a t e c e l l s y s t e m .
Acknowledgments We
are
gratefully
Garrison, Mrs. V . Mrs.
acknowledging
the
P a r c e l l s , Mr. C.
skillful
K„ G i l s o n . M i c r o p h o t o g r a p h s w e r e k i n d l y
debted t o Mrs. Carmel Bierwirth Previously States
Army
National
unpublished Medical
Institute
of
Allergy
assistance
of
Mrs.
M. and
s u p p l i e d b y G . M a r t i n e . We are i n -
f o r her c o m p e t e n t a s s i s t a n c e w i t h t h e t y p e s c r i p t .
studies
Research
technical
M u l l e n , M r s . E. G i l s o n , M r s . M. M a l h o i t ,
of
and and
the
authors
Development Infectious
were
supported
Command
Diseases
by
the United
(DADA-17-12-C-2170),
(PHS-RO-1-AI
1 0 9 8 4 ) , The
World H e a l t h O r g a n i z a t i o n , and The R o c k e f e l l e r F o u n d a t i o n .
VII.
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a n d I m m u n o l o g y (Ε. W e i s s , e d . ) ,
Chapter 2
TICK TISSUE CULTURE AND ARBOVIRUSES J . Rehacek
I.
Introduction
21
II.
Organ c u l t u r e a n d i t s a p p l i c a t i o n i n v i r o l o g y
22
1. General c o n s i d e r a t i o n s
22
2 . P r e p a r a t i o n of o r g a n c u l t u r e
22
3 . C u l t i v a t i o n of a r b o v i r u s e s i n o r g a n c u l t u r e
23
III.
IV.
4. Prospects of organ c u l t u r e in v i r o l o g i c a l research
23
Hemocyte culture and i t s application in virology
23
1. General considerations
23
2. P r e p a r a t i o n of h e m o c y t e c u l t u r e
23
3 . C u l t i v a t i o n of a r b o v i r u s e s i n t i c k h e m o c y t e s in vitro
24
4 . P r o s p e c t s of h e m o c y t e c u l t u r e i n v i r o l o g y
24
Primary t i s s u e c u l t u r e a n d i t s a p p l i c a t i o n i n v i r o l o g y
24
1 . General c o n s i d e r a t i o n s
24
2 . Source of t i s s u e s a n d c e l l s
24
3. P r e p a r a t i o n of t i s s u e s a n d c e l l s f o r c u l t u r e s
V.
VI.
25
5. C e l l c o m p o s i t i o n a n d g r o w t h i n c u l t u r e
25
6. E l e c t r o n m i c r o s c o p e s t u d y of t i c k c e l l s in vitro
26
7. U t i l i z a t i o n of t h e a m i n o a c i d s a n d s u g a r s b y t i c k c e l l s in vitro
26
8. U s e o f p r i m a r y c e l l c u l t u r e s f o r t h e c u l t i v a t i o n o f v i r u s e s
26
9. P r o s p e c t s o f t i c k p r i m a r y c u l t u r e s i n v i r o l o g y
29
Passage of t i c k c e l l c u l t u r e s
29
1 . General c o n s i d e r a t i o n s
29
2. C u l t u r e of e m b r y o n i c c e l l s
30
3 . C e l l c u l t u r e f r o m o v a r i e s of f e m a l e t i c k s
30
4. C u l t i v a t i o n of v i r u s e s i n t i c k e m b r y o n a l c e l l s
30
5. P r o s p e c t s o f t i c k p a s s a g e c u l t u r e s i n v i r o l o g y
30
C e l l lines from t i c k t i s s u e s
31
1 . General c o n s i d e r a t i o n s
31
2. Cell lines from the t i c k Rhipicephalus appendiculatus
31
3 . C u l t i v a t i o n of a r b o v i r u s e s i n R. a p p e n d i c u l a t u s c e l l l i n e s
31
4 . P r o s p e c t s of t i c k c e l l l i n e s i n v i r o l o g y
32 2
VII. Conclusions
3
V I 11.References I.
24
4. Effects of media c o m p o s i t i o n on t i s s u e c u l t u r e g r o w t h
Introduction T i c k s are v e c t o r s of many m i c r o o r g a n i s m s ( v i r u s e s , r i c k e t t s i a e a n d p r o t o z o a n p a r a -
sites)
pathogenic
t o man a n d a n i m a l s . There
21
is no d o u b t , t h a t t h e c u l t u r e o f t i c k
J. REHACEK t i s s u e s and c e l l s in vitro can be e f f e c t i v e in i n v e s t i g a t i o n s of a number of i n t e r e s t ing and important problems c o n c e r n i n g t h e r e l a t i o n s h i p s b e t w e e n t h e s e p a t h o g e n s and t h e i r man and animal h o s t s . The purpose o f t h i s lecture is to f a m i l i a r i s e you w i t h the present s t a t u s of t i c k c e l l and t i s s u e c u l t u r e in a r b o v i r u s i n v e s t i g a t i o n s . II.
Organ c u l t u r e and i t s a p p l i c a t i o n in v i r o l o g y
1)
General
considerations.
Very l i t t l e a t t e n t i o n has been p r e v i o u s l y p a i d to t h e use of organ c u l t u r e s f r o m t i c k s for c u l t i v a t i o n of v i r u s e s . It is p r o p o s e d t h a t s u c h c u l t u r e s o f f e r a r e a s o n a b l e s u b s t r a t e f o r s t u d i e s of many i n t e r e s t i n g problems in c e l l - v i r u s r e l a t i o n s h i p s . 2)
Preparation
of organ
culture.
A t t e m p t s at the c u l t i v a t i o n of t i c k organs in vitro s u c c e e d e d o n l y in their temporary s u r v i v a l . The f i r s t e x p e r i m e n t s on m a i n t a i n i n g t i c k t i s s u e s in vitro were reported by Weyer, 1952. He c u l t i v a t e d e x p i a n t s of c o n n e c t i v e t i s s u e s and p r o b a b l y t h o s e of other organs of Rhipicephalus bursa (for c u l t i v a t i o n o f r i c k e t t s i a e ) by t h e hanging drop method in a medium c o n s i s t i n g of human or r a b b i t p l a s m a w i t h t h e a d d i t i o n of r a b b i t s p l e e n or t e s t e s e x t r a c t s . C u l t u r e s were k e p t a t 31 - 3 2 ° C . The organs w e r e v i a b l e o n l y f o r a very short t i m e . Organ c u l t u r e s f r o m t i c k s were a l s o d e v e l o p e d as a medium f o r c u l t i v a t i o n of Eastern Equine E n c e p h a l o m y e l i t i s (EEE) v i r u s (Rehâcek, 1 9 5 8 a , b , Rehâcek and Pesek, 1960). The v a r i o u s organs of h a l f - engorged f e m a l e s of Dermacentor pictusr Dermacentor marginatus and Ixodes ricinus c u l t i v a t e d in TC 199 medium at 2 5 ° C ret a i n e d t h e i r v i a b i l i t y for a b o u t 30 d a y s . The organ c u l t u r e s were d e v e l o p e d f u r t h e r f r o m h a l f - e n g o r g e d f e m a l e s of D. p i c t u s for the s t u d y of Coxiella burneti d e v e l o p m e n t from i t s f i l t e r a b l e par» a n d /.ricinus t i d e s (Kordovâ and Rehâcek, 1959). T h e media used in t h e s e e x p e r i m e n t s w e r e Parker 199, and Parker 199 c o n t a i n i n g 25% heated horse serum w i t h or w i t h o u t hemolymph from c o c k r o a c h e s . C o n t r a c t i o n of o v a r i e s and M a l p i g h i a n t u b u l i were o b s e r v e d f o r a p e r i o d of 10 d a y s and a s l i g h t p r o l i f e r a t i o n of f i b r o b l a s t - l i k e c e l l s w a s obs e r v e d in a f e w o v a r y c u l t u r e s . A s e r i e s of e x p e r i m e n t s w i t h o r g a n c u l t u r e s of a d u l t Rhipicephalus appendiculatus t i c k s were performed by M a r t i n and V i d i e r , 1962. The e x p i a n t s of t i c k organs w e r e m a i n t a i n e d in a medium c o n s i s t i n g of H a n k s ' b a lanced s a l t s o l u t i o n , a m i n o a c i d s and v i t a m i n s of Eagle's basal medium w i t h 20% ox serum. Some of t h e e x p i a n t s s u r v i v e d w i t h o u t a c t i v e g r o w t h up to 170 d a y s . The organs of several hard t i c k s p e c i e s - Hya/omma anatolicum excavatum, Hyalomma dromedarii, Rhipicephalus sanguineus, Rhipicephalus evertsi, Boophilus microplus, Boophilus decoloratus and Boophilus annulatus w e r e prepared for s t u d i e s on the maintenance of p i r o p l a s m s in vitro (Hoffmann and K ö h l e r , 1 9 6 8 , Hoffmann et al%v 1970). The e x p i a n t s of o v a r i e s s u r v i v e d in the medium composed of H a n k s ' s o l u t i o n and a m o d i f i e d m i x t u r e of v i t a m i n s and a m i n o a c i d s ( a f t e r Eagle) a t 2 8 ° C f o r 28 - 8 2 d a y s ; s a l i v a r y g l a n d s for 26 - 56 d a y s ; i n t e s t i n a l t r a c t f o r 12 - 46 d a y s a n d M a l p i g h i a n t u b u l i f o r 13 - 17 d a y s . anderThe e x p i a n t s prepared from male a n d f e m a l e g e n i t a l g l a n d s of Dermacentor son/ and B. microplus s u r v i v e d in H L H medium or E a g l e ' s m e d i u m , both w i t h 10% f e -
22
T I C K TISSUE C U L T U R E A N D A R B O V I R U S E S
t a l c a l f serum f o r as long as t w o months and both s e r v e d as t h e s o u r c e s of f i b r o b l a s t and e p i t h e l i a l c e l l s (Rehécek, 1971). 3)
Cultivation
of arboviruses
in organ
cultures.
O n l y t h e EEE a n d N e w c a s t l e D i s e a s e ( N D V ) v i r u s e s w e r e c u l t i v a t e d in s u r v i v i n g e x p i a n t s of v a r i o u s organs of t h r e e s p e c i e s of t i c k s (Rehàcek, 1 9 5 8 a , Rehécek a n d P e s e k , 1960, B l a s k o v i c and Rehàcek, 1962). N o m u l t i p l i c a t i o n of the EEE v i r u s ocand /. ricinus t i c k s i n o c u l a t e d c u r r e d in organ c u l t u r e s of D. pictus, D. marginatus w i t h 1 θ 4 T C I D 5 0 of v i r u s , but v i r u s s u r v i v e d in an a l m o s t u n d i m i n i s h e d t i t e r f o r s i x d a y s . When the c u l t u r e s were i n o c u l a t e d w i t h 1 0 2 . 5 0f 1 0 ^ · ^ . T C I D 5 0 of v i r u s , i t s m u l t i p l i c a t i o n w a s noted in t h e c o n n e c t i v e t i s s u e and h y p o d e r m i s of D. pictus and /. ricinus, w h e r e a s the o t h e r t i s s u e s o n l y s l o w e d d o w n t h e v i r u s i n a c t i v a t i o n . The same amounts of N D V i n o c u l a t e d i n t o t i c k organ c u l t u r e s d i d not m u l t i p l y . The f a c t t h a t i t w a s p o s s i b l e t o d e m o n s t r a t e EEE in t h e c o n n e c t i v e t i s s u e s and hypod e r m i s of D. pictus in s u c h a s m a l l d o s e of t h e v i r u s w h i c h w a s not d e t e c t a b l e in h i g h l y s u s c e p t i b l e s u b s t r a t e s as icer i n o c u l a t e d mice or c h i c k embryo c e l l s , m i g h t be u s e f u l in v i r u s i s o l a t i o n e x p e r i m e n t s . 4)
Prospects
of organ culture
in virological
research.
Most v i r o l o g i s t s l o o k f o r the most c o n v e n i e n t s u b s t r a t e f o r t h e i r w o r k i.e. s u f f i c i e n t amount of a v a i l a b l e c e l l s , h i g h s e n s i t i v i t y and e c o n o m y . In our o p i n i o n , the t i c k organ c u l t u r e s d e s p i t e t h e i r l a b o r i o u s p r e p a r a t i o n are s u c h a s u b s t r a t e , s u i t a b l e namely f o r s p e c i a l s t u d i e s as o b s e r v a t i o n s of how t h e v i r u s p a r t i c l e s p a s s t h r o u g h the gut c e l l s and how t h e y enter t h e c e l l s in w h i c h t h e y m u l t i p l y . A l t h o u g h t i c k organ c u l t u r e s have been r e c e n t l y e s s e n t i a l l y a b a n d o n e d , I propose t h a t f o r e x p e r i m e n t s on the p a t h o g e n i t y o f v a r i o u s v i r u s e s f o r t i c k s , t h e i s o l a t e d organs m a i n t a i n e d in vitro m i g h t be recommended.
III.
Hemocyte c u l t u r e a n d i t s a p p l i c a t i o n in v i r o l o g y
1 ) General considerations. H e m o c y t e s of t i c k s in vitro have been r a r e l y used a s a s u b s t r a t e f o r t h e c u l t i v a t i o n of p a t h o g e n s . The reason f o r t h i s w a s p r o b a b l y t h e s m a l l amount of a v a i l a b l e cells. 2)
Preparation
of hemocyte
culture.
papilliS u r v i v i n g hemocytes of s e v e r a l a r g a s i d t i c k s (Ornithodoros lahorensis, 0. pes and Argas persicus) f o r in vitro s t u d i e s of p h a g o c y t o s i s of b a c t e r i a w e r e used b y S i d o r o v , 1960. In t h e s e e x p e r i m e n t s h e m o c y t e s s u r v i v e d o n l y f o r s e v e r a l d a y s in hemolymph o b t a i n e d from the same t i c k s p e c i e s . The f i r s t hemocyte c u l t u r e in g r e a t e r v o l u m e w a s p r e p a r e d f r o m Hyalomma asiaticum d e v e l o p i n g a d u l t s (Rehàcek, 1 9 6 3 ) . The c e l l s w e r e c u l t i v a t e d in a medium c o m p o s e d of equal parts of E a g l e ' s and V a g o and C h a s t a n g ' s media p l u s 3% dext r a n e or 10% c a l f s e r u m . The c e l l s d i d not m u l t i p l y b u t s u r v i v e d in good c o n d i t i o n f o r about one w e e k . D. andersoni f e m a l e s , h a l f - engorged on r a b b i t s , s e r v e d as a good s o u r c e of h e m o c y t e s in t h e e x p e r i m e n t s of C o r y and Yunker, 1 9 7 1 . The h e m o c y t e s s e t up in HLH medium w i t h 10% f e t a l c a l f serum s u r v i v e d for 72 - 74 d a y s . They became f i b r o b l a s t - l i k e , s p i n d l e shaped or r o u n d e d , b e i n g w e l l d i s p e r s e d or in c l u m p s . The same r e s u l t s were a c h i e v e d in our l a b o r a t o r y .
23
J. R E H A C E K
3)
Cultivation
of arboviruses
in tick
hemocyte s in
The c u l t u r e s prepared from h e m o c y t e s of
vitro.
H. asiaticum
showed m u l t i p l i c a t i o n of
TBE v i r u s , but t o a lower e x t e n t than t h e c u l t u r e s from t i s s u e s of d e v e l o p i n g a d u l t s c o n t a i n i n g e p i t h e l i a l and f i b r o b l a s t - I ike c e l l s (Rehâcek, 1963) andersoni hemocyte c u l t u r e s were used s u c c e s s f u l l y f o r t h e c u l t i Dermacentor v a t i o n of C o l o r a d o t i c k f e v e r (CTF) v i r u s . E x t r a c e l l u l a r v i r u s w a s r e c o v e r e d f r o m c u l t u r e s as long as 62 and 71 d a y s after i n o c u l a t i o n . T w o p e a k s of v i r u s p r o l i f e r a t i o n were d e m o n s t r a t e d . F i r s t , t h e higher peak o c c u r r e d b e t w e e n the 1st - 3rd w e e k s a n d the s e c o n d , lower peak o c c u r r e d 6 w e e k s a f t e r i n o c u l a t i o n . T h e v i r u s d i d not c a u s e any c y t o p a t h i c c h a n g e s in h e m o c y t e s . V i r u s growth c u r v e s r e s e m b l e d t h o s e seen in CTF v i r u s i n f e c t e d primary c u l t u r e s of d e v e l o p i n g a d u l t s (Yunker and C o r y , 1 9 6 7 ) , but t h e y were lower t h a n in t h e l a t t e r c u l t u r e s c a u s e d p r o b a b l y by a l e s s e r amount of c e l l s in c u l t u r e or by absence of c e l l s able t o m u l t i p l y t h e v i r u s . 4)
Prospects
of hemocyte
culture
in
virology.
Because of h e m o c y t e s long v i a b i l i t y a n d good s e n s i t i v i t y t o v i r u s e s , w e recommend t h e i r use f o r a l s o g r o w i n g other pathogens such as p r o t o z o a n p a r a s i t e s and r i c k e t t s i a e . H o w e v e r , t h e l i m i t e d number of c e l l s a v a i l a b l e w i l l a l w a y s i n t e r f e r e w i t h a broader use of hemocyte c u l t u r e s in m i c r o b i o l o g y . IV.
Primary t i s s u e c u l t u r e and i t s a p p l i c a t i o n in v i r o l o g y
1) General
considerations.
This t y p e of c u l t u r e i n c l u d e s the c u l t i v a t i o n of c e l l s and t i s s u e s f r o m donors w h e n s i g n i f i c a n t m u l t i p l i c a t i o n o c c u r s for an u n d e t e r m i n e d t i m e p e r i o d . It r e q u i r e s more s p e c i f i c a l l y d e f i n e d c o n d i t i o n s than t h o s e mentioned a b o v e . 2)
Source of tissues
and
cells.
Most of the e x p e r i m e n t s c o n c e r n e d w i t h primary t i c k c e l l c u l t u r e e m p l o y d e v e l o p ing t i s s u e s of a d u l t s w i t h i n nymphs undergoing m e t a m o r p h o s i s . T h i s m a t e r i a l c o n s i s t s of t i s s u e s , c e l l s and h e m o c y t e s of t h e w h o l e p r e i m a g i n a l t i c k o r g a n i s m w i t h t h e e x c e p t i o n of c e l l s and t i s s u e s of M a l p i g h i a n t u b u l i and d i g e s t i v e t r a c t w h i c h are d i s c a r d e d d u r i n g d i s s e c t i o n . T h i s m a t e r i a l is e i t h e r used as an e x p i a n t o f t h e t o t a l body c o n t e n t , or the c e l l s are separated b y g e n t l e p i p e t t i n g , t h e use of t r y p s i n , or b y a c o m b i n a t i o n of both m e t h o d s . The b e s t s o u r c e of t h e h i g h e s t number of g r o w i n g c e l l s is the engorged nymph in w h i c h the d e v e l o p i n g a d u l t is c l e a r l y v i s i b l e , e . g . the d e v e l o p i n g f r o n t a l p a r t of the imaginai body and t h e l e g s . Because of t h e long t i m e , n e c e s s a r y f o r c o m p l e t i o n of t h e t i c k l i f e c y c l e , nymphs a r e not a l w a y s a v a i l a b l e w h e n needed. E x p e r i m e n t s w i t h D. andersoni t i c k s indic a t e d t h a t metamorphosing nymphs h e l d at - 1 1 ° C f o r many w e e k s p r o v i d e no less g r o w i n g t i s s u e s than unrefr ige rated t i s s u e s (Yunker and C o r y , 1 9 6 5 ) . 3)
Preparation
of tissues
and cells
for
cultures.
Engorged nymphs are in r u n n i n g w a t e r , d i s i n f e c t e d b y i m m e r s i o n in 7 0 % e t h a n o l f o r a f e w m i n u t e s and r e p e a t e d l y w a s h e d in s t e r i l w a t e r . Then the d e v e l o p i n g a d u l t s are s e p a r a t e d f r o m the metamorphosing n y m p h s , w a s h e d in s t e r i l e s a l i n e and c u t i n t o s m a l l p i e c e s . A f t e r t r e a t m e n t w i t h 0.25% t r y p s i n the t i s s u e s are m a g n e t i c a l l y s t i r r e d in 0.25% t r y p s i n prewarmed t o 2 7 ° C f o r 10 m i n u t e s (Varma and W a l l e r s , 1965 and Varma and Pudney, 1969a) a t room t e m p e r a t u r e . The t i s s u e f r a g m e n t s are a g i t a t e d g e n t l y u n t i l t h e f l u i d s h o w s even c l o u d i n e s s , w i t h o n l y nerve g a n g l i a r e m a i n i n g c o n s p i c u o u s . The s u s p e n s i o n is s e d i m e n t e d by c e n t r i f u g a t i o n a t 8 0 0 r . p .
24
TICK TISSUE C U L T U R E A N D A R B O V I R U S E S
m. f o r 7 m i n u t e s , the t r y p s i n removed w i t h t h e supernatant and a f t e r w a s h i n g in s a l i n e the c e l l s are ready f o r s e e d i n g . 4)
Effects
of media composition
on tissue
culture
growth.
The f i r s t d e s c r i p t i o n of p r o l i f e r a t i o n of f i b r o b l a s t - l i k e c e l l s from t i c k e x p i a n t s w a s o b s e r v e d in D. marginatus (Rehacek, 1 9 5 8 , a , b . ) . The h a n g i n g drop method and a temperature of 25 - 2 8 ° C w a s used in t h i s e x p e r i m e n t . The e x p i a n t s were m o s t l y c u l t i v a t e d in T r a g e r ' s medium and H a n k s ' m e d i u m p l u s 0.5% l a c t a l b u m i n h y d r o l y s a t e with 0 . 1 % yeast hydrolysate. A t t e m p t s were done to improve the c o m p o s i t i o n o f t h e n u t r i e n t medium b y a d d i n g sera or embryonal e x t r a c t s f r o m s e v e r a l i n v e r t e b r a t e s and t o prepare s y n t h e t i c media composed of t h e same c o n s t i t u e n t s f o u n d in t h e t i c k h e m o l y m p h . Because o f h i g h g l y c o p r o t e i n c o n t e n t of t i c k s e r u m , v a r i o u s sugars and p o l y s a c h a r i d e s w e r e added t o the medium. Marked improvement i n t h e g r o w t h of t i c k t i s s u e s w a s found i n s y n t h e t i c Eagle p l u s V a g o and C h a s t a n g ' s medium used i n a r a t i o of 1 : 1 , and in t h e same medium t o w h i c h 5% d e x t r a n (M.W. 60.000) or t i c k egg e x t r a c t w a s added (Rehäcek and H â n a , 1961). Further improvement w a s a c h i e v e d by e n r i c h m e n t o f t h e media w i t h 10% c a l f serum (Rehâcek, 1 9 6 2 , Varma and Pudney, 1967). Yunker a n d C o r y , 1967 s u c c e s s f u l l y e x a m i n e d the HLH medium (Grand I s l a n d B i o l . C o . ) t o w h i c h 10% heat - i n a c t i v a t e d normal r a b b i t s e r u m , T0% w h o l e c h i c k e n - egg u l t r a f i l t r ä t e , a n d 10 m g / m l b o v i n e plasma a l b u m i n were added. C e l l u l a r o u t g r o w t h w a s o b s e r v e d i n t h i s medium up t o 246 d a y s and s u r v i v a l of t i s s u e s , as e v i d e n c e d by c o n t r a c t i o n s , f o r as long as 263 d a y s . We have prepared a new medium f o r t h e c u l t i v a t i o n of t i c k c e l l s based on the r e s u l t s of c h e m i c a l a n a l y z e s of t h e a m i n o a c i d s , sugars and s a l t s in the hemol y m p h of B.microplus, Argas lagenoplastis and H. dromedarii containing vitamins o f the Β c o m p l e x , organic a c i d s , 1 % A n t h e r a e a p e r n y i h e m o l y m p h a n d 1 % b o v i n e p l a s m a . The c e l l s of R. sanguineus t i c k w e r e v i a b l e in t h i s medium f o r n i n e months (Rehécek and B r z o s t o w s k i , 1969a). Varma and Pudney, 1973 c u l t i v a t e d s u c c e s s f u l l y t i s s u e s and c e l l s f r o m d e v e l o p i n g a d u l t s of R. appendiculatus in a medium VP 12 o f t h e i r o w n f o r m u l a t i o n . The c e l l s remained h e a l t h y up t o 4 months and w e r e s u b c u l t u r e d four t i m e s . ticks, When t e s t e d medium in e x p e r i m e n t s w i t h D. marginatus and H. dromedarii o n l y n e g a t i v e r e s u l t s were o b t a i n e d ( f t e h a c e k , u n p u b l . r e s u l t s ) . A l s o V P 12 med i u m in w h i c h p r i m a r y c e l l c u l t u r e s of H. dromedarii w e r e grown s u c c e s s f u l l y , w a s p r o v e d u n s a t i s f a c t o r y f o r the growth o f /?. appendiculatus and B. microplus c e l l s . On the c o n t r a r y L - 15 m e d i u m , in w h i c h c e l l s of R. appendiculatus and B. microplus grew w e l l p r o v e d l e s s s a t i s f a c t o r y f o r c e l l s f r o m H. dromedarii (Varma et al., 1975). It is e v i d e n t t h a t t h e s u c c e s s f u l g r o w t h of t i c k c e l l s depends not o n l y on the media but p r e d o m i n a n t l y on the t i c k s p e c i e s u s e d . 5)
Cell
composition
and growth
in
culture.
O b s e r v a t i o n by Yunker and C o r y , 1967 i n d i c a t e t h a t the p r o d u c t i o n o f o u t g r o w t h i s b i p h a s i c . The i n i t i a l c e l l u l a r r e s p o n s e is t h e m i g r a t i o n of h e m o c y t e s and t h e i r a d h e r e n c e t o t h e v e s s e l w a l l . These c e l l s s u r v i v e f o r a f e w w e e k s and are g r a d u a l l y overgrown by c e l l s a r i s i n g f r o m organs and t i s s u e s , w h i c h p r o d u c e s h e e t - l i k e complexes. Martin and V i d i e r , 1962 r e c o g n i z e d f o u r c e l l t y p e s in the c u l t u r e of R. appendiculatus. The f i b r o b l a s t - l i k e c e l l s w e r e seen in c u l t u r e s f r o m 24 hours up t o 167
25
J. R E H A C E K
d a y s after s e e d i n g . The slender e l o n g a t e t y p e c o n t i n u e d to appear in v a r y i n g numbers f o r about 60 - 70 d a y s . The t h i r d t y p e of c e l l s o b s e r v e d w e r e s m a l l polymorp h i c e p i t h e l i a l - l i k e c e l l s t h a t began to appear in c u l t u r e s f r o m the 1 0 t h t o t h e 3 5 t h - 40th day and d i s a p p e a r e d by the 5 0 t h - 8 5 t h d a y . The f o u r t h t y p e of c e l l s w a s of e p i t h e l i a l c h a r a c t e r , a p p e a r i n g in c u l t u r e s after 1 1 - 2 7 d a y s and c o n t i n u i n g up t o 175 d a y s . In c e l l c u l t u r e from d e v e l o p i n g a d u l t s of D. andersoni ticks different types of v e s i c l e s were seen from the f i r s t d a y a f t e r s e e d i n g the e x p l a n t s (Rehàcek, 1971). 6)
Electron
microscope
study of tick cells
in
vitro.
C e l l c u l t u r e s prepared f r o m d e v e l o p i n g a d u l t s of R. sanguineus t i c k s were inv e s t i g a t e d w i t h t h e e l e c t r o n m i c r o s c o p e . The purpose of t h i s s t u d y w a s t o determine w h a t ultra structura I d i f f e r e n c e s , if a n y , e x i s t between t i c k c e l l s and other i n v e r t e b r a t e c e l l s and a l s o t o e s t a b l i s h w h e t h e r t i c k c e l l s of t h i s s p e c i e s act as c a r r i e r s of v a r i o u s t i c k s p e c i f i c v i r u s e s or other p a t h o g e n s . I t w a s s h o w n in one w e e k o l d c u l t u r e s t h a t the c e l l s t e s t e d d o not p o s s e s s s p e c i a l arrangements o f o r g a n e l l e s and in general d o not d i f f e r in u l t r a s t r u c t u r e f r o m in vitro c u l t u r e s of other arthropod c e l l s . No i n c l u s i o n s , v i r a j p a r t i c l e s or other p a t h o g e n s w e r e f o u n d in any of the c e l l s e x a m i n e d (F i l s h i e and Rehàcek, u n p u b l . r e s u l t s ) . 7)
Utilization
of the aminoacids
and sugars
by tick cells
in
vitro.
The s t u d y of u t i l i z a t i o n of the a m i n o a c i d s and sugars in the medium by the c e l l s of R. s a n g u i n e u s has s h o w n t h a t a m i n o a c i d s c a n be d i v i d e d into t h o s e w h i c h w e r e u t i l i z e d t o a s i g n i f i c a n t e x t e n t and those in w h i c h no c h a n g e , or an increase in c o n c e n t r a t i o n o c c u r r e d . The f i r s t group c o n s i s t e d of l e u c i n e , m e t h i o n i n e , t h r e o n i n e , p h e n y l a l a n i n e , p r o l i n e , g l u t a m i c and a s p a r t i c a c i d s . The second one c o n s i s t e d of i s o l e u c i n e , v a l i n e , c y s t i n e , t y r o s i n e , a r g i n i n e , h i s t i d i n e , l y s i n e , g l y c i n e , s e r i n e and a l a n i n e . Sugars in the medium were u t i l i z e d b y t h e c e l l s in c u l t u r e , g l u c o s e d i s a p peared to a greater e x t e n t than i n o s i t o l - 4 3 , 5 % of g l u c o s e w a s m e t a b o l i z e d over 10 d a y p e r i o d compared to 3 0 , 1 % of t h e i n o s i t o l (Rehàcek and B r z o s t o w s k i , 1969b, Rehàcek, 1969). Varma and Pudney (1969a) in measuring the uptake of g l u c o s e a s a p o s s i b l e i n d i c a t o r of growth and m e t a b o l i s m of t h e t i c k c e l l s in vitro f o u n d t h a t t h e g l u c o s e uptake w a s p r o p o r t i o n a l to the number of c e l l s . When c o m p a r i n g the uptake of g l u c o s e by c e i l s g r o w i n g in tubes w i t h or w i t h o u t c o v e r s l i p s , t h e y f o u n d t h a t t h e c e l l s grown under a c o v e r g l a s s u t i l i z e d s i g n i f i c a n t l y larger amounts of g l u c o s e ; b y s i x d a y s , 50% of the g l u c o s e w a s used up and by the 12 d a y s t h i s had i n c r e a s e d o n l y t o 70%. These r e s u l t s i n d i c a t e t h a t a l t h o u g h t h e g r o w t h rate w a s l o w , the c e l l s in were a c t i v e l y m e t a b o l i z i n g the amino a c i d s and s u g a r s . 8)
Use of primary a)
cell culture
for the cultivation
of
vitro
viruses.
C u l t i v a t i o n of d i f f e r e n t v i r u s e s .
The marked m u l t i p l i c a t i o n of TBE v i r u s in c u l t i v a t e d H. dromedarii c e l l s (Rehàcek, 1962) s t i m u l a t e d further s t u d i e s w i t h t h i s and other v i r u s e s in t i s s u e c u l t u r e s f r o m t i c k s . It w a s found t h a t mosquito - borne v i r u s e s , namely WEE, EEE, S i n d b i s and very w e l l at a p p r o x i m a S e m l i k i Forest v i r u s m u l t i p l i e d in c u l t u r e s of H. dromedarii t e l y s i m i l a r rate of 0,5 - 1 log u n i t per d a y . U s i n g s m a l l amounts of v i r u s as inoc u l u m ( 1 - 1 0 mouse LD50 of CPD50), by t h e 8th day of c u l t i v a t i o n t h e a m o u n t s 4 5 w e r e 1 0 - 1 0 t i m e s greater. L a n g a t (TP 2 1 ) , Japanese e n c e p h a l i t i s , S t . L o u i s enc e p h a l i t i s and y e l l o w f e v e r v i r u s e s m u l t i p l i e d in t h e s e c u l t u r e s t o a l e s s e r e x t e n t . 26
T I C K TISSUE C U L T U R E A N D A R B O V I R U S E S
Kyasanur Forest D i s e a s e , P o w a s s a n , O m s k hemorrhagic f e v e r , a n d West N i l e v i r u s e s m u l t i p l i e d r e l a t i v e l y w e l l and both s u b t y p e s of TBE v i r u s and l o u p i n g - i l l v i r u s m u l t i p l i e d very w e l l . The h i g h e s t t i t r e s of t h e v i r u s e s w e r e again o b t a i n e d w i t h s m a l l i n o c u l a (1-10 mouse LD50 p e r 0 , 0 3 m l ) , t h e v i r u s increment being a p p r o x i m a t e l y o f the order of 0,5 - 1 log u n i t per d a y . V i r u s e s other t h a n a r b o v i r u s e s ( E M C , p o l i o , v a c c i n i a , N D V and P s e u d o r a b i e s ) , d i d not m u l t i p l y in t i c k t i s s u e c u l t u r e s . A n e x c e p t i o n w a s LCM v i r u s w h i c h m u l t i p l i e d in t i c k c e l l s v e r y w e l l , s i m i l a r t o v i r u s e s t r a n s m i t t e d in nature by t i c k s (Rehacek, 1965b). The r e s u l t s of c u l t i v a t i o n of CTF v i r u s in c u l t u r e s of D. andersoni have s h o w n about a 4Î4 log u n i t s increase of e x t r a c e l l u l a r v i r u s f r o m 6 - 1 0 d a y s up t o 4 - 5 w e e k s of c u l t i v a t i o n . The v i r u s was r e c o v e r e d in d i m i n i s h i n g q u a n t i t i e s f o r as long a s 159 d a y s in the medium and 166 d a y s in t r i t u r a t e d t i s s u e s (Yunker and C o r y , 1967). T i t e r s in w h o l e nymphs f e d on v i r e m i c hamsters remained a b o u t t h e same l e v e l from drop - off to m o l t i n g , but t i t e r s in c u l t u r e s prepared f r o m t h e s e t i c k s i n c r e a s e d about f i v e log u n i t s 2 w e e k s a f t e r s e e d i n g . I t w a s s h o w n t h a t a t i s s u e c u l t u r e f r o m D. andersoni i s a v e r y s e n s i t i v e s y s t e m f o r t h e d e t e c t i o n of s m a l l amounts of CTF v i r u s , because l e s s than 0,1 of s u c k l i n g mouse i c e r LD50 can be p r o p a g a t e d t o h i g h t i t e r s . The 98 and 124 d a y s p r o p a g a t i o n of the CTF v i r u s ( s t r a i n F l o r i o 2) in t i c k t i s s u e c u l t u r e s d i d not a l t e r the v i r u l e n c e of t h e v i r u s . H. dromedarii t i s s u e c u l t u r e s w e r e a l s o s u c c e s s f u l l y used f o r c u l t i v a t i o n o f t h e T r i b e c v i r u s (Kemerovo group of a r b o v i r u s e s ) . S t a r t i n g f r o m t h e second d a y p o s t i n f e c t i o n t h e v i r u s t i t e r had i n c r e a s e d a b o u t 4 log u n i t s . T h e s e v a l u e s w e r e d e t e c t a b l e u n t i l t h e 14th d a y s ( t h e c o n c l u s i o n o f t h e e x p e r i m e n t ) . A c l e a r - c u t s p e c i f i c f l u o r e s c e n c e w a s seen in t h e c y t o p l a s m of both e p i t h e l i a l - l i k e and f i b r o b l a s t l i k e c e l l s from t h e s e c o n d to s e v e n t h d a y a f t e r i n f e c t i o n . A t t h e b e g i n n i n g o n l y a f e w f l u o r e s c i n g g r a n u l e s w i t h p e r i n u c l e a r l o c a l i z a t i o n w e r e f o u n d , b u t by t h e f o u r t h day t h e y increased in number and b l e n d e d t o g e t h e r into l a r g e r , b r i g h t f l u o r e s c i n g m a s s e s , f i l l i n g the w h o l e c y t o p l a s m (Rehacek et a/.c 1969) ( F i g . 1). The L a n j a n v i r u s propagated in c u l t u r e s of H. dromedarii i n c r e a s e d f r o m 1,4 log u n i t s on the 1st d a y a f t e r i n o c u l a t i o n t o 4,8 log u n i t s o n the 10th d a y a f t e r i n o c u l a t i o n . Twenty d a y s after i n f e c t i o n w h e n the experiment w a s d i s c o n t i n u e d , t h e v i r u s t i t r e w a s 3,5 l o g u n i t s . Q u a r a n f i l v i r u s increased in t h e same c u l t u r e from a t i t r e o f 1,2 log u n i t s at 2 d a y s a f t e r i n o c u l a t i o n to 3,5 log u n i t s on 10th day after inoc u l a t i o n . A t 20 d a y s a f t e r i n f e c t i o n t h e v i r u s t i t r e w a s s t i l l 2,4 l o g u n i t s (Varma and Pudney, 1969b). In a l l e x p e r i m e n t s w i t h the v i r u s e s m e n t i o n e d a b o v e , t h e appearance of t i c k t i s s u e c u l t u r e s w a s not a f f e c t e d by v i r a l i n f e c t i o n s as shown by t h e a b s e n c e of CPE and i n c l u s i o n s . In c o m p a r i s o n w i t h v e r t e b r a t e t i s s u e c u l t u r e s a l l t e s t e d v i r u s e s m u l t i p l i e d s l o w l y in t i c k t i s s u e c u l t u r e s perhaps due to t h e low temperature of c u l t i vation. b) C u l t i v a t i o n of t h e TBE v i r u s , w e s t e r n t y p e . Since t h e TBE v i r u s , w e s t e r n t y p e , is spread in s e v e r a l p a r t s of Europe and somet i m e s c a u s e s s e r i o u s d i s e a s e , much a t t e n t i o n w a s p a i d to t h i s v i r u s in e x p e r i m e n t s . No d i f f e r e n c e s w e r e noted b e t w e e n t h e rate of m u l t i p l i c a t i o n of v i r u s s t r a i n s tise i t h e r adapted or not adapted to HeLa c e l l s in H. dromedarii and D. marginatus s u e c u l t u r e s , from w h i c h o n l y t h e l a t t e r t i c k s p e c i e s is k n o w n as a v e c t o r of t h i s v i r u s (Rehacek, 1963).
27
J.
REHACEK
In many e x p e r i m e n t s t h e dose of v i r u s used s u c c e s s f u l l y f o r t h e i n o c u l a t i o n o f t i c k t i s s u e c u l t u r e s w a s s o s m a l l t h a t it w a s n o t d e t e c t a b l e in icer i n o c u l a t e d m i c e or HeLa c e l l s . Therefore t h e s e n s i t i v i t y of c h i c k embryo c e l f s , w h i c h appeared t o be t h e h i g h e s t f o r t h e p r o p a g a t i o n of the TBE v i r u s , w a s compared w i t h t h a t of p r i m a r y t i c k t i s s u e c u l t u r e s . It w a s f o u n d t h a t w i t h i n o c u l a of 30 and 3 IFD5Q, the percenta g e , of i n f e c t e d c h i c k embryo c e l l s and t i c k c e l l c u l t u r e s w a s a b o u t e q u a l . With i n o c u l a c o n t a i n i n g 0,3 and 0 , 0 3 IFD5Q of t h e v i r u s , the r e s p e c t i v e p e r c e n t a g e s of i n f e c t e d t i c k c e l l c u l t u r e s were 76 and 1 8 , w h e r e a s t h a t of c h i c k embryo c e l l c u l t u r e s were 3 4 and 3 (Rehàcek and K o z u c h , 1964). The v i r u s p r o p a g a t i o n o c c u r r e d o n l y in t h e c y t o p l a s m of c e l f s and w a s c o n c e n t r a t e d around the n u c l e u s . The pos i t i v e f l u o r e s c e n c e in t i c k c e l l s as w e l l as t h e v i r u s y i e l d s f r o m t h e s e c e l l s were in d i r e c t r e l a t i o n t o the t i m e f o l l o w i n g i n f e c t i o n and t h e d o s e of i n o c u l a t e d v i r u s ( F i g . 2 and Table 1.) (Rehàcek, 1965c). The r e s u l t s i n d i c a t e t h a t t i c k c e l l c u l t u r e s are a c t u a l l y one of t h e most s u s c e p t i b l e s y s t e m s for d e t e c t i n g s m a l l amounts of t h i s v i r u s . c)
I s o l a t i o n of the TBE v i r u s in t i c k cel'ls in vitro f r o m v a r i o u s m a t e r i a l s c o l l e c t ed in nature.
The t e s t m a t e r i a l s from nature ( b l o o d and s u s p e n s i o n s of b r a i n s o f v a r i o u s a n i m a l s and t i c k s ) were s e p a r a t e l y i n o c u l a t e d into 2 - 5 d a y s o l d t i c k c e l l c u l t u r e s ( w i t h o u t w a s h i n g or c h a n g e of medium f o l l o w e d by 7 - 9 d a y s i n c u b a t i o n ) w i t h c h i c k embryo c e l l c u l t u r e s , t h e m a t e r i a l s w e r e left t o adsorb f o r 2 h o u r s , then w a s h e d , and supp l i e d w i t h f r e s h medium and i n c u b a t e d for 5 d a y s . F o l l o w i n g the s e l e c t e d i n t e r v a l s , t h e c u l t u r e f l u i d s f r o m e i t h e r t i c k or c h i c k embryo c e l l c u l t u r e s w e r e i n t r a c e r e b r a l ^ i n o c u l a t e d into s u c k l i n g m i c e , w h i c h w e r e o b s e r v e d for d e v e l o p i n g symptoms of i n f e c t i o n . Of a t o t a l 187 s a m p l e s , f i v e s t r a i n s of TBE v i r u s w e r e i s o l a t e d : one from t h e blood of Ta Ipa europaea, one from the b l o o d of Apodemus flavicollis a n d three t i c k s . A l l the s t r a i n s w e r e i s o l a t e d by b o t h m e t h o d s . T o x i c e f f e c t s f r o m /. ricinus o f mammalian b l o o d or b r a i n and t i c k s u s p e n s i o n s w e r e not o b s e r v e d in t i c k or in c h i c k embryo c e l l c u l t u r e s . The r e s u l t s o b t a i n e d i n d i c a t e t h a t t i c k t i s s u e c u l t u r e s are as s u s c e p t i b l e t o TBE v i r u s and as s u i t a b l e f o r i s o l a t i o n e x p e r i m e n t s as c h i c k embryo c e l l c u l t u r e s (Rehàcek and K o z u c h , 1969).
t a b l e
Dectection
of TBE virus
Inoculum
in H, dromedarii
6,5
tissue
cultures
Virus yields and immunofluorescence at days f o l l o w i n g i n f e c t i o n 2
++
1.
5
9
+
3,5+
5,5
1,5+
4,0+
1,5
0,5
3,0+
4,5+
0,5
neg
2,0+
4,5+
2,5
5,5+ 6,5+
0,05
neg
neg
5,0+
0,005
neg
neg
neg
++ l o g L D 5 Q / 0 , 0 3 m l ( m o u s e i c e r )
+ positive immunofluorescence
28
T I C K TISSUE C U L T U R E A N D A R B O V I R U S E S
F ig.
1. C e l l f r o m H. dromedarii
(IMF
method).
Fig. 2 . C e l l f r o m H. dromedarii (IMF method)
d)
primary c u l t u r e , 6 days after t h e infection w i t h Tribec v i r u s
p r i m a r y c u l t u r e , 10 d a y s a f t e r t h e i n f e c t i o n w i t h TBE v i r u s
M a i n t e n a n c e of t h e TBE v i r u s i n t i c k c e l l c u l t u r e s .
T i c k t i s s u e c u l t u r e s of H. dromedarii were used as a s u b s t r a t e f o r t h e l a b o r a t o r y m a i n t e n a n c e of t h e w e s t e r n t y p e of TBE v i r u s . T h i r t y four p a s s a g e s a t w e e k l y interv a l s w e r e performed d u r i n g a one y e a r p e r i o d . T h e v i r u s t i t r e s v a r i e d b e t w e e n 3,0 and 6,5 l o g u n i t s d u r i n g t h e p a s s a g e s . T h e v i r u s t i t e r s a t t h e 6,10 a n d 15th p a s s a g e s were at the same l e v e l , a t t h e 2 0 t h p a s s a g e t h e t i t e r in s u b c u t a n e o u s I y i n j e c t e d m i c e w a s 0,5 l o g u n i t h i g h e r , but i t d e c r e a s e d a b o u t one l o g u n i t a t t h e 2 5 t h and 3 0 t h p a s s a g e . It c a n be c o n c l u d e d f r o m t h e s e r e s u l t s t h a t TBE v i r u s c a n m a i n t a i n e d in t i c k t i s s u e c u l t u r e s as a v i r u s s t o c k f o r laboratory e x p e r i m e n t s w i t h o u t d e t e c t a b l e c h a n g e in i t s v i r u l e n c e (Rehàéek, 1973). e)
Mixed
infection
w i t h TBE a n d Kemerovo v i r u s e s
in t i c k
tissue cultures.
The problem of d u a l i n f e c t i o n w i t h a r b o v i r u s e s of t h e same b i o l o g i c a l v e c t o r a r o s e w i t h the d i s c o v e r y o f m i x e d f o c i o f a r b o v i r u s i n f e c t i o n s . The w e s t e r n t y p e of TBE v i r u s and the v i r u s o f Kemerovo c o m p l e x b o t h o c c u r in / . ricinus t i c k s in S l o v a k i a . T h i s f i n d i n g prompted us to e x a m i n e w h e t h e r t h e s e v i r u s e s can c a u s e i n f e c t i o n o f t i c k c e l l s and t i s s u e s in vitro. V i r u s amounts u s e d a s i n o c u l u m w e r e 1 0 ^ o f TBE where the viruses v i r u s and 10^ of Kemerovo v i r u s . In c u l t u r e s of H. dromedarii w e r e i n o c u l a t e d at i n t e r v a l s of 3 - 7 d a y s no i n t e r f e r e n c e w a s d e m o n s t r a t e d . H o w e v e r , a marked mutual i n t e r f e r e n c e in m u l t i p l i c a t i o n o f both v i r u s e s as d o c u m e n t e d b y growth c u r v e s and d e c r e a s e d p e r c e n t a g e o f i n f e c t e d c e l l s w a s f o u n d in c u l t u r e s i n o c u l a t e d s i m u l t a n e o u s l y . T h i s e f f e c t seems t o be r e l a t e d t o the p e r i o d o f v i r u s a d s o r p t i o n on c e l l s u r f a c e s or to t h e p i n o c y t o s i s phase and is c e r t a i n l y not r e l a t e d t o t h e p r o d u c t i o n of i n t e r f e r o n ( L i b i k o v a and Rehadek, 1974). 9.
Prospects
of tick primary
cultures
in
virology
A l m o s t a l l p r o b l e m s c o n c e r n i n g i n t e r a c t i o n s b e t w e e n c e l l s and v i r u s e s c a n be i n v e s t i g a t e d in p r i m a r y t i c k c u l t u r e s , h o w e v e r , t h e same p r o b l e m s can be s t u d i e d in i n t i c k a l l l i n e s a n d t h e p r e f e r e n t i a l use o f the l a t t e r is e x p e c t e d in t h e f u t u r e . V.
Passage of t i c k c e l l c u l t u r e s .
/ ) Genera I c ons
/derations.
A t t e m p t s t o e s t a b l i s h c o n t i n u o u s t i c k c e l l l i n e s had been u n s u c c e s s f u l f o r a long t i m e . One of the most p r o m i s i n g t y p e s of c u l t u r e f o r e s t a b l i s h i n g c e l l l i n e s w a s p r o p o s e d to be e m b r y o n ic t i s s u e s and c e l l s , or c e l l s from male and f e m a l e g e n i t a l g l a n d s ; both o f f e r i n g an a b u n d a n c e of c e l l s in an a c t i v e m i t o t i c s t a t e .
29
J. R E H A C E K
2)
Culture
of embryonic
cells.
O n l y t w o c o n t r i b u t i o n s are c o n c e r n e d w i t h t h e e s t a b l i s h m e n t o f e m b r y o n i c c e l l c u l t u r e s . The f i r s t c u l t u r e w a s prepared from t i s s u e s and c e l l s t a k e n f r o m H. asiaticum eggs (Medvedeeva et al.c 1972). The best source of c e l l s w a s t h o u g h t to be t h e e g g s ι kept a t 18 - 2 0 ° C f o r 1 0 - 1 4 d a y s a f t e r o v i p o s i t i o n . The c u l t u r e s p r i m a r i l y o f c e l l s in s u s p e n s i o n in the medium of M i t s u h a s h i and Maramorosch w e r e s u b c u l t u r e d 10 t i m e s before d y i n g off ( M e d v e d e e v a , p e r s . c o m . ) . T h e s e c o n d e m b r y o n i c c e l l c u l t u r e w a s reported f r o m B. microplus t i c k s (Pudney et al., 1973). The c e l l s were o b t a i n e d from t h e eggs l a i d by f e m a l e s t h e s u r f a c e of w h i c h were s t e r i l i z e d before o v i p o s i t i o n w i t h a 1 : 10 s o l u t i o n of Roccal c o n t a i n i n g 1 % b e n z a l k o n i u m c h l o r i d e . The c e l l s w e r e c u l t i v a t e d in H L H medium w i t h 10% f e t a l c a l f serum. A b o u t a w e e k a f t e r s e e d i n g , a l m o s t a l l t i s s u e e x p i a n t s had a t t a c h e d to v e s s e l s u r f a c e and large g r a n u l a r f i b r o b l a s t - t y p e c e l l s appeared b e t w e e n t h e e x p i a n t s . V e s i c l e s and tube l i k e f o r m s w e r e common in the c u l t u r e s . F o u r w e e k s l a t e r the s u r f a c e of the v e s s e l s were c o v e r e d w i t h c l o s e l y p a c k e d , d i s t i n c t , round or f l a t t e n e d e p i t h e l i a l - t y p e c e l l s . S u b c u l t u r e s were done s u c c e s s f u l l y in L e i b o v i t z L - 15 medium s u p p l e m e n t e d w i t h 10% t r y p t o s e phosphate broth and 15% f e t a l c a l f s e r u m . Most c e l l s had d i p l o i d chromosome numbers, 21 f o r male and 22 f o r f e m a l e . The c e l l s d i e d o f f after 20 s u b c u l t u r e s . 3)
Cell culture
from ovaries
of female
ticks.
A m o n g the v a r i o u s k i n d s of t i s s u e s and c e l l s of a d u l t t i c k s t e s t e d in vitro, most a t t e n t i o n has been p a i d to o v a r i a l t i s s u e s , w h i c h p r o d u c e w e l l - formed m u l t i p l y i n g epithelial cells. (Rehacek, 1 9 7 1 ) , I n v e s t i g a t i o n s on t h e c u l t i v a t i o n of o v a r i e s from D. andersoni were done r e c e n t l y in our l a b o r a t o r y . The medium used D. pictus and D. marginatus w a s H L H w i t h 10% f e t a l c a l f serum p r e v i o u s l y heated at 5 6 ° C f o r 60 m i n u t e s . The s m a l l p i e c e s of o v a r i e s t a k e n f r o m p a r t i a l l y engorged f e m a l e s w e r e c u l t i v a t e d a t 2 8 - 30OC in Τ f l a s k s . A f t e r m i g r a t i o n of a f e w f i b r o b a l s t - t y p e c e l l s d u r i n g t h e f i r s t d a y s , d i s c r e t e s m a l l c o l o n i e s of e p i t h e l i a l - t y p e c e l l s appeared o n d a y s 7 1 0 , w h i c h f u r t h e r s l o w l y i n c r e a s e d in t h e number. Two - f o u r w e e k s later t h e s e c o l o n i e s became very d e n s e c o v e r i n g a l m o s t c o m p l e t e l y t h e s u r f a c e of the c u l t u r e v e s s e l s ( F i g s 3 - 4 ) . Most of t h e s e c e l l s w e r e s u b c u l t u r e d 5 - 9 t i m e s , b u t , a f t e r t h e 7 t h - 9 t h p a s s a g e t h e c e l l s c e a s e d to m u l t i p l y and became v e r y large and granular and d i e d off. 4)
Cultivation
of viruses
in tick
embryonal
cells.
Of the c u l t u r e s mentioned a b o v e , o n l y embryonal c e l l s of B. microplus employed i n v i r u s r e p l i c a t i o n s t u d i e s w e r e f o u n d t o support p r o p a g a t i o n o f Dugbe v i r u s . The c h r o n i c i n f e c t i o n w i t h t h i s v i r u s w a s e s t a b l i s h e d w i t h o u t v i s i b l e i n c l u s i o n s in t h e c e l l s and w i t h o u t v i s i b l e c y t o p a t h i c e f f e c t ( D a v i d - V e s t , 1974). 5)
Prospects
of tick passage
cultures
in
virology.
O b t a i n i n g c e l l s for repeated s u b c u l t u r e s is r e l a t i v e l y e a s y and h o l d s p r o m i s e of a n emergent c e l l l i n e . The e s t a b l i s h m e n t of p e r f e c t monolayers of t h e s e c e l l s in c u l t u r e v e s s e l s offers a s u i t a b l e s u b s t r a t e f o r the c u l t i v a t i o n o f v a r i o u s p a t h o g e n s a s w e l l as v i r u s e s . H o w e v e r , most v i r o l o g i s t s w i l l prefer to use the s i m p l e s t t y p e o f c u l t u r e s , i.e. e s t a b l i s h e d c e l l l i n e s .
30
TICK TISSUE C U L T U R E A N D A R B O V I R U S E S
Fig.
VI.
3-4.
D. pictus
cell culture from ovaries, 7th passage.
C e l l l i n e s from t i c k t i s s u e s .
1 ) G enera I c ons id erat ions. Most a t t e m p t s to c u l t i v a t e t i c k t i s s u e s and c e l l s in vitro b l i s h m e n t of c e l l I i n e s . 2)
Cell
lines
from the tick R.
i s d i r e c t e d t o the e s t a -
appendiculatus.
The f i r s t t i c k c e l l l i n e s were e s t a b l i s h e d from R. appendiculatus t i c k s (Varma et aL, 1975). The method used f o r t h e p r e p a r a t i o n o f t i s s u e s a n d c e l l s f o r t h e e s t a b l i s h m e n t of c e l l l i n e s w a s t h e same as used f o r p r i m a r y c u l t u r e s . Of a t o t a l 24 p r i m a r y c u l t u r e s s e e d e d , 3 became e s t a b l i s h e d a s c e l l l i n e s , i.e. TTC - 2 1 9 , TTC - 243 and TTC - 2 5 7 . They are m a i n t a i n e d in L e i b o v i t z L-15 medium s u p p l e m e n t e d w i t h 10% t r y p t o s e phosphate broth and 10% f e t a l c a l f s e r u m . In March 1974 ( t h e time w h e n t h e paper c o n c e r n i n g the e s t a b l i s h m e n t of c e l l l i n e s w a s subm i t t e d f o r p u b l i c a t i o n ) t h e c e l l s were at the 5 4 t h , 4 1 s t and 34th s u b c u l t u r e s . In t h e e a r l y s u b c u l t u r e s the e p i t h e l i a l c e l l s w e r e p r e d o m i n a n t . W i t h p r o g r e s s i v e s u b c u l t u r e s t h e c e l l s tended to aggregate in dense c l u m p s w h i c h p r o v i d e d most of t h e c e l l s f o r f u r t h e r p a s s a g e s . The c u l t u r e s c o n s i s t of round or e p i t h e l i a l - t y p e c e l l s w i t h irregular o u t l i n e s and s p i n d l e - shaped c e l l s . Chromosome p r e p a r a t i o n s o f c e l l s showed mixed p l o i d y . Most of c e l l s namely in TTC - 243 w e r e d i p l o i d w i t h t h e male chromosome c o m p l e m e n t of 2n = 21 and the f e m a l e c o m p l e m e n t of 2n = 22 chromosomes. 3)
Cultivation Preliminary
of arboviruses
in R. appendiculatus
cell
r e s u l t s of c u l t i v a t i o n showed that in c e l l
31
lines. l i n e TTC - 243 West N i l e
J. R E H A C E K
v i r u s i n c r e a s e d in t i t e r from 1 0 ^ 4 no t h e 1st d a y to 1 θ 3 , 7 on t h e 10th d a y a f t e r i n o c u l a t i o n , the Langat v i r u s f r o m 1 0 ^ / 4 on the 1 s t day to 1 0 ^ , 4 n 0 the 4 - 6th day, t h e l o u p i n g - i l l v i r u s from 1 0 ^ ^ on t h e 1st day t o 1 0 ^ , 6 n0 the 4 t h d a y , and t h e Q u a r a n f i l v i r u s from 1θ2,8 on the 1st day to 1 0 4 , 6 o n the 4 t h day. A l l t h e s e v i r u s e s m u l t i p l i e d in c e l l s w i t h o u t p r o d u c i n g any d e t e c t a b l e c y t o p a t h i c e f f e c t . 4)
Prospects
of tick cell
lines
in
virology.
The e s t a b l i s h m e n t of t h r e e c e l l l i n e s by Varma et al., 1 9 7 5 , p r o v i d e s e v i d e n c e that i s p r o m i s i n g f o r the d e v e l o p m e n t of t i c k c e l l l i n e s f o r use in i n v e s t i g a t i n g c e l l i n t e r a c t i o n s w i t h a v a r i e t y of m i c r o o r g a n i s m s .
VII.
Conclusions
T i s s u e c u l t u r e s prepared from b l o o d - s u c k i n g arthropods have p r o g r e s s e d as a t o o l i n a r b o v i r o l o g y d u r i n g t h e l a s t f e w y e a r s . T h i s has been a c c o m p l i s h e d m o s t l y by t h e e s t a b l i s h m e n t of s e v e r a l mosquito c e l l l i n e s and d u r i n g t h i s y e a r by t h e e s t a b l i s h ment of t i c k c e l l l i n e s . The p r e f e r e n t i a l use o f arthropod c e l l c u l t u r e s in a r b o v i r o l o g y i s supported by t h e i r e a s e of p r e p a r a t i o n a n d h i g h s e n s i t i v i t y to p a t h o g e n s . A t p r e s e n t most s p e c i a l i s t s in i n v e r t e b r a t e c e l l c u l t u r e r e s e a r c h h a v e abandoned t h e use of organ and primary c u l t u r e s because of t h e l a b o r i o u s methods or preparat i o n . More a t t e n t i o n is d i r e c t e d toward t h e d e v e l o p m e n t w i t h c e l l l i n e s . A l t h o u g h t h e y are ideal from the s t a n d p o i n t of e a s i e r c u l t i v a t i o n m e t h o d s , t h e g e n u i n e c h a r a c t e r of a r t h r o p o d c e l l s has often c h a n g e d d u r i n g t h e s e r i a l p a s s a g e of c e l l s . Sometimes s u c h changes may not have an e f f e c t on the c u l t i v a t i o n of v i r u s e s , but i t may be proposed t h a t to e x p l a i n t h e r e l a t i o n s h i p of v e c t o r s t o p a t h o g e n s , it w o u l d be more p l a u s i b l e to use e x p l a n t e d organs rather than d i s p e r s e d and p o s s i b l y t r a n s formed c e l l s . H o w e v e r , a t p r e s e n t there is no s c i e n t i f i c b a s i s f o r t h i s s u g g e s t i o n . A l t h o u g h good primary c u l t u r e s have often been prepared f r o m v a r i o u s t i c k spec i e s , t h e y may not be w i d e l y used in m i c r o b i o l o g i c a l research b e c a u s e of preferenc e for e s t a b l i s h e d c e l l l i n e . S i g n i f i c a n t progress has been noted in t h e area of t i c k t i s s u e c u l t u r e w i t h regard t o v i r u s s t u d i e s . Perhaps t h e m o s t p r o m i s i n g from t h e s t a n d p o i n t of p r a c t i c a l v i r o l o g y are t h e f i n d i n g s of in vitro t i c k c u l t u r e s t h a t c o u l d support t h e r e p r o d u c t i o n of v i r u s e s and t h e i r higher s e n s i t i v i t y to v i r u s e s in c o m p a r i s o n t o t h a t of v e r t e b r a t e s t i s s u e s and c e l l s in vivo and in vitro. The use of t i c k t i s s u e c u l t u r e in v i r o l o g y appears p r o m i s i n g f o r t h e i s o l a t i o n o f v i r u s e s from natural s o u r c e s , for e v e n t u a l d i f f e r e n t i a t i o n among arbo and other v i r u s e s , for the i n v e s t i g a t i o n of e v e n t u a l changes in the p r o p e r t i e s o f v i r u s e s f o l l o w i n g t h e i r c u l t i v a t i o n in t i c k t i s s u e s and f o r t h e e s t a b l i s h m e n t of a t t e n u a t e d s t r a i n s o f v i r u s e s for p o s s i b l e v a c c i n e p r o d u c t i o n . In m u l t i p l i c a t i o n of agents to be used f o r t h e p r e p a r a t i o n o f a n t i g e n s and for o t h e r p r a c t i c a l a p p l i c a t i o n c o u l d i n c l u d e c u l t i v a t i o n of t i c k or o t h e r arthropod p a t h o g e n s w h i c h might be of v a l u e in arthropod c o n t r o l programs. It is most l i k e l y t h a t t h e t i c k t i s s u e c u l t u r e s , ( p r i m a r i l y t i c k c e l l l i n e s ) , w i l l assume a p o s i t i o n in modern v i r o l o g y and t h e r e is o p t i m i s m t h a t many i n v e s t i g a t i o n s u t i l i s i n g t h e s e t e c h n i q u e s w i l l y i e l d important r e s u l t s in t h e v a r i o u s areas of m i crobiology.
32
TICK TISSUE C U L T U R E A N D
ARBOVIRUSES
V I I I . References Biological
B l a s k o v i c , D., and Rehécek, J . (1962). Press, Inc. , N e w Y o r k , p. 1 3 5 . C o r y , J . , a n d Y u n k e r , C . E . (1971 ). Ann. D a v i d - W e s t , T . S . ( 1 9 7 4 ) . Arch.
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P u d n e y , M . , V a r m a , M . G . R . , a n d L e a k e , C J . ( 1 9 7 3 ) . J. Med. Ent. R e h é c e k , J . ( 1 9 5 8 a ) . Ph. D. R e h é c e k , J . ( 1 9 5 8 b ) . Acta
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H o f f m a n , G . , S c h e i n , E., a n d J a g o w , M. ( 1 9 7 0 ) . Ζ. Jropenmed. K o r d o v â , Ν . , a n d R e h é c e k , J . ( 1 9 6 9 ) . A eta virol.
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33
686. 20.
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Chapter 3
MORPHOGENESIS OF SINDBIS VIRUS IN CULTURED MOSQUITO CELLS
D.T. Brown, J . F . S m i t h , J . B . G l i e d m a n , B. R i e d e l , D. F i l t z e r , and D. Renz
I.
Introduction
35
II.
Results and d i s c u s s i o n
35
III.
Conclusion
47
IV.
References
47
I.
Introduction
We have undertaken an e x t e n s i v e c o m p a r a t i v e study of t h e d e v e l o p m e n t of t h e group A a r b o v i r u s S i n d b i s in c u l t u r e d v e r t e b r a t e and i n v e r t e b r a t e c e l l s . T h i s study w h i c h began n e a r l y f o u r y e a r s ago i s p r e s e n t l y e x a m i n i n g the g e n e t i c s , b i o c h e m i s t r y , m o l e c u l a r b i o l o g y , and morphology of v i r u s i n f e c t e d t i s s u e c u l t u r e d c e l l s of t h e v e r t e b r a t e and i n v e r t e b r a t e h o s t s . S i n d b i s v i r u s is i d e a l l y s u i t e d f o r a compar a t i v e study of t h i s t y p e as t h i s p a r t i c u l a r group A v i r i o n has been e x t e n s i v e l y s t u d i e d in t h e v e r t e b r a t e c e l l s (Pfefferkorn and Shapiro, 1 9 7 4 ) . Our e x p e r i m e n t s have been c a r r i e d out w i t h c u l t u r e d c h i c k embryo f i b r o b l a s t (CEF) and baby h a m s t e r k i d n e y (BHK-21) c e l l s as r e p r e s e n t a t i v e s of t h e v e r t e b r a t e s y s t e m , and c o n t i o r i g i n a l l y prepared by Singh (1967) nuous c u l t u r e d l a r v a e c e l l s of Aedes albopictus and p r o v i d e d by D r s . I. Snyder (Walter Reed) and S. B u c k l e y (Yale U n i v e r s i t y ) and The A m e r i c a n Type C u l t u r e C o l l e c t i o n . In each c a s e t h e o b s e r v a t i o n s f o r t h e t w o v e r t e b r a t e c e l l s y s t e m s w e r e s i m i l a r as w e r e g e n e r a l l y t h e o b s e r v a t i o n s made in the m o s q u i t o c e l l s o b t a i n e d from t h e t h r e e s o u r c e s . Some d i f f e r e n c e s w e r e d e t e c t e d in t h e i n v e r t e b r a t e c e l l l i n e s p r i m a r i l y w i t h r e s p e c t t o g r o w t h rate and t h e amount of v i r u s p r o d u c e d . The r e a s o n s f o r t h e s e d i f f e r e n c e s are not c l e a r . In an a t t e m p t t o p l a c e t h e f o l l o w i n g d i s c u s s i o n in an a p p r o p r i a t e frame of r e f e r e n c e i t w i l l be n e c e s s a r y to f i r s t b r i e f l y s u m m a r i z e r e s u l t s o b t a i n e d in our l a b o r a t o r y and by o t h e r s w i t h vertebrate host c e l l s . IL
Results and d i s c u s s i o n
S i n d b i s i s a s t r u c t u r a l l y and c h e m i c a l l y s i m p l e v i r u s w h i c h has s i n g l e s t r a n d e d RNA as i t s g e n e t i c m a t e r i a l . The RNA genome is c o n t a i n e d w i t h i n a c a p s i d w h i c h has i c o s a h e d r a l symmetry and i s c o m p o s e d of 9 2 i d e n t i c a l s u b u n i t s ( A c h e s o n and Tamm, 1967; Brown and G l i e d m a n , 1973) w h i c h c o n t a i n m u l t i p l e c o p i e s of a s i n g l e p r o t e i n h a v i n g a m o l e c u l a r w e i g h t of 30,000 ( S c h e e l e and P f e f f e r k o r n , 1 9 7 0 ) . The c a p s i d is i t s e l f e n c l o s e d w i t h i n a membrane, t h e l i p i d m o i e t y of w h i c h i s d e r i v e d from the host membranes. The v i r a l membrane in turn c o n t a i n s t w o " e n v e l o p e p r o t e i n s " w h i c h are c o d e d for by t h e v i r u s and are g l y c o s y l a t e d in a p a t t e r n w h i c h
35
D. T. B R O W N et al.
s u g g e s t s t h a t t h e sugars are added by host e n z y m e s (Sefton and Bürge, 1 9 7 4 ) . The e n v e l o p e p r o t e i n s r e s i d e on t h e outer s u r f a c e of t h e v i r a l e n v e l o p e and are a n c h o r e d i n t o the e n v e l o p e by a s m a l l h y d r o p h o b i c r e g i o n w h i c h p r o t r u d e s i n t o t h e outer member of t h e e n v e l o p e b i l a y e r (Utermann and Simons, 1974) . The morphogen e s i s of S i n d b i s v i r i o n s in v e r t e b r a t e h o s t c e l l s t a k e s p l a c e in t h r e e d i s t i n c t s t e p s (see F i g . 1 ) . The v i r a l p o l y p e p t i d e s are s y n t h e s i z e d from a s p e c i e s of RNA w h i c h has t h e same p o l a r i t y as t h e RNA c o n t a i n e d in t h e i n f e c t i n g v i r i o n but i s o n l y one t h i r d t h e s i z e (26 S or i n t e r j a c e n t RNA) (Simmons et al., 1 9 7 2 ) . The s t r u c t u r a l p r o t e i n s are p r o d u c e d as a s i n g l e p o l y p e p t i d e from w h i c h t h e c a p s i d p r o t e i n is c l e a v e d before t h e n a s c e n t p o l y p e p t i d e i s r e l e a s e d from t h e p o l y r i b o s o m e c o m p l e x (Scheele and P f e f f e r k o r n , 1970; S c h l e s i n g e r and S c h l e s i n g e r , 1 9 7 3 ) . The c a p s i d p r o t e i n i s r a p i d l y i n c o r p o r a t e d i n t o v i r a l c o r e s w h i c h are seen f r e e in t h e c e l l c y t o p l a s m in t h e e l e c t r o n m i c r o s c o p e ( A c h e s o n and Tamm, 1967; Brown a n d S m i t h , 1 9 7 5 ) . The remainder of the p o l y p e p t i d e i s d e s t i n e d to be s e q u e n t i a l l y c l e a v e d and g l y c o s y l a t e d , and the p r o d u c t s are i n s e r t e d i n t o t h e plasma membrane of t h e i n f e c t ed c e l l ( S c h l e s i n g e r and S c h l e s i n g e r , 1973; S c h l e s i n g e r et ah, 1972; Sefton and Bürg e , 1974; Sefton et aL, 1 9 7 3 ) . The i n s e r t i o n of v i r u s s p e c i f i c p r o t e i n s i n t o t h e v e r t e b r a t e plasma membrane o c c u r s r a p i d l y and can be d e t e c t e d by a n t i g e n i c p r o b e s and h e a m a d s o r b t i o n hours before i n f e c t i o u s v i r u s is r e l e a s e d . The membrane of t h e i n f e c t e d c e l l c o n t a i n s one of t h e p r o t e i n s r e c o v e r e d in t h e v i r a l e n v e l o p e (E-j) and a p r e c u r s o r t o the other (PE2) ( J o n e s et al., 1974; Sefton et al., 1 9 7 3 ) . A f t e r t h e i n s e r t i o n of t h e p a r t i a l l y p r o c e s s e d v i r u s p r o t e i n into t h e p l a s m a membrane t h e v i ral n u c l e o c a p s i d s m i g r a t e to the inner s u r f a c e of t h i s s t r u c t u r e and a t t a c h to some u n i d e n t i f i e d v i r u s s p e c i f i c component a s s o c i a t e d w i t h i t (Brown and S m i t h , 1 9 7 5 ) . The a s s o c i a t i o n of the n u c l e o c a p s i d w i t h t h e inner s u r f a c e of t h e h o s t plasma membrane seems to be s t r o n g as c a p s i d - m e m b r a n e c o m p l e x e s can be i s o l a t e d from c e l l s in w h i c h the a t t a c h e d n u c l e o c a p s i d s remain bound to t h e membrane a f t e r p u r i f i c a t i o n by d e n s i t y g r a d i e n t c e n t r i f u g a t i o n (Brown and S m i t h , 1 9 7 5 ) . The f i n a l stage in v i r u s m o r p h o g e n e s i s o c c u r s as the v i r u s n u c l e o c a p s i d is w r a p p e d in a f r a g m e n t of the m o d i f i e d h o s t membrane as i t i s r e l e a s e d i n t o t h e s u r r o u n d i n g media ( A c h e s o n and Tamm, 1967; Brown et aL, 1972; Brown and S m i t h , 1 9 7 5 ) . L i t t l e i s known r e g a r d i n g t h e m e c h a n i s m of t h i s " b u d d i n g " p r o c e s s . A f i n a l c l e a v a g e of one of the v i r u s p r e c u r s o r p o l y p e p t i d e s (PE2 > E2) ( J o n e s et al., 1974; S c h l e s i n g e r and S c h l e s i n g e r , 1972; Sefton et al., 1973) and t h e d i s p l a c e m e n t of host-membrane p r o t e i n s in t h e p o r t i o n of t h e v i r a l membrane d e s t i n e d to become the v i r a l e n v e l o p e seen to accompany b u d d i n g ( F i g s . 1,2) (Brown et al., 1 9 7 2 ) . The a t t a c h m e n t of the c a p s i d to the plasma membrane does not in i t s e l f c a u s e t h e f i n a l p r o c e s s i n g c l e a v a g e of the p r e c u r s o r p o l y p e p t i d e (PE2) or t h e d i s p l a c e m e n t of h o s t membrane p r o t e i n s (Brown et al., 1972; Brown and S m i t h , 1975; J o n e s et al., 1 9 7 4 ) . The r e a c t i o n s f o l l o w i n g t h e a t t a c h m e n t of t h e n u c l e o c a p s i d to t h e plasma membrane appear to move s e q u e n t i a l l y around t h e n u c l e o c a p s i d m o v i n g i t from a p o s i t i o n in t h e c e l l c y t o p l a s m to one o u t s i d e of t h e c e l l . T h i s p r o c e s s i s a c c o m p a n i e d by t h e c l e a v a g e of the p r e c u r s o r p o l y p e p t i d e , the d i s p l a c e m e n t of t h e h o s t membrane p r o t e i n s and t h e i n t i m a t e a s s o c i a t i o n of the c a p s i d p r o t e i n w i t h t h e d e v e l o p i n g v i r a l e n v e l o p e . The g r o w t h of S i n d b i s v i r u s in c u l t u r e d v e r t e b r a t e c e l l s p r o d u c e s g r o s s c y t o p a t h i c e f f e c t s and t h e r e p l i c a t i v e c y c l e i s t e r m i n a t e d by death and l y s i s of t h e h o s t c e l l 14-20 hours after i n f e c t i o n . A number of i n v e s t i g a t i o n s of A r b o v i r u s g r o w t h in c u l t u r e d m o s q u i t o c e l l s , h a v e r e v e a l e d the rate and amount of v i r u s p r o d u c e d in t h i s s y s t e m to be s i m i l a r t o t h a t o b s e r v e d in c u l t u r e d v e r t e b r a t e c e l l s . The c u l t u r e d m o s q u i t o c e l l s , u n l i k e t h e i r v e r t e b r a t e c o u n t e r p a r t s , are g e n e r a l l y a b l e t o s u r v i v e i n f e c t i o n by A r b o v i r u s e s and 36
MORPHOGENESIS OF S I N D B I S V I R U S IN C U L T U R E M O S Q U I T O C E L L S
/. S c h e m a t i c r e p r e s e n t a t i o n o f S i n d b i s v i r u s m o r p h o g e n e s i s in BHK or CEF ( v e r t e b r a t e Fig. c e l l s ) . 1 . N u c l e o c a p s i d s are a s s e m b l e d in t h e c y t o p l a s m o f t h e i n f e c t e d c e l l . 2 . C o m p l e t e d n u c l e o c a p s i d s a t t a c h t o inner s u r f a c e o f h o s t p l a s m a membrane w h i c h h a s b e e n a l t e r e d b y t h e a d d i t i o n o f v i r u s p r o t e i n s ( s p i k e s ) , t h e membrane c o n t a i n s i n t e r i o r membrane h o s t - g l y c o p r o t e i n b e a d s . 3 . C a p s i d b e g i n s t o bud t h r o u g h p l a s m a membrane w h i c h s t i l l c o n t a i n s g l y c o p r o t e i n b e a d s . 4 . A more a d v a n c e d s t a g e of b u d d i n g , i n t e r i o r membrane b e a d s a r e m i s s i n g f r o m some a r e a s o f t h e d e v e l o p i n g e n v e l o p e . 5. P a r t i a l r e l e a s e o f v i r i o n b y f u s i o n o f inner l e a f l e t o f e n v e l o p e . V i r i o n i s s t i l l a t t a c h e d t o h o s t b y c o n t i n u i t y o f o u t e r l e a f l e t . 6 . Free v i r i o n interior of envelope membrane is free of g l y c o p r o t e i n beads. Compare t o F i g s . 2 a n d 1 2 . i n f e c t i o n u l t i m a t e l y r e s u l t s in t h e e s t a b l i s h m e n t of a p e r s i s t e n t l y i n f e c t e d c e l l p o p u l a t i o n w h i c h , in terms of i t s g r o w t h c h a r a c t e r i s t i c s , i s s i m i l a r to n o n - i n f e c t e d c e l l s ( B u c k l e y , 1969; P e l e g , 1969; S t e v e n s , 1970; Raghow et al., 1 9 7 3 ) . In our l a b o r a t o r y maximum y i e l d s of v i r u s ( c a . 4,000 P F U / c e l l ) w e r e o b t a i n e d from t h e m o s q u i t o c e l l s at 21-36 hours p o s t i n f e c t i o n . A f t e r t h i s i n i t i a l b u r s t of v i r u s p r o d u c t i o n t h e t i t e r of t h e c u l t u r e media f a l l s by about one log and r e m a i n s at t h i s l o w e r l e v e l for s e v e r a l d a y s . A n i n v e s t i g a t i o n of t h e m o r p h o g e n e s i s of S i n d b i s v i r u s in t h e c u l t u r e d m o s q u i t o c e l l s by e l e c t r o n m i c r o s c o p y w a s u n d e r t a k e n in t h e hope t h a t the l a c k of c y t o c i d a l e f f e c t s of v i r u s i n f e c t i o n in t h e s e c e l l s c o u l d be in part e x p l a i n e d by d i f f e r e n c e s in t h e morphology of t h e i n f e c t e d c e l l s at t h e ultrastructural level. The f i r s t n o t i c e a b l e m o r p h o l o g i c a l e v e n t o c c u r r i n g in t h e i n f e c t e d m o s q u i t o c e l l s w a s t h e appearance of membrane-rich v e s i c u l a r s t r u c t u r e s in w h i c h , as t i m e p r o g r e s s e d , v i r a l n u c l e o c a p s i d s c o u l d be f o u n d ( F i g s . 3 , 4 ) . The l a r g e numbers of c y t o p l a s m i c n u c l e o c a p s i d s r e a d i l y f o u n d in i n f e c t e d v e r t e b r a t e h o s t c e l l s ( A c h e s o n and Tamm, 1967; Brown and S m i t h , 1965) w e r e not f o u n d in t h e i n f e c t e d m o s q u i t o c e l l s . F r e e z e - e t c h i n g of t h e v e s i c u l a r s t r u c t u r e s in t h e i n f e c t e d m o s q u i t o c e l l s r e v e a l e d t h a t t h e i n t e r n a l membranes c o n t a i n e d t h e c l a s s i c a l d i s t r i b u t i o n of i n t e rior membrane g l y c o p r o t e i n beads ( M a r c h e s i et al., 1972; T i l l a c k et al., 1972) ( F i g . 5 ) . A s t i m e p r o g r e s s e d t h e v e s i c l e s w e r e f o u n d t o c o n t a i n many mature v i r i o n s as w e l l as p a r t i a l l y mature forms and f r e e n u c l e o c a p s i d s ( F i g . 6 ) . Envelopment of the n u c l e o c a p s i d s seemed t o o c c u r through i n t e r a c t i o n of t h e c a p s i d s w i t h t h e membranes in t h e v e s i c l e s and not by b u d d i n g of c y t o p l a s m i c n u c l e o c a p s i d s i n t o t h e vac u o l e s . A t t i m e s l a t e r than 20 hours a f t e r i n f e c t i o n some c e l l s c o n t a i n e d many e l e c tron dense v e s i c l e s ( F i g . 7) w h i c h when e x a m i n e d at high m a g n i f i c a t i o n p r o v e d to c o n t a i n large numbers of mature v i r i o n s ( F i g . 8 ) . The c o m p o s i t i o n or o r i g i n of t h e
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Fig. 2. A n e l e c t r o n m i c r o g r a p h o f a c h i c k e m b r y o f i b r o b l a s t c e l l a f t e r i n f e c t i o n w i t h S i n d b i s v i r u s . The c l e a v e d p l a s m a membrane (oL) h a s many n e a r l y m a t u r e v i r i o n s ( 1 , 2 , 3 ) w h i c h a r e s t i l l attached t o t h e cell by t h e c o n t i n u i t y of their envelopes w i t h t h e cell membrane. A number of d e v e l o p i n g v i r a l b u d s are a p p a r e n t ( b ) . A t t h i s t i m e i n d e v e l o p m e n t t h e i n t e r i o r o f t h e membrane of t h e b u d s t i l l c o n t a i n s t h e g l y c o p r o t e i n beads w h i c h a r e c h a r a c t e r i s t i c of t h e r e s t o f t h e p l a s m a m e m b r a n e . The b e a d s are n o t p r e s e n t i n t h e i n t e r i o r s o f t h e v i r a l e n v e l o p e s ( 1 , 2 , 3 ) . C c y t o p l a s m N , n u c l e u s , N p n u c l e o p o r e . M a g n i f i c a t i o n b a r i s 0 . 5 yum, from Brown et al. 1972 b y p e r m i s s i o n o f t h e A m e r i c a n S o c i e t y o f M i c r o b i o l o g y . e l e c t r o n dense m a t e r i a l w h i c h c o m p l e t e l y surrounds and p a r t i a l l y o b s c u r e s t h e mature v i r i o n s in t h e s e v a c u o l e s i s not u n d e r s t o o d . The e l e c t r o n d e n s e , v i r u s - c o n t a i n i n g v a c u o l e s d e s c r i b e d here are s i m i l a r t o t h o s e d e s c r i b e d by Raghow and c o w o r k e r s in m o s q u i t o c e l l s i n f e c t e d w i t h S e m l i k i f o r e s t v i r u s and Ross River v i r u s (both group A v i r i o n s ) (Raghow et a/.., 1 9 7 3 ) . Raghow and c o w o r k e r s s u g g e s t e d that t h e s e v i r i o n s do n o t c o n t r i b u t e t o e x t r a c e l l u l a r t i t r e s as they are d e s t r o y e d when the v e s i c l e f u s e s w i t h l y s o z o m a l structures» We f o u n d no e v i d e n c e for d e s t r u c t i o n of t h e s e v e s i c l e s but r a t h e r f o u n d t h a t t h e v i r u s p r o d u c ed in t h e s e s t r u c t u r e s i s r e l e a s e d from the h o s t by f u s i o n of t h e v e s i c u l a r s t r u c t u r e w i t h t h e p l a s m a membrane. A s t h e v e s i c l e s f u s e w i t h t h e c e l l s u r f a c e t h e u n i f o r m i n t e r i o r appeared to break up r e l e a s i n g v i r i o n s w i t h a c o a t i n g of t h e e l e c t r o n d e n s e
38
MORPHOGENESIS OF S I N D B I S V I R U S IN C U L T U R E M O S Q U I T O CELLS
Fig. 3. 1 jjm,
A n u n i n f e c t e d Aedes
albopictus
c e l l a f t e r u l t r a t h i n s e c t i o n i n g . M a g n i f i c a t i o n bar i s
at 4 Fig. 4. U l t r a t h i n s e c t i o n o f v e s i c l e s f o u n d in t h e c y t o p l a s m o f a n Aedes albopictus hours post i n f e c t i o n w i t h Sindbis v i r u s . The v e s i c l e s contain many t r a n s v e r s e membranes a n d r i b o s o m e s . T h e m e m b r a n e s t o t h e r i g h t o f t h e p h o t o g r a p h a r e n o t y e t c o n t a i n e d in a l i m i t i n g m e m b r a n e . M a g n i f i c a t i o n b a r = 0.5 ,um. T h i s a n d a l l s u b s e q u e n t e x p e r i m e n t s w e r e made w i t h a m u l t i p l i c i t y o f 50 PFU v i r u s / c e l l and w e r e i n c u b a t e d at 2 8 ° C . m a t e r i a l ( F i g . 9 ) . T h i s e l e c t r o n d e n s e m a t e r i a l w a s not s t r o n g l y bound t o t h e new v i r i o n s as e x a m i n a t i o n of t h e p a r t i c l e s before and a f t e r d e n s i t y g r a d i e n t p u r i f i c a t i o n r e v e a l e d them t o be f r e e of s u r f a c e c o n t a m i n a t i o n ( F i g . 1 0 ) . The v i r i o n s p r o d u c e d in t h e f i r s t f o r t y hours a f t e r i n f e c t i o n w e r e m o r p h o l o g i c a l l y i n d i s t i n g u i s h a b l e from v i r u s p r o d u c e d from BHK or CEF c e l l s . A t l a t e r t i m e s t h e i n f e c t e d c e l l s p r o d u c e d , in a d d i t i o n to the normal s i z e v i r i o n , t w o s m a l l e r p a r t i c l e s 80% and 59% t h e s i z e of t h e normal v i r i o n (Brown and G l i e d m a n , 1 9 7 3 ) . These p a r t i c l e s w e r e f o u n d t o be n o n i n f e c t u o u s f o r both BHK and m o s q u i t o c e l l s when p u r i f i e d away from t h e normal p a r t i c l e s . These p a r t i c l e s c o u l d not be f o u n d in u n i n f e c t e d m o s q u i t o c e l l s in s p i t e of a number of a t t e m p t s to i n d u c e them c h e m i c a l l y and by i r r a d i a t i o n . I n f e c t i o n of
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D. T. BROWN er al.
Fig. 5. A c y t o p l a s m i c m e m b r a n e - c o n t a i n i n g v e s i c l e s i m i l a r t o t h o s e in F i g . 4 a f t e r f r e e z e e t c h i n g . The m e m b r a n e s w i t h i n t h e v e s i c l e c o n t a i n a t y p i c a l d i s t r i b u t i o n of i n t e r i o r g l y c o p r o t e i n b e a d s ( c o m p a r e w i t h F i g . 2 ) . M a g n i f i c a t i o n bar = 1 u r n .
Fig. c e l l s a t 12 h o u r s a f t e r i n f e c t i o n w i t h 6. A v e s i c l e f o u n d in i n f e c t e d Aedes albopictus S i n d b i s v i r u s . The v a c u o l e c o n t a i n s v i r u s n u c l e o c a p s i d s ( C ) , p a r t i a l l y e n v e l o p e d v i r i o n s (E) a n d m a t u r e v i r i o n s ( V ) . M a g n i f i c a t i o n bar = 100 n m .
40
MORPHOGENESIS OF SINDBIS V I R U S IN C U L T U R E M O S Q U I T O C E L L S
Fig. 7. Aedes albopictus c e l l s typical of those found at 14-21 hours after i n f e c t i o n . The c e l l c o n t a i n s a large number o f e l e c t r o n d e n s e b o d i e s w h i c h e x a m i n e d a t h i g h e r m a g n i f i c a t i o n c o n t a i n e d large n u m b e r s of v i r i o n s ( a r r o w s ) . M a g n i f i c a t i o n bar = 1 ,um.
Fig. 8. A h i g h m a g n i f i c a t i o n o f a v e s i c l e o f t h e t y p e s e e n i n F i g . 7 . T h e v e s i c l e i s f i l l e d w i t h e l e c t r o n d e n s e m a t e r i a l in w h i c h mature v i r i o n s are s e e n p a c k e d i n a p a r a c r y s t a l i n e a r r a y . M a g n i f i c a t i o n bar = 1 0 0 n m . t h e Aedes albopictus c e l l s w i t h a number of S i n d b i s t e m p e r a t u r e s e n s i t i v e m u t a n t s at n o n p e r m i s s i v e t e m p e r a t u r e p r o d u c e d no p a r t i c l e s of t h i s t y p e s u g g e s t i n g t h a t S i n d b i s is not p r o v i d i n g a h e l p e r f u n c t i o n f o r some l a t e n t v i r u s - l i k e p a r t i c l e „ Att e m p t s t o e s t a b l i s h t h e p r e s e n c e of RNA in t h e s m a l l e r v i r i o n s by l a b e l i n g w i t h u r i d i n e h a v e been n e g a t i v e . We have not been a b l e to d e t e r m i n e if t h e s e p a r t i c l e s p l a y any r o l e in t h e e s t a b l i s h m e n t of t h e p e r s i s t e n t s t a t e of i n f e c t i o n or if t h e y are in any w a y r e l a t e d to t h e i n t e r f e r i n g agent p r o d u c e d by t h i s i n f e c t e d c e l l s y s tem ( S t o l l a r and Shenk, 1 9 7 3 ) . In l e s s than one p e r c e n t of t h e c e l l s e x a m i n e d in t h i s s t u d y a f e w c y t o p l a s m i c n u c l e o c a p s i d s w e r e d e t e c t e d and o c c a s i o n a l l y c a p s i d s c o u l d be f o u n d near t h e s u r f a c e of t h e c e l l in a c o n f i g u r a t i o n w h i c h w a s s u g g e s t i v e of b u d d i n g (Fig„ 1 1 ) .
41
D. T . BROWN et al.
Fig. 9. A v i r u s c o n t a i n i n g v e s i c l e f u s i n g w i t h t h e c e l l s u r f a c e ( a r r o w ) . The u n i f o r m appeara n c e o f t h e v e s i c l e i s b r o k e n up d u r i n g t h e p r o c e s s o f v i r u s r e l e a s e . The v i r i o n s appear c o a t e d w i t h t h e e l e c t r o n dense c o n t e n t s o f t h e v e s i c l e . M a g n i f i c a t i o n bar = 100 n m .
10. Mature S i n d b i s v i r i o n s f r o m Aedes albopictus c e l l s a t 2 4 h o u r s after i n f e c t i o n . T h e Fig. v i r i o n s w e r e p u r i f i e d by i s o p y c n i c d e n s i t y g r a d i e n t c e n t r i f u g a t i o n in p o t a s s i u m t a r t r a t e , n e g a t i v e l y s t a i n e d w i t h p h o s p h o t u n g s t i c a c i d . M a g n i f i c a t i o n bar = 1 0 0 n m . A n e x h a u s t i v e search f o r t r u e b u d d i n g f i g u r e s u t i l i z i n g f r e e z e - e t c h i n g t e c h n i q u e s w a s n e g a t i v e . C o n s i d e r i n g t h e ease w i t h w h i c h t h i s p r o c e s s i s d e t e c t e d in verteb r a t e c e l l s u t i l i z i n g e i t h e r f r e e z e - e t c h i n g ( F i g . 12) (Brown et ai,, 1 9 7 2 ) , or t h i n s e c t i o n i n g ( A c h e s o n and Tamm, 1967; Brown and S m i t h , 1975) w e c o n c l u d e t h a t under c o n d i t i o n s e m p l o y e d in our l a b o r a t o r y l i t t l e v i r u s i s p r o d u c e d by t h i s r o u t e . The r e l e a s e of mature v i r i o n s from the i n f e c t e d c e l l by f u s i o n of t h e v e s i c u l a r s t r u c t u r e s w i t h the c e l l s u r f a c e and t h e o b s e r v a t i o n t h a t e n v e l o p m e n t of t h e v i r i o n s t a k e s p l a c e in t h e s e v e s i c l e s p r e s e n t e d us w i t h t h e p o s s i b i l i t y t h a t d u r i n g the p r o c e s s of v i r u s r e l e a s e p a r t i a l l y e n v e l o p e d s t r u c t u r e s m i g h t a l s o be r e l e a s e d from t h e c e l l s . E x a m i n a t i o n of v i r u s s t r u c t u r e s d i s c h a r g e d i n t o t h e g r o w t h medium in t h e e l e c t r o n m i c r o s c o p e r e v e a l e d a v a r i e t y o f i n t e r m e d i a t e s in t h e p r o c e s s o f e n v e l o p m e n t ( F i g . 1 3 ) . These s t r u c t u r e s w e r e f o u n d in rather large numbers in t h e
42
MORPHOGENESIS OF S I N D B I S V I R U S IN C U L T U R E M O S Q U I T O CELLS
Fig. 11. Thin s e c t i o n of Aedes albopictus c e l l s w i t h s u r f a c e - a s s o c i a t e d v i r i o n s , a) T w o i n f e c t e d c e l l s . Mature v i r i o n s are s e e n i n t h e s p a c e b e t w e e n t h e m . The l o w e r c e l l h a s n u c l e o c a p s i d s in t h e c y t o p l a s m . O n e of w h i c h i s a s s o c i a t e d w i t h t h e p l a s m a membrane a s t h o u g h b u d d i n g , b) A n a p p a r e n t " l a t e s t a g e " in b u d d i n g o f v i r i o n s f r o m t h e s u r f a c e o f a n i n f e c t e d Aedes albopictus c e l l . M a g n i f i c a t i o n b a r = 100 nm. g r o w t h medium and c o u l d be c o n c e n t r a t e d in p o t a s s i u m t a r t r a t e d e n s i t y g r a d i e n t s on t h e " l i g h t " s i d e of t h e major v i r u s b a n d . Large f r a g m e n t s of membranes w e r e seen w i t h a t t a c h e d n u c l e o c a p s i d s . In some i n s t a n c e s t h r e e s i z e s of c a p s i d s c o u l d be f o u n d a t t a c h e d t o one s i d e of t h e membrane ( F i g . 1 3 a ) . These s t r u c t u r e s p r o b a b l y c o r r e s p o n d t o t h e c a p s i d s of t h e t h r e e m o r p h o l o g i c a l t y p e s of v i r u s p r o d u c e d by t h i s c e l l s y s t e m (Brown and G l i e d m a n , 1 9 7 3 ) . The membrane s u r f a c e away from t h e s i d e of c a p s i d a t t a c h m e n t w a s f r e e of s p i k e - l i k e s t r u c t u r e s s u g g e s t i n g t h a t e i ther t h e m o r p h o l o g i c a l change in t h e membrane r e s u l t i n g in t h e a p p e a r a n c e of s p i k e s has not t a k e n p l a c e or t h a t they had been l o s t from t h e membrane d u r i n g p u r i f i c a t i o n . M e m b r a n e - n u c l e o c a p s i d c o m p l e x e s c o u l d a l s o be f o u n d w h i c h seemed t o be in a somewhat more a d v a n c e d s t a t e of m a t u r a t i o n ( F i g . 1 3 b ) . These s t r u c t u r e s d i f f e r e d from t h a t d e s c r i b e d above in t h a t t h e n u c l e o c a p s i d s w e r e p a r t i a l l y e n c l o s e d in t h e membrane f r a g m e n t as though e n v e l o p m e n t had been i n t e r r u p t e d . In a d d i t i o n , the outer s u r f a c e of the membrane w a s c o a t e d w i t h s p i k e s t r u c t u r e s o v e r i t s e n t i r e s u r f a c e i n c l u d i n g t h o s e areas w h e r e no e n v e l o p m e n t of v i r u s w a s t a k i n g p l a c e . Large numbers of c l o s e d membraneous s t r u c t u r e s c o n t a i n i n g more than one n u c l e o c a p s i d w e r e a l s o f o u n d ( F i g . 13c) s u g g e s t i n g t h a t t h e p r o c e s s of e n v e l o p ment in t h e m o s q u i t o v e s i c l e s i s not as p r e c i s e as t h e b u d d i n g p r o c e s s o c c u r r i n g on the s u r f a c e of v e r t e b r a t e host c e l l s w h i c h do not p r o d u c e m u l t i c a p s i d s t r u c t u r e s under normal c o n d i t i o n s . The t o t a l a l t e r a t i o n of t h e s u r f a c e morphology of t h e v e s i c u l a r membranes a f t e r t h e a t t a c h m e n t of t h e n u c l e o c a p s i d s s u g g e s t e d t h a t in t h i s s y s t e m , u n l i k e t h e vert e b r a t e s y s t e m , t o p o l o g i c a l a l t e r a t i o n of t h e membranes c o u l d t a k e p l a c e in areas not a c t i v e l y i n v o l v e d in v i r a l e n v e l o p m e n t . That t h i s i s t h e c a s e w a s a l s o s u g g e s t ed by f r e e z e - e t c h i n g of t h e membranes in t h e v i r u s - i n d u c e d v e s i c l e s a t t i m e s l a t e after i n f e c t i o n ( F i g . 1 4 ) . Some of t h e s t a c k e d membranes c o u l d be seen p o s s e s s i n g larger than normal numbers of t h e i n t e r i o r membrane beads w h i l e o t h e r i n t e r i o r membrane s u r f a c e s w e r e c o m p l e t e l y f r e e of t h e p a r t i c l e s . E x a m i n a t i o n of v i r u s p u r i f i e d from t h e m o s q u i t o s y s t e m r e v e a l e d t h a t l i k e t h e v e r t e b r a t e s y s t e m t h e i n t e r i o r of t h e v i r a l e n v e l o p e i s f r e e of t h e s e i n t e r i o r membrane p a r t i c l e s (not s h o w n ) . These o b s e r v a t i o n s s u g g e s t t h a t u n l i k e t h e v e r t e b r a t e c e l l s y s t e m d e s 43
D. T . BROWN et al.
Fig. 12. A f r e e z e - e t c h r e p l i c a of t h e p l a s m a membrane of a c h i c k e m b r y o f i b r o b l a s t c e l l a f t e r i n f e c t i o n w i t h S i n d b i s v i r u s . A t t h e edge o f t h e c e l l a l a r g e number o f n e a r l y m a t u r e v i r i o n s c a n b e seen ( a ) . On t h e i n t e r i o r s u r f a c e o f t h e h o s t p l a s m a m e m b r a n e many large s t a l k s (s) ( F i g s , a + b) c a n be d e t e c t e d . T h e s e r e p r e s e n t p o i n t s o f a t t a c h m e n t o f t h e v i r a l e n v e l o p e t o t h e p l a s m a m e m b r a n e . They a r e left o n t h e s u r f a c e o f t h e i n n e r l e a f l e t o f t h e membrane b i l a y e r a f t e r t h e o u t e r l e a f l e t i s r e m o v e d by t h e c l e a v i n g p r o c e s s . T h e s e s t a l k s c a n a l s o be s e e n a t t h e b a s e o f some o f t h e c l e a v e d p e r i p h e r a l v i r i o n s . M a g n i f i c a t i o n bar a = 1.0 jum, b = 2 0 0 n m , f r o m Brown et al. 1972 b y p e r m i s s i o n o f t h e A m e r i c a n S o c i e t y of M i crobiology.
c r i b e d above an e n t i r e membrane s u r f a c e can be r e o r g a n i z e d by t h e a p p e a r a n c e of s p i k e s on i t s s u r f a c e and t h e c o m p l e t e l o s s of p r e e x i s t i n g membrane p r o t e i n s from the membrane i n t e r i o r . A s s u g g e s t e d at the o u t s e t of t h i s d i s c u s s i o n , one w o u l d l i k e to be a b l e to corr e l a t e t h e s e d e s c r i b e d m o r p h o l o g i c a l o b s e r v a t i o n s ( s u m m a r i z e d in F i g . 15) to t h e a b i l i t y of t h e m o s q u i t o c e l l s to s u r v i v e i n f e c t i o n by A r b o v i r u s e s and t o t h e e s t a b l i s h m e n t of t h e p e r s i s t e n t l y i n f e c t e d c e l l p o p u l a t i o n . In t h i s r e s p e c t t h e p r o d u c t i o n of progeny v i r i o n s in i n t e r n a l l y l o c a t e d v e s i c l e s is a p p e a l i n g as i t may i s o l a t e some of t h e b i o c h e m i c a l p r o c e s s e s w h i c h are e s s e n t i a l to v i r u s p r o d u c t i o n b u t
44
M O R P H O G E N E S I S OF SINDBIS V I R U S IN C U L T U R E M O S Q U I T O C E L L S
Fig. 13. Immature membrane c a p s i d c o m p l e x e s r e l e a s e d f r o m S i n d b i s i n f e c t e d Aedes albopictus c e l l s , a) Large m e m b r a n e f r a g m e n t w i t h a t t a c h e d n u c l e o c a p s i d s . T h e c a p s i d s h a v e 3 d i s t i n c t s i z e s a n d are made up o f d i s t i n c t s u b u n i t s ( i n s e c t ) , b) Membrane f r a g m e n t w i t h p a r t i a l l y enveloped c a p s i d s and s p i k e s , c) A m u l t i c a p s i d c o n t a i n i n g v i r a l envelope. Magnif i c a t i o n bars = 100 n m . t o x i c t o t h e h o s t - c e l l . The p r e s e n c e of r i b o s o m e s in t h e v e s i c u l a r s t r u c t u r e s s u g g e s t s t h a t v i r a l p r o t e i n s y n t h e s i s c o u l d o c c u r in t h e s e s t r u c t u r e s . It has a l s o been s u g g e s t e d t h a t t h e p r e s e n c e of d o u b l e - s t r a n d e d RNA in c e l l s may be r e s p o n s i b l e f o r t h e c y t o p a t h i c e f f e c t s p r o d u c e d by RNA v i r u s e s ( C o r d e l I-Stewart and T a y l o r , 1971; Garwes et al., 1 9 7 5 ) . T h u s , t h e s e q u e s t e r i n g of r e p l i c a t i v e i n t e r m e d i a t e s t o e n c l o s e d f a c t o r i e s in t h e i n f e c t e d c e l l c y t o p l a s m c o u l d m i n i m i z e t h i s t o x i c e f f e c t . The r e s t r i c t i o n of t h e p r o c e s s of e n v e l o p m e n t of v i r a l n u c l e o c a p s i d s to t h e v e s i c u lar s t r u c t u r e s c o u l d a l s o e l i m i n a t e t h e n e c e s s i t y of t h e m o d i f i c a t i o n of t h e h o s t p l a s m a membranes by i n s e r t i o n of v i r u s p r o t e i n s , a p r o c e s s w h i c h o c c u r s e a r l y in i n f e c t e d v e r t e b r a t e c e l l s and m i g h t c o n t r i b u t e to t h e o v e r a l l c y t o p a t h i c p r o c e s s . We have f o u n d t h a t m o s q u i t o c e l l s a c t i v e l y p r o d u c i n g S i n d b i s v i r u s do not heamadsorb goose red c e l l s under c o n d i t i o n s in w h i c h s i m i l a r l y i n f e c t e d BHK or CEK c e l l s r e a d i l y do so. Electron m i c r o s c o p y c o n f i r m e d t h a t n e a r l y a l l of t h e c e l l s in t h e s e e x p e r i m e n t s w e r e i n f e c t e d , s u g g e s t i n g t h a t t h e s u r f a c e of t h e i n f e c t e d mosq u i t o c e l l s does not c o n t a i n t h e v i r a l h e a m a d s o r b i n g p r o t e i n in d e t e c t a b l e q u a n t i ties.
45
D. T . B R O W N et al.
F ig. 14. A f r e e z e - e t c h r e p l i c a o f a v i r u s i n d u c e d c y t o p l a s m i c v a c u o l e 2 0 h o u r s a f t e r i n f e c t i o n w i t h Sindbis v i r u s . One o f t h e v e s i c u l a r membranes h a s a large number o f interior membrane beads ( s i n g l e arrow) w h i l e another i s smooth (double arrow) (compare t o F i g . 5 ) . M a g n i f i c a t i o n bar = 1 ;um.
F ig. 15. A s c h e m a t i c d i a g r a m m o f t h e p o s s i b l e s e q u e n c e o f e v e n t s o c c u r r i n g a f t e r i n f e c t i o n o f Aedes albopictus. Membranes are found w i t h i n t h e c y t o p l a s m ( A ) and are s u b s e q u e n t l y e n c l o s e d in a l i m i t i n g m e m b r a n e (B) f o r m i n g a v e s i c l e i n w h i c h r i b o s o m e s a n d a f e w n u c l e o c a p s i d s a t t a c h t o t h e m e m b r a n e s i n t h e v e s i c l e s (1) a n d t h e m e m b r a n e s u n d e r g o a t o p o l o g i c a l rearrangement r e s u l t i n g in t h e appearance o f v i r a l s p i k e s ( 2 ) . The a t t a c h e d n u c l e o c a p s i d s are e n v e l o p e d b y t h e m o d i f i e d m e m b r a n e (3) p r o d u c i n g m a t u r e v i r i o n s . T h e v i r u s - i n d u c e d v e s i c l e f u s e s w i t h t h e c e l l p l a s m a membrane (D) r e l e a s i n g m a t u r e v i r i o n s ( 4 ) , m e m b r a n e s w i t h attached nucleocapsids (5), and intermediates in envelopment.
A p r o p o s a l t h a t s e q u e s t e r i n g of v i r u s p r o d u c t i o n t o i n t e r n a l v e s i c l e s a c c o u n t s f o r the ability
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i m p o r t a n c e of t h e s e c e l l s
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46
occasional
a n d w h a t appear t o be v i r i o n s
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p r o c e s s of v i r u s p r o d u c t i o n .
MORPHOGENESIS OF S I N D B I S V I R U S IN C U L T U R E M O S Q U I T O C E L L S
Fig. 16. A r e a o f an Aedes albopictus c e l l a f t e r i n f e c t i o n f o r 20 h o u r s w i t h S i n d b i s v i r u s in t h e p r e s e n c e o f c o l c e m i d . N u m e r o u s n u c l e o c a p s i d s a r e s e e n i n t h e c e l l c y t o p l a s m a (a f e w are m a r k e d w i t h a r r o w s ) . M a g n i f i c a t i o n bar = 0 . 5 jum. I t is p o s s i b l e , as Raghow and c o w o r k e r s h a v e s u g g e s t e d ( Raghow et aL, 1 9 7 3 ) , t h a t most of t h e c e l l s s y n t h e s i z e c y t o p l a s m i c n u c l e o c a p s i d s and t h a t t h e y are i m m e d i a t e l y and e f f i c i e n t l y t r a n s p o r t e d out of t h e c y t o p l a s m by b u d d i n g t h r o u g h the p l a s m a membrane. T h i s e n t i r e p r o c e s s w o u l d have to o c c u r much more r a p i d l y than in t h e v e r t e b r a t e h o s t , in order to reduce a p p r e c i a b l y t h e p r o b a b i l i t y of o b s e r v i n g t h i s p r o c e s s in t h e e l e c t r o n m i c r o s c o p e . F o l l o w i n g t h i s l o g i c t h e f e w c e l l s in w h i c h t h i s p a t h w a y is m o r p h o l o g i c a l l y s u g g e s t e d m i g h t r e p r e s e n t a s i t u a t i o n in w h i c h t h i s p r o c e s s i s s l o w e d or a r r e s t e d f o r p h y s i o l o g i c a l r e a s o n s . On t h e other h a n d , to s u g g e s t t h a t t h e c o n s i d e r a b l e amount of v i r u s t h a t is a s s e m b l e d in v e s i c l e s , w h i c h are e a s i l y d e t e c t e d in t h e e l e c t r o n m i c r o s c o p e , d o e s not c o n t r i b u t e to t h e e x t r a c e l l u l a r v i r u s , is t o i m p l y t h a t a s y s t e m w h i c h i s so e f f i c i e n t on the one hand in m a t u r i n g c y t o p l a s m i c n u c l e o c a p s i d s by b u d d i n g t h r o u g h t h e p l a s m a membrane i s c o m p l e t e l y w a s t e f u l on a m a j o r p o r t i o n of t h e t o t a l v i r u s i t p r o d u c e s . One m u s t , of c o u r s e , c o n s i d e r m o d e l s w h i c h p r o p o s e t h a t both p r o c e s s e s of v i r u s p r o d u c t i o n do o c c u r and t h a t w h e t h e r one p a t h w a y of v i r u s p r o d u c t i o n or t h e o t h e r p r e v a i l s in a p a r t i c u l a r c e l l i s d e c i d e d by some u n k n o w n f a c t o r s . In t h i s regard w e h a v e f o u n d t h a t t r e a t m e n t of S i n d b i s v i r u s i n f e c t e d m o s q u i t o c e l l s w i t h c o l c e m i d r e s u l t s in t h e p r o d u c t i o n of many f r e e c y t o p l a s m i c n u c l e o c a p s i d s in m o s t of t h e c e l l s e x a m i n e d ( F i g . 1 6 ) . T h i s t r e a t m e n t , h o w e v e r , d i d not i n c r e a s e t h e f r e q u e n c y of v i r u s b u d d i n g from t h e c e l l s u r f a c e , but d i d r e s u l t in some r e d u c t i o n in t h e t o t a l virus yields. ill.
Conclusion
A n e l e c t r o n m i c r o s c o p e s t u d y of i n f e c t e d c e l l s by p u l s e c h a s e a u t o r a d i o g r a p h y w i t h p r o t e i n and RNA p r e c u r s o r s i s in p r o g r e s s . H o p e f u l l y , s u c h a program w i l l c l a r i f y t h e l o c a t i o n of p r o t e i n and RNA s y n t h e s i s in t h e i n f e c t e d c e l l s and f u r t h e r e s t a b l i s h t h e i n t r a c e l l u l a r f a t e of t h e s e p r o d u c t s a f t e r s y n t h e s i s . Acknowledgments T h i s r e s e a r c h w a s s u p p o r t e d by t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t IV.
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12, 1 5 3 4 .
SFB 7 4 .
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B r o w n , D.T., W a i t e , M.R.F. and P f e f f e r k o r n , E.R., ( 1 9 7 2 ) . J. B r o w n , D.T., a n d S m i t h , J . F . (1975) . J. ViroL B u c k l e y , S . M . ( 1 9 6 9 ) . Proc.
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C o r d e l l - S t e w a r t , B . , a n d T a y l o r , M.W. (1 971 ) . Proc.
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G a r w e s , D . J . , W r i g h t , P . J . a n d C o o p e r , P.D. ( 1 9 7 5 ) . J. Gen. J o n e s , K.M., W a i t e , M.R.F. a n d B o s e , H.R. ( 1 9 7 4 ) . J.
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M a r c h e s i , V . T . , J a c k s o n , R.L., S e g r e s t , J . P . a n d K a l t a n e , I. ( 1 9 7 2 ) . Federation
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P f e f f e r k o r n , E.R., a n d C l i f f o r d , R.L. ( 1 9 6 4 ) . Virology P f e f f e r k o r n , E.R., a n d S h a p i r o ,
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Raghow, R.S., G r a c e , T . D . C . , F i l s h i e , B„K., B a r t e l y , W. a n d D a l g a r n o , L. (1973) . J. Gen. 21, 1 0 9 . C M . , and P f e f f e r k o r n , E.R. (1970) . J.
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S c h l e s i n g e r , M . J . , a n d S c h l e s i n g e r , S. ( 1 9 7 3 ) . J . ViroL
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S c h l e s i n g e r , M . J . , S c h l e s i n g e r , S. a n d B ü r g e , B.W. ( 1 9 7 2 ) . Virology S c h l e s i n g e r , S., and S c h l e s i n g e r , M . J . (1972) . J. S e f t o n , B.M., and B ü r g e , B.W. (1974) . J. Virol.
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S i m m o n s , D . T . , and S t r a u s s , J . H . , J r . ( 1 9 7 2 ) . J. Mol. K.R.P. ( 1 9 6 7 ) . Current
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T i l l a c k , T . W . , S c o t t , R.E., and M a r c h e s i , V . E . ( 1 9 7 2 ) . J . Exptl. Utermann,
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S t o l l a r , V . , a n d S h e n k , T . E . ( 1 9 7 3 ) . . / . Virol. Stevens,
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Chapter 4
ESTABLISHED MOSQUITO CELL LINES AND THE STUDY OF TOGAVIRUSES
V . S t o l l a r , T. E. Shenk, R Koo, A . I g a r a s h i , and R. W. S c h l e s i n g e r
I.
Introduction
II.
The a c u t e i n f e c t i o n of A. albopictus
„
.
c e l l s w i t h Sindbis virus
1 . G r o w t h c u r v e of S i n d b i s v i r u s i n A. albopictus
49
.
50
cells
50
2. L a c k o f s i a l i c a c i d i n S i n d b i s v i r u s g r o w n in m o s q u i t o c e l l s I I I . A. albopictus
cells persistently infected with Sindbis virus
50 „
55
1. Generation of temperature-sensitive small plaque mutant virus
56
2. Resistance t o superinfection w i t h the homologous v i r u s IV.
A c e l l f u s i n g a g e n t p r e s e n t i n c u l t u r e s of Aedes
V.
Conclusions
VI.
References
I.
Introduction
aegypti
,
(Peleg)
57 60 64
ο «,
66
Those members of t h e t o g a v i r u s group w h i c h are t r a n s m i t t e d by i n s e c t s have t h e r e m a r k a b l e and uncommon a b i l i t y of b e i n g a b l e to m u l t i p l y in s p e c i e s as f a r apart i n t h e e v o l u t i o n a r y s c a l e as m o s q u i t o e s and m a n . The b i t e o f an i n f e c t e d m o s q u i t o and t h e s i m u l t a n e o u s t r a n s m i s s i o n of v i r u s can r e s u l t in s e r i o u s i l l n e s s and somet i m e s d e a t h , in man as w e l l as in o t h e r mammalian s p e c i e s . E x a m p l e s o f d i s e a s e s c a u s e d by t o g a v i r u s e s and t r a n s m i t t e d b y m o s q u i t o e s are Eastern and Western eq u i n e e n c e p h a l i t i s , among t h e a l p h a v i r u s subgroup a n d d e n g u e , y e l l o w f e v e r , and J a p a n e s e e n c e p h a l i t i s among t h e f l a v i v i r u s subgroup. D u r i n g t h e p a s t y e a r s much has been l e a r n e d about t h e s t r u c t u r e , t h e r e p l i c a t i o n , t h e m o l e c u l a r b i o l o g y , and t h e m o r p h o g e n e s i s of t h e t o g a v i r u s e s , e s p e c i a l l y a b o u t c e r t a i n of t h e a l p h a v i r u s e s . In t h e l a b o r a t o r y , S i n d b i s and S e m l i k i Forest v i r u s e s have p r o v e n v e r y useful model s y s t e m s and have t h e a d v a n t a g e of b e i n g r e l a t i v e l y a v i r u l e n t for man 0 In a r e c e n t r e v i e w , Pfefferkorn a n d Shapiro (1974) have c o l l e c t e d a n d s u m m a r i z e d w h a t is known of t h e s t r u c t u r e and r e p l i c a t i o n of t h e t o g a v i r u s e s . Most of t h e e x p e r i m e n t a l s y s t e m s , h o w e v e r , have e m p l o y e d v e r t e b r a t e c e l l h o s t s , e i t h e r w h o l e a n i m a l s s u c h as m i c e , or c u l t u r e d c e l l s d e r i v e d from o n e of s e v e r a l d i f f e r e n t s p e c i e s . R e l a t i v e l y s p e a k i n g , much l e s s i s known about t h e i n t e r a c t i o n between t o g a v i r u s e s and t h e m o s q u i t o h o s t . The e s t a b l ishement b y S i n g h , a n d b y and Aedes aegypti represented P e l e g of c e l l l i n e s d e r i v e d from Aedes albopictus a g r e a t step f o r w a r d , and one t h a t made it p o s s i b l e to u s e i n s e c t t i s s u e c u l t u r e s y s t e m s f o r the s t u d y of t h e r e p l i c a t i o n of t o g a v i r u s e s . The a v a i l a b i l i t y o f t h e s e mosquito c e l l l i n e s w i l l a l s o u n d o u b t e d l y p r o v e useful f o r the study and i d e n t i f i c a t i o n of new v i r a l a g e n t s , some of w h i c h w i l l l i k e l y p r o ve p a t h o g e n i c f o r man.
49
V . S T O L L A R et al.
Results and d i s c u s s i o n The s y s t e m w e h a v e c h o s e n t o s t u d y most i n t e n s i v e l y i s t h a t o f S i n d b i s v i r u s r e p l i c a t i o n in t h e Aedes albopictus c e l l l i n e o f Singh ( 1 9 6 7 ) . Some r e f e r e n c e w i l l a l s o b e made to e x p e r i m e n t s w i t h t h e Aedes aegypti c e l l l i n e o f Peleg ( 1 9 6 8 ) . Our S i n d b i s v i r u s s t o c k s , o r i g i n a l l y d e r i v e d f r o m t h e HR s t r a i n of Bürge and Pfeff e r k o r n ( 1 9 6 6 ) , have been p l a q u e - p u r i f i e d and grown i n c h i c k c e l l s a t a l o w i n i t i a l m u l t i p l i c i t y in order to e x c l u d e t h e p r o d u c t i o n of d e f e c t i v e v i r a l p a r t i c l e s . c e l l l i n e (Singh) d e r i v e d from m o s q u i t o l a r v a e w a s m a i n t a i n The Aedes albopictus e d o r i g i n a l l y in t h e medium d e v i s e d by M i t s u h a s h i and Maramorosch (1964) (MM med i u m ) but more r e c e n t l y has been a d a p t e d to a medium c o m p o s e d of 9 p a r t s E a g l e ' s m i n i m a l medium ( E a g l e , 1959) ( i n c l u d i n g n o n - e s s e n t i a l amino a c i d s ) and 1 p a r t MM m e d i u m . In each c a s e f e t a l c a l f serum w a s added to a f i n a l c o n c e n t r a t i o n o f 5 t o 10%. II.
The a c u t e i n f e c t i o n of A . a l b o p i c t u s c e l l s w i t h S i n d b i s v i r u s .
/.
Growth curve of Sindbis
virus
in Aedes
albopictus
cells.
It has g e n e r a l l y been o b s e r v e d t h a t in t h e f a c e o f h i g h v i r u s y i e l d s , a n d v i r a l a n t i g e n in t h e m a j o r i t y of the c e l l s , i n f e c t e d m o s q u i t o c e l l c u l t u r e s i n f e c t e d w i t h a l p h a v i r u s e s c o n t i n u e to g r o w , and do not show any o b v i o u s c y t o p a t h i c e f f e c t (CPE), ( S t e v e n s , 1970). Figure 1 d e m o n s t r a t e s a g r o w t h c u r v e o f S i n d b i s v i r u s in A. albopictus cells m a i n t a i n e d a t 2 8 ° C . The l a t e n t p e r i o d , t h e k i n e t i c s o f r e p l i c a t i o n , and t h e f i n a l y i e l d do not d i f f e r s u b s t a n t i a l l y f r o m w h a t w e o b s e r v e w h e n v i r u s is g r o w n in c h i c k embryo f i b r o b l a s t s (CEF) or in hamster c e l l s . N o c y t o p a t h i c e f f e c t w a s v i s i b l e , in k e e p i n g w i t h p r e v i o u s o b s e r v a t i o n s made on m o s q u i t o c e l l s i n f e c t e d w i t h S i n d b i s v i r u s or other a l p h a v i r u s e s ( D a v e y et aL, 1973). Other e x p e r i m e n t s in our l a b o r a t o r y h a v e shown 1) t h a t t h e i n f e c t e d m o s q u i t o c e l l s c o n t i n u e to grow at a rate comparab l e to t h a t o f t h e u n i n f e c t e d c e l l s , and 2) t h a t by 24 hours a f t e r i n f e c t i o n at l e a s t 75-80% of t h e c e l l s c o n t a i n v i r a l a n t i g e n as d e m o n s t r a t e d by t h e f l u o r e s c e n t a n t i body method, Since the i n f e c t e d mosquito c e l l s are not k i l l e d but c o n t i n u e to g r o w , a c h r o n i c a l l y i n f e c t e d c u l t u r e r e s u l t s , w i t h c o n t i n u o u s v i r u s p r o d u c t i o n . Such c h r o n i c a l l y i n f e c t e d c u l t u r e s have been m a i n t a i n e d in our l a b o r a t o r y f o r up to 2 y e a r s . Some propert i e s o f t h e s e c u l t u r e s and of t h e v i r u s p r o d u c e d by them w i l l be d e s c r i b e d in a later section* 2.
Lack of sialic
acid in Sindbis
virus grown in mosquito
cells.
Toga v i r u s e s are composed of R N A , p r o t e i n , c a r b o h y d r a t e in t h e f o r m of sugar r e s i d u e s on t h e e n v e l o p e g l y c o p r o t e i n s , and l i p i d . The v i r a l RNA and p r o t e i n are both s p e c i f i e d by t h e v i r a l genome. In c o n t r a s t , t h e e v i d e n c e is good t h a t both t h e l i p i d and c a r b o h y d r a t e components of t h e v i r a l membrane are d e t e r m i n e d l a r g e l y by t h e h o s t c e l l . To e x p a n d , p r e c i s e l y w h i c h sugar r e s i d u e s are added t o t h e v i r a l g l y c o p r o t e i n s is p r o b a b l y a f u n c t i o n both o f t h e nature and t h e a c t i v i t y o f t h e c e l l u l a r g l y c o s y l t r a n s f e r a s e e n z y m e s . It m i g h t be e x p e c t e d t h a t c o m p a r a t i v e s t u d i e s o f S i n d b i s v i r u s grown in m o s q u i t o and v e r t e b r a t e c e l l s w o u l d show t h e e f f e c t o f s u c h h o s t m o d i f i c a t i o n s much more d r a m a t i c a l l y than if one compared S i n d b i s v i r u s g r o w n in 2 d i f f e r e n t v e r t e b r a t e s p e c i e s . 50
E S T A B L I S H E D M O S Q U I T O C E L L LINES A N D T H E S T U D Y OF T O G A V I R U S 9
io F
| 05 |
"
1
,
,
,
.
.
5
10
15
20
25
1
TIME (hours) Fig. 1. G r o w t h o f S i n d b i s v i r u s i n Aedes w a s a p p r o x i m a t e l y 30 p f u / c e l l .
albopictus
c e l l s at 2 8 ° . The input m u l t i p l i c i t y
Warren (1963) and S c h l e s i n g e r et al* (1961) have s h o w n t h a t in c o n t r a s t t o v e r t e b r a t e c e l l s , n e a r l y a l l i n v e r t e b r a t e c e l l s a n d t i s s u e s e x a m i n e d l a c k s i a l i c a c i d . If t h e a d d i t i o n o f s i a l i c a c i d t o v i r a l g l y c o p r o t e i n is a " h o s t f u n c t i o n " it seemed l i k e l y t h a t S i n d b i s v i r u s grown i n m o s q u i t o c e l l s w o u l d a l s o l a c k s i a l i c a c i d and t h u s d i f f e r r e m a r k a b l y from v i r u s g r o w n in BHK21 or c h i c k c e l l s . Before e x a m i n i n g v i r u s , w e w i s h e d to be c e r t a i n t h a t t h e c u l t u r e d m o s q u i t o c e l l s i n d e e d d i d l a c k s i a l i c a c i d . In t h e f i r s t e x p e r i m e n t , A. albopictus c e l l s , and hamst e r c e l l s (BHK-21) w e r e grown in t h e p r e s e n c e of r a d i o a c t i v e g l u c o s a m i n e (a g o o d p r e c u r s o r of s i a l i c a c i d in v e r t e b r a t e c e l l s ) (Kraemer, 1967). The c e l l s w e r e h a r v e s t e d , w a s h e d , and t h e n i n c u b a t e d w i t h n e u r a m i n i d a s e . T h e r e l e a s e o f T C A - s o l u b l e c o u n t s (above t h e c o n t r o l v a l u e ) w a s t a k e n as e v i d e n c e f o r t h e p r e s e n c e o f s i a l i c a c i d . In t h e c a s e of t h e hamster c e l l s , s u b s t a n t i a l l y more T C A s o l u b l e c o u n t s w e r e r e l e a s e d upon i n c u b a t i o n w i t h n e u r a m i n i d a s e t h a n w e r e r e l e a s e d in buffer o n l y ; w h e n mosquito c e l l s were t r e a t e d under s i m i l a r c o n d i t i o n s , t h e r e w a s no d i f f e r e n c e i n t h e TCA s o l u b l e r a d i o a c t i v i t y r e l e a s e d from c o n t r o l c e l l s and from c e l l s i n c u b a t e d
51
V 0 S T O L L A R et al.
Fig. 2. S u c r o s e - D 20 e q u i l i b r i u m g r a d i e n t s o f S i n d b i s v i r u s f r o m BHK c e l l s and A. albopictus cells: BHK c e l l s w e r e i n f e c t e d a t an i n p u t m u l t i p l i c i t y o f a p p r o x i m a t e l y 5 0 p f u / c e l l a n d m a i n t a i n e d a t 3 7 ° . C u l t u r e m e d i u m w a s h a r v e s t e d a f t e r 22 hourSc A. albopictus c e l l s were a l s o i n f e c t e d w i t h a n i n p u t m u l t i p l i c i t y of 5 0 p f u / c e l l , b u t i n t h i s case t h e medium w a s changed several t i m e s so a s to c o l l e c t t h e v i r u s y i e l d s between 1 and 1 8 h o u r s , 2 2 and 40 h o u r s , and 5 4 and 7 2 h o u r s . A l l v i r u s s a m p l e s were e x t r a c t e d once w i t h G e n e t r o n ( t r i c h l o r o t r i f l u o r o e t h a n e ) a n d c l a r i f i e d by low speed centrifugation. A s a m p l e ( 0 . 8 m l ) of each p r e p a r a t i o n w a s t h e n l a y e r e d over 11.2 m l 14-40% s u c r o s e ( w / w ) i n TNE b u f f e r made up in ϋ 2 0 (Shenk a n d S t o l l a r , 1 9 7 3 ) . C e n t r i f u g a t i o n w a s in t h e SW-41 r o t o r a t 1 0 ° f o r 15 h o u r s a t 3 1 , 0 0 0 r p m . F r a c t i o n s w e r e then c o l l e c t e d (0.6 m l ) a n d a s s a y e d for p l a q u e f o r m a t i o n on BHK-21 c e l l m o n o l a y e r s . w i t h n e u r a m i n i d a s e (Table 1 ) . A l t h o u g h t h e s e r e s u l t s were c o n s i s t e n t w i t h t h e a b s e n c e of s i a l i c a c i d in m o s q u i t o c e l l s , f o r c o n f i r m a t i o n w e t u r n e d to a more direct chemical assay. BHK-21 and A. albopictus c e l l s were h a r v e s t e d , w a s h e d three t i m e s w i t h PBS, and t h e n a s s a y e d for s i a l i c a c i d u s i n g t h e t h i o b a r b i t u r i c a c i d method of Warren ( 1 9 5 9 ) . BHK c e l l s c o n t a i n e d 0.37 j j m / m l p a c k e d c e l l s (Table 2 ) , a v a l u e s i m i l a r t o t h a t o b t a i n e d by others w i t h t h e s e c e l l s . T u r n i n g to t h e A. albopictus c e l l s , we found t h a t even a f t e r t h r e e w a s h i n g s w i t h PBS, s i a l i c a c i d w a s u s u a l l y p r e s e n t w h e n t h e c e l l s were grown in MM medium c o n t a i n i n g 10% f e t a l c a l f s e r u m . Since t h e serum c o m p o n e n t of t h e medium c o n t a i n s high l e v e l s of s i a l i c a c i d , w e t h o u g h t i t l i k e l y t h a t t h e " c e l l u l a r " s i a l i c a c i d w e were measuring w a s d e r i v e d from t h e serum a n d w a s perhaps t i g h t l y bound to t h e c e l l s . C o n s i s t e n t w i t h t h i s idea w a s t h e o b s e r v a -
52
E S T A B L I S H E D M O S Q U I T O C E L L LINES A N D T H E S T U D Y OF T O G A V I R U S TABLE 1 Release
of acid soluble
Enzyme
used
radioactivity
from glucosamine
labeled
cells
TCA s o l u b l e cpm released from BHK cells
A. albopictus
CI. perfringens
neuraminidase
63,696 60,384
159,077 157,378
Vibrio
neuraminidase
48,494 52,502
160,994 152,539
22,036 22,903
163,922 156,509
cholerae
Buffer o n l y
cells
4
BHK c e l l s a n d A. albopictus c e l l s w e r e l a b e l e d w i t h ' C - 1 - g l u c o s a m i n e m e d i u m f o r 48 1 4 h o u r s . The C - 1 - g l u c o s a m i n e ( 5 6 . 5 m c / m M ) w a s a d d e d t o a f i n a l c o n c e n t r a t i o n o f 1 / j c / m l . C e l l s w e r e s c r a p e d from p e t r i p l a t e s a n d w a s h e d 5 t i m e s w i t h c o l d p h o s p h a t e b u f f e r e d s a l i n e , u n t i l t h e c o u n t s in t h e s u p e r n a t a n t d i d n o t d e c r e a s e f u r t h e r . C e l l s w e r e then s u s p e n d e d in 8 ml of b u f f e r A ( 0 . 5 M s o d i u m a c e t a t e p H 5.5 . 1 5 M N a C I , . 0 0 9 M C a C I 2) a n d d u p l i c a t e 1 m l s a m p l e s w e r e i n c u b a t e d w i t h CI. perfringens neuraminidase (Worthington Biochemical Corp.) (0.1 m g ) , V. pholerae n e u r a m i n i d a s e ( B e h r i n g D i a g n o s t i c s ) ( 0 . 1 ml s t o c k s o l u t i o n ) , or b u f f e r a l o n e . A s m e a s u r e d b y T C A p r e c i p i t a b l e c p m , t h e A. albopictus cell suspension contained a p p r o x i m a t e l y 4 t i m e s a s m u c h r a d i o a c t i v i t y a s t h e s y s p e n s i o n o f BHK21 c e l l s . A f t e r 30 m i n u t e s a t 3 7 ° t h e c e l l s w e r e p e l l e t e d , a n d T C A added t o t h e s u p e r n a t a n t ( f i n a l c o n c e n t r a t i o n 5%). The T C A t r e a t e d s u p e r n a t a n t w a s a p p l i e d t o a g l a s s f i b e r f i l t e r a n d t h e f i l t r a t e c o l l e c t e d . O n e m l o f f i l t r a t e w a s added t o 10 m l o f c o u n t i n g l i q u i d (1 v o l . Triton-X and 2 v o l . P e r m a f l u o r - P a c k a r d ) a n d c o u n t e d in a s c i n t i l l a t i o n c o u n t e r .
t i o n t h a t if the serum c o n c e n t r a t i o n i n t h e medium w a s r e d u c e d , t h e l e v e l of t h e ' c e l l u l a r " s i a l i c a c i d a l s o f e l l . T h i s w a s not so i n t h e c a s e of t h e BHK-21 c e l l s . T h e s i t u a t i o n w a s f u r t h e r c l a r i f i e d a f t e r it w a s f o u n d t h a t t h e A. albopictus cells c o u l d be grown in MM medium w i t h o u t any s e r u m . Under s u c h c o n d i t i o n s , t h e r a t e o f growth w a s s l o w e r , and there w a s a marked t e n d e n c y of t h e c e l l s t o a g g r e g a t e ; n e v e r t h e l e s s , t h e c e l l s d i d c o n t i n u e to grow for s e v e r a l w e e k s w h i c h w a s as long a s we a t t e m p t e d to m a i n t a i n t h e m . A n a l y s i s of t h e s e c e l l s s h o w e d no d e t e c t a b l e sialic acid. From t h e s e e x p e r i m e n t s , a n d t h o s e in w h i c h t h e c e l l s were l a b e l e d w i t h r a d i o a c t i v e g l u c o s a m i n e , w e c o n c l u d e t h a t t h e c u l t u r e d A. albopictus c e l l s like nearly all i n v e r t e b r a t e c e l l s do not c o n t a i n s i a l i c a c i d . If c e l l s do not c o n t a i n s i a l i c a c i d , it seemed p r o b a b l y t h a t t h e y w o u l d a l s o l a c k s i a l y l t r a n s f e r a s e , t h e enzyme w h i c h a t t a c h e s s i a l i c a c i d m o i e t i e s c o v a l e n t l y o n t o g l y c o p r o t e i n . T h i s w a s indeed t h e c a s e . In c o n t r a s t t o c h i c k c e l l s no s i a l y l t r a n s f e r a s e a c t i v i t y w a s f o u n d in c u l t u r e d A. albopictus c e l l s . T h i s was so w h e t h e r t h e enzyme i n c u b a t i o n was c a r r i e d o u t at 2 8 ° C or at 3 7 ° C (data not s h o w n ) . We next proceeded to see w h e t h e r S i n d b i s v i r u s grown in m o s q u i t o c e l l s c o n t a i n e d s i a l i c a c i d . T h i s was done by g r o w i n g v i r u s in t h e p r e s e n c e o f 1 4 o i a b e l e d g l u c o s a m i n e and t e s t i n g f o r t h e r e l e a s e by n e u r a m i n i d a s e of T C A - s o l u b l e r a d i o a c t i v i t y 0 In the c a s e of v i r u s grown in c h i c k or hamster c e l l s between 9% and 1 1 % o f t h e i n p u t TCA p r e c i p i t a b l e r a d i o a c t i v i t y w a s r e l e a s e d in T C A = s o l u b l e form ( T a b l e 3 ) ,
53
V. S T O L L A R et al. TABLE 2 Sialic
acid content
of media and
cells μ Μ / m l medium or p a c k e d c e l l s
Materials assayed Fetal c a l f s e r u m not d i a l y s e d dialysed
5.5 4.8
4% b o v i n e s e r u m a l b u m i n
.01
MM m e d i u m w i t h 5% f e t a l c a l f s e r u m
.32 .37 < .01
MM m e d i u m w i t h o u t s e r u m BHK c e l l s g r o w n w i t h 10% s e r u m overnight w i t h 0 . 1 % serum o v e r n i g h t w i t h 0.2% BSA
0.37 0.37 0.32
A. albopictus c e l l s grown w i t h 5% FCS o v e r n i g h t w i t h 0.2% FCS
0.17 0.09
A. albopictus c e l l s grown w i t h o u t serum in s u s p e n s i o n c u l t u r e s as m o n o l a y e r s
< .01 < .01
C e l l s t o be a s s a y e d f o r s i a l i c a c i d w e r e h a r v e s t e d by s c r a p i n g , w a s h e d 3 t o 5 t i m e s i n PBS and f i n a l l y p e l l e t e d at 1 2 0 0 rpm f o r 5 m i n u t e s .
In sharp c o n t r a s t , no s i g n i f i c a n t c o u n t s were r e l e a s e d f r o m S i n d b i s v i r u s g r o w n in m o s q u i t o c e l l s . T h u s , g l u c o s a m i n e d i d not l a b e l any m o i e t i e s in t h e m o s q u i t o grown v i r u s w h i c h w e r e h y d r o l y z a b l e by e i t h e r of t h e 2 n e u r a m i n i d a s e p r o p o r t i o n s t e s t e d . T h e s e r e s u l t s t a k e n t o g e t h e r are s t r o n g e v i d e n c e t h a t t h e p r e s e n c e or a b s e n c e o f s i a l i c a c i d in t h e S i n d b i s v i r i o n , and b y a n a l o g y t h a t of o t h e r sugars as w e l l , is d i c t a t e d by t h e c a p a b i l i t i e s of t h e host c e l l . 3.
Heterogeneity
of Sindbis
virus produced
in A. albopictus
cells.
What of the p h y s i c a l p r o p e r t i e s of t h e v i r u s p r o d u c e d in m o s q u i t o c e l l s ? P r e v i o u s e x p e r i m e n t s in our laboratory h a v e s h o w n t h a t t h e progeny v i r u s from h a m s t e r or CEF i n f e c t e d w i t h our w i l d t y p e v i r u s banded s h a r p l y w i t h a d e n s i t y c l o s e to 1.20 g / c c in s u c r o s e - D 2 0 e q u i l i b r i u m g r a d i e n t s . Such v i r u s c o u l d be m o n i t o r e d by r a d i o a c t i v e label (e,g„ 3 H - u r i d i n e ) h e m a g g l u t i n a t i n g a c t i v i t y o r i n f e c t i v i t y . When S i n d b i s v i r u s grown in m o s q u i t o c e l l s w a s e x a m i n e d in a s i m i l a r w a y , a rather d i f f e r e n t p i c ture w a s s e e n . Figure 2 s h o w s s u c r o s e ^ O g r a d i e n t s o f S i n d b i s v i r u s , in w h i c h i n f e c t i v i t y w a s used to i d e n t i f y v i r u s . A s noted a b o v e , v i r u s f r o m c h i c k c e l l s gave a sharp homogeneous p e a k . V i r u s t a k e n f r o m m o s q u i t o c e l l s s h o w e d i n c r e a s i n g h e t e r o g e n e i t y w i t h t i m e after i n f e c t i o n . A t 18 h o u r s , t h e main i n f e c t i v e p e a k w a s a t 1.20 g / c c , w i t h l e s s e r p e a k s of both greater and l e s s e r d e n s i t y . Much t h e same p i c t u r e w a s seen at 40 h o u r s . By 72 h o u r s , t h e predominant p e a k w a s a t 1.17 g / c c w i t h o n l y s m a l l peaks a t d e n s i t i e s of 1.20 g / c c and 1.22 g / c c . O b s e r v a t i o n s b y Brown and G l i e d m a n (1973) d e s c r i b i n g s i z e h e t e r o g e n e i t y o f v i r u s p a r t i c l e s from A. albopictus c e l l s i n f e c t e d w i t h S i n d b i s v i r u s may b e r e l a t e d
54
ESTABLISHED MOSQUITO C E L L LINES A N D T H E STUDY OF T O G A V I R U S
TABLE 3 Release
of acid soluble
radioactivity
O r i g i n of V i r u s
Neuraminidase
from glucosamine
BHK C e l l s a
used
TCA precipitable
labeled
Sindbis
C h i c k Embryo Fibroblasts
virus
A. albopictus Cells
c< >
P( b )
C
Ρ
C
3014
3147
3074
3354
5623
Ρ 6212
cpm added
Δ
307 50
422 49
312 42
378 47
00 LO CO CO
A c i d s o l u b l e + Enz. CPM r e l e a s e d —Enz.
86 31
257
373
270
331
3
55
8.3
11.9
% of i n p u t c p m r e l e a s e d
SV-C (PFU/ml)
by Sindbis (a)
4
A. albo.
(CFA)
A . a l b o . ( C F A ) 24.
5.8
141.6
Hanks Buffered Saline S o l u t i o n for V e r t e b r a t e s eel Is
G. E C H A L I E R TABLE III D22 Medium f o r Drosophila
melanogaster
embryonic c e l l s mM
For 1 l i t e r
gm
P o t a s s i u m G l u t a m a t e (I H 2 O )
24.5
Sodium G l u t a m a t e (I H 20 )
42.7
Glycine
67
5.00
4.47
0.9
MgCI2
. 6 H 20
M g S 0 4 . 7 H 20
13.6
N a H 2 P 0 4 . 2 H 20
2.74
C a C I 2 (to be d i s s o l v e d a p a r t )
7.3
Sodium A c e t a t e , 3 H 2 0
0.17
4.975 7.98
3.36 0.43 0.8 0.023
Succinic Acid
0.055
Malic Acid
0.6
Glucose
1.8
Lactalbumin Hydrolysate (Difco) Difco Yeastolate
13.6 1.36
V i t a m i n s Β (see T. G r a c e 1962) Streptomycin Penicillin
0.1 250,000 I.U.
A d u s t t o pH 6 . 6 - 6 . 7 w i t h Ν KOH
L i k e w i s e , the v a r i o u s c a t i o n i c c o n c e n t r a t i o n s are c o n s i d e r e d , e s p e c i a l l y the char a c t e r i s t i c N a / K r a t i o ( 1 : 4 ) . Because it is w e l l known t h a t C I is not the d o m i n a n t a n i o n in the body f l u i d of many i n s e c t s , s u c h as Drosophila, and t h a t it is o f t e n r e p l a c e d by organic a n i o n s , the b a s i c s a l i n e s o l u t i o n of our medium D 2 2 is e s s e n t i a l l y a m i x t u r e of s o d i u m and p o t a s s i u m g l u t a m a t e s and g l y c i n a t e s - a c c o r d i n g t o a s u g g e s t i o n by Shaw ( 1 9 5 6 ) . A h i g h c o n t e n t of f r e e a m i n o - a c i d s is s u p p l i e d by l a c t a l b u m i n h y d r o l y s a t e and a l l u n k n o w n n u t r i t i o n a l r e q u i r e m e n t s are c o v e r e d by a d d i t i o n of y e a s t e x t r a c t , some v i t a m i n s Β and organic a c i d s . G l u c o s e is p r e s e n t in t h e c o n c e n t r a t i o n of 1.8 gm per liter. B e s i d e s , most of the media have to be s u p p l e m e n t e d w i t h 10 t o 20% of d e c o m p l e mented f e t a l c a l f s e r u m . In t h e c a s e of long e s t a b l i s h e d l i n e s , t h i s serum c o n t e n t may be lowered t o 5% or, s o m e t i m e s , 2 % . We have e v e n s e l e c t e d a s u b l i n e f r o m our l i n e K, t h a t g r o w s in medium D 2 2 w i t h o u t serum or a n y other p r o t e i n s u p p l e m e n t a t i o n . It is v e r y e c o n o m i c a l w h e n large amounts of c e l l s are needed f o r b i o c h e m i c a l i n v e s t i g a t i o n s (see b e l o w ) . U n f o r t u n a t e l y , t h e k a r y o t y p e of t h i s s e r u m - i n d e p e n d e n t s t r a i n w a s found t o be rather u n s t a b l e . N o c o n n e c t i o n b e t w e e n t h i s a n e u p l o i d y and the a b s e n c e of serum w a s e s t a b l i s h e d . N e v e r t h e l e s s , medium D22 c a n n o t be c o n s i d e r e d as a c h e m i c a l l y d e f i n e d b e c a u s e of its l a c t a l b u m i n and y e a s t e x t r a c t c o n t e n t s . V a r i o u s s i m p l i f i e d have been s u c c e s s f u l l y used for short l a b e l l i n g e x p e r i m e n t s , b u t nobody f a r , s u c c e e d e d to grow Drosophila c e l l s in a t r u l y " ' s y n t h e t i c " medium f o r f i c a n t l y long p e r i o d .
136
medium, versions h a s , so a signi-
L I N E S OF DROSOPHILA
CELLS
To c o n c l u d e , it must be e m p h a s i z e d t h a t the e x t r e m e d i v e r s i t y of the f o r m u l a e w h i c h are proposed f o r the c u l t u r e of Drosophila t i s s u e s of c e l l s , p r o v e s in i t s e l f t h a t there is not y e t a v a i l a b l e a p e r f e c t , p o l y v a l e n t m e d i u m , w h i c h might be used f o r any t i s s u e of Drosophila and that w o u l d promote c e l l m u l t i p l i c a t i o n as w e l l as in vitro d i f f e r e n t i a t i o n . It is q u i t e l i k e l y t h a t s u c h a " u n i v e r s a l " medium s i m p l y does not e x i s t , because every t y p e o f s p e c i a l i z e d c e l l must h a v e its s p e c i f i c n u t r i t i o n a l and e n v i r o n m e n t a l r e q u i r e m e n t s . 3 . Cloning
method
It is n e c e s s a r y t o c l o n e f r e q u e n t l y c e l l s for g e n e t i c i n v e s t i g a t i o n s , t o a s s u r e homogenous c e l l p o p u l a t i o n s . C l o n i n g of i n s e c t c e l l s has been s u c c e s s f u l l y c a r r i e d o u t in several l a b o r a t o r i e s in r e c e n t years ( M c i n t o s h & R e c h t o r i s , 1 9 7 4 ; M c i n t o s h , 1975; N a k a j i m a & M i y a k e , 1 9 7 5 ) , but t h e c l o n i n g of Drosophila c e l l s remains d i f f i c u l t . The s i m p l e d i l u t i o n t e c h n i q u e , s o e x t e n s i v e l y used f o r mammalian c e l l s d o e s not w o r k h e r e . Some years a g o , n e v e r t h e l e s s , w e s u c c e e d e d in i s o l a t i n g a f e w clones using the d i l u t i o n method, provided that dispersed c e l l s were covered w i t h an agar s o l i d i f i e d medium ( E c h a l i e r , 1971). The p l a t i n g e f f i c i e n c y w a s so l o w t h a t t h i s method c o u l d not be used a s a standard t e c h n i q u e . In c l o s e c o n n e c t i o n w i t h A . O h a n e s s i a n ' g r o u p , w e have r e c e n t l y d e v i s e d a c l o n i n g method w h i c h is an a d a p t a t i o n of t h e " f e e d e r - l a y e r " t e c h n i q u e c o n t r i v e d b y Puck f o r mammalian c e l l s . S i n g l e c e l l s are h e l p e d to grow and m u l t i p l y into s m a l l c o l o n i e s , w h e n t h e y are d i s p e r s e d among large p o p u l a t i o n s of " f e e d e r " c e l l s , t h a t is c e l l s w h o s e c a p a c i t y to d i v i d e has been d e s t r o y e d w i t h a s u f f i c i e n t d o s e of X - r a y s , b u t w h i c h keep on m e t a b o l i z i n g and " c o n d i t i o n i n g " t h e c u l t u r e medium. The q u a n t i t a t i v e a s p e c t s of t h e t e c h n i q u e w i l l be d e s c r i b e d by O h a n e s s i a n a n d R i c h a r d - M o l a r d in 1975 ( p e r s o n a l c o m m u n i c a t i o n ) . The s a f e d o s e s of i r r a d i a t i o n for Drosophila c e l l s vary w i t h d i f f e r e n t s t r a i n s , but were found t o be much h i g h e r t h a n f o r mammalian c e l l s . Our l i n e Κ can w i t h s t a n d a t l e a s t 25,000 r a d s , w i t h gamma r a y s . In p r a c t i c e , t h e c e l l s to be c l o n e d are d i l u t e d in s t a n d a r d m e d i u m , a n d d i s t r i b u t e d i n t o t h e small w e l l s of a Falcon p l a s t i c d i l u t i o n p l a t e (on an a v e r a g e , one c e l l per w e l l ) . T h e n , some 5 0 , 0 0 0 i r r a d i a t e d c e l l s are added t o each w e l l . A f t e r 4 - 6 w e e k s c o l o n i e s reach a s u f f i c i e n t s i z e t o be s u b c u l t u r e d . To a s c e r t a i n t h e i r c l o n a l nature the p r o c e s s has t o be r e p e a t e d s u c c e s s i v e l y t w o or t h r e e t i m e s .
I I I . Experimental p o s s i b i l i t i e s o f f e r e d by Drosophila
cell cultures
Four main a p p r o a c h e s w i l l be d e s c r i b e d , f o r the s i m p l i c i t y o f p r e s e n t a t i o n . But it s h o u l d be c l e a r l y understood t h a t they a l l are i n t e r r e l a t e d and r e p r e s e n t v a r i o u s f a c e t s of t h e same endeavour: t o t r y t o u n d e r s t a n d s o m e t i n g a b o u t t h e s t r u c t u r e and t h e f u n c t i o n a l m o d u l a t i o n of the g e n e t i c m a t e r i a l of e u k a r y o t i c c e l l s of h i g h e r organisms. /- Basic biochemical
investigations
To grow in vitro large amounts o f Drosophila c e l l s is an easy w a y t o overcome t h e l i m i t i n g f a c t o r w h i c h i s , for b i o c h e m i c a l w o r k , t h e s m a l l s i z e of t h e Drosophila organism. In order t o i l l u s t r a t e i t , w e w i l l b r i e f l y m e n t i o n s e v e r a l t y p e s of research c a r r i e d out in v a r i o u s l a b o r a t o r i e s w i t h mass c u l t u r e s of Drosophila cells:
137
G. E C H A L I E R
- Thomas and c o l l a b o r a t o r s , in B o s t o n , are s t u d y i n g t h e D N A of Drosophila melanogaster, by a n a l y s i n g t h e fragments o b t a i n e d from d i g e s t i o n of t h i s D N A by s e v e ral r e s t r i c t i o n e n z y m e s ( M a n t u e i l , personal c o m m u n i c a t i o n ) . By u s i n g v a r i o u s c e l l l i n e s w i t h d e f i n i t e and s t a b l e k a r y o t y p e s , t h e y hope to i n d e n t i f y f r o m w h i c h chromosome come some s p e c i f i c f r a g m e n t s and perhaps to a s s i g n t o t h e m some p h y s i o l o g i c a l f u n c t i o n s . G a r e n ' s g r o u p , in New H a v e n , has been p r e p a r i n g Drosophila RNA p o l y m e r a s e . They are m a i n l y i n t e r e s t e d in t h e p r o t e i n s t h a t b i n d t o D N A , e s p e c i a l l y c e r t a i n minor s p e c i e s t h a t r e q u i r e large amounts of c e l l m a t e r i a l f o r t h e i r i s o l a t i o n ( p e r s o n a l c o m m u n i c a t i o n ) . They w i s h t o compare t h i s c l a s s of p r o t e i n s in t h e c u l t u r e c e l l s w i t h t h e c o r r e s p o n d i n g p r o t e i n s of e m b r y o n i c s t a g e s (where t h e y a r e s u s p e c t e d t o p l a y a d e c i s i v e r o l e in t h e m e c h a n i s m s of c e l l d e t e r m i n a t i o n ) . It w a s t h o u g h t t h a t it m i g h t be r e l a t i v e l y e a s y , on a c c o u n t of t h e s i m p l e chromosomal set of Drosophila, to i s o l a t e , on a large s c a l e , / c metaphase chromosomes from c e l l c u l t u r e s and e v e n t u a l l y , t o s e p a r a t e e v e r y chromosome p a i r . T h i s e x c i t i n g p r o j e c t seems t o encounter t e c h n i c a l d i f f i c u l t i e s , a p p a r e n t l y b e c a u s e of an unusual s t i c k i n e s s of the m i t o t i c /a s p i n d l e . H o w e v e r , a p r e p a r a t i v e method w a s d e s c r i b e d by Hanson and H e a r s t ( 1 9 7 3 ) . J o r d a n , in M a r s e i l l e , is s t u d y i n g t h e s t r u c t u r e and m a t u r a t i o n of r i b o s o m a l RNAs from Drosophila c u l t u r e d c e l l s . H e is e s t a b l i s h i n g t h e n u c l e o t i d e sequence of 5S RNA t o compare i t to t h o s e of other o r g a n i s m s . B e s i d e s , he has d i s c o v e r e d a new s p e c i e s of r i b o s o m a l RNA, " 2 8 " , w h i c h seems to be not c o - v a l e n t l y l i n k e d to 26S RNA ( J o r d a n , 1974). In T o u l o u s e , Z a l t a has d e v i s e d a t e c h n i q u e f o r p r e p a r i n g a very pure n u c l e o l a r f r a c t i o n from Drosophila c e l l s (personal communication). The above I i s t c o m p r i s e s o n l y a f e w s i g n i f i c a n t e x a m p l e s and demands are now r a p i d l y i n c r e a s i n g f o r c e l l l i n e s e s p e c i a l l y f o r our s t r a i n t h a t r e q u i r e s no s e r u m . 2 - Genetics
of somatic
cells
Several t e c h n i c a l o b s t a c l e s must be o v e r c o m e before Drosophila c e l l s c a n become a s t a n d a r d t o o l of g e n e t i c i s t s . The aim i s not s i m p l y to r e p e a t , w i t h i n s e c t c e l l s , w h a t has a l r e a d y been a c h i e v e d w i t h mammalian c e l l s . There w o u l d be no p o i n t in u s i n g s o m a t i c h y b r i d i z a t i o n f o r m a p p i n g genes in Drosophila, a s i t i s c u r r e n t l y and s u c c e s s f u l l y done w i t h murine or human c e l l s . But rather w e s h o u l d t a k e a d v a n t a g e of the s p e c i f i c p r o b l e m s w h i c h can be s o l v e d by t h i s i n s e c t m a t e r i a l . a) Selection
of genetic
markers
O n l y f e a t u r e s w h i c h are e x p r e s s e d a t t h e c e l l u l a r level and under in vitro c o n d i t i o n s may be used as m a r k e r s . M u t a t i o n s d i s t u r b i n g t h e main m e t a b o l i c p a t h w a y s are s u i t a b l e f o r t h i s p u r p o s e . F o l l o w i n g t h e e s t a b l i s h i n g of new Drosophila cell l i n e s , w o r k e r s engaged in s u c h s t u d i e s w i l l a v a i l t h e m s e l v e s in t h e near f u t u r e of t h e r i c h c o l l e c t i o n of Drosophila mutants: - Morphological l i a b l e markers.
features
of t h e c e l l s are t o o u n s t a b l e in c u l t u r e in vitro
t o be re-
- Chromosomal markers are e x t e n s i v e l y used in g e n e t i c s of s o m a t i c c e l l s . When one t h i n k s of t h e hard work w h i c h i s r e q u i r e d f o r t h e i d e n t i f i c a t i o n of t h e v a r i o u s chromosomal p a i r s of any v e r t e b r a t e c e l l , it w i l l be e a s i l y a d m i t t e d t h a t t h e s i m p l i c i t y of t h e k a r y o t y p e of Drosophila melanogaster, w i t h i t s four r e c o g n i z a b l e p a i r s of chromosomes i s a g r e a t asset. A n e x t e n s i v e k a r y o l o g i c a l a n a l y s i s has been c a r r i e d out b y F a c c i o - D o l f i n i a n d o t h e r members of t h e I n s t i t u t o d i G e n e t i c a at M i l a n o , I t a l y , on our main c e l l l i n e s 138
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CELLS
as w e l l as on new ones t h e y had r e c e n t l y g r o w n . The p o l y m o r p h i s m of chromosomal f o r m u l a e in c e l l p o p u l a t i o n s w a s c a r e f u l l y s t u d i e d ( D o l f i n i , 1 9 7 1 , 1973; F a c c i o D o l f i n i , 1974b; Mosna and D o l f i n i , 1972). Moreover, i n f o r m a t i o n w a s c o l l e c t e d , w i t h a u t o r a d i o g r a p h i c m e t h o d s , on t h e f i n e s t r u c t u r e of m i t o t i c chromosomes and espec i a l l y on D N A r e p l i c a t i o n in e u c h r o m a t i c and h e t e r o c h r o m a t i c s e c t i o n s ( B a r i g o z z i et al., 1967; H a l f e r and B a r i g o z z i , 1972; F a c c i o - D o l f i n i , 1 9 7 4 a ) . In r e c e n t y e a r s , t e c h n i q u e s of d i f f e r e n t i a l s t a i n i n g ( " b a n d i n g " ) of s p e c i f i c chromosome s e g m e n t s , and m o s t l y f l u o r e s c e n c e t e c h n i q u e ( f i g . 2) p e r m i t t e d s p e c i a l i z e d s t u d i e s on p u r i n e m e t a b o l i s m (Becker, 1970) and of f l u o r e s c e n c e p a t t e r n s (Zuffardi et al.c 1 9 7 1 ) .
Fig. 2. F l u o r e s c e n c e p a t t e r n of Drosophila c e l l s . Q u i n a c r i n e ( l e f t ) a n d o r c e i n ( r i g h t ) , (a) a n d (b) Embryonic m a l e c e l l o f t h e w i l d - t y p e s t o c k V a r e s e . B r i g h t f l u o r e s c e n c e on the Y (four b l o c k s ) a n d on t h e c h r o m o s o m e s I V , w e a k f l u o r e s c e n c e on t h e t e r m i n a l p o r t i o n of t h e X a n d on t h e c e n t r o m e r i c r e g i o n s of t h e III p a i r . ( c ) a n d (d) E s t a b l i s h e d l i n e in vitro: f e m a l e c e l l of t h e K c l i n e w i t h o n l y o n e I V c h r o m o s o m e . (e) a n d (f) E s t a b l i s h e d l i n e ( C o u r t e s y of Dr. S. F a c c i o in vitro: m a l e c e l l of t h e C a l i n e w i t h o n l y o n e I V c h r o m o s o m e . D o l f i n i a n d Dr. C. H a l f e r )
- Drug resistance a n d e s p e c i a l l y r e s i s t a n c e to several p u r i n e and p y r i m i d i n e analogues p r o v i d e d c o n v e n i e n t g e n e t i c markers of mammalian s o m a t i c c e l l s , b e c a u s e t h e e n z y m a t i c d e f i c i e n c i e s , by w h i c h most of them are a c c o u n t e d f o r , may b e used in e f f i c i e n t " s e l e c t i v e s y s t e m s " for s o r t i n g out h y b r i d c e l l s (see b e l o w t h e s p e c i a l paragraph on t h i s q u e s t i o n ) . - Isozymes are e x c e l l e n t n a t u r a l markers ( s e e t h e d e s c r i p t i o n s d e v o t e d t o i s o z y m e s and f u n c t i o n a l s t a t e of in vitro c e l l s . )
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b)
Cell
fusion
G e n e t i c s of s o m a t i c c e l l s began w i t h t h e i n i t i a l o b s e r v a t i o n of B a r s k i et al ( I 9 6 0 ) t h a t , when t w o r e c o g n i z a b l e s t r a i n s of murine c e l l s are grown in the same c u l t u r e v e s s e l , a f e w c e l l s may a c c i d e n t a l l y f u s e t h e i r c y t o p l a s m , then t h e i r n u c l e i a n d g i v e r i s e to v i a b l e h y b r i d c e l l s . T h i s e v e n t remains rare and s e v e r a l m e t h o d s had to be d e v i s e d to i n c r e a s e t h e c h a n c e s of c e l l f u s i o n and t o s o r t out s u c h h y b r i d c e l l s from the parental p o p u l a t i o n s . Spontaneous c e l l f u s i o n o c c u r s most p r o b a b l y in Drosophila c e l l c u l t u r e s as w e l l . F o r t u i t o u s l y , D o l f i n i ( B a r i g o z z i 1971) happened to o b s e r v e k a r y o l o g i c a l f i g u r e s w h i c h are r e a s o n a b l e e v i d e n c e of s u c h an e v e n t : t h e y w e r e t e t r a p l o i d metaphases w i t h 3X chromosomes and IY c h r o m o s o m e , w h i c h can o n l y b e e x p l a i n e d by t h e f u s i o n , amid a mixed p o p u l a t i o n , of a " f e m a l e " c e l l w i t h a " m a l e " o n e . Those " a n d r o g y n e " c e l l s w e r e o n l y rarely o b s e r v e d , however. - The t r e a t m e n t w i t h U V i n a c t i v a t e d v i r i o n s of Sendai p a r a - i n f l u e n z a v i r u s - the c l a s s i c a l method d e v i s e d by H a r r i s and W a t k i n s (1965) t o induce p o l y k a r y o c y t o s i s in v e r t e b r a t e c e l l s - has no e f f e c t on Drosophila c e l l s . We were u n a b l e t o f i n d any r e c e p t o r for a d s o r p t i o n of Sendai v i r u s on t h e s u r f a c e of Drosophila cells. There i s , n e v e r t h e l e s s , in the l i t t é r a t u r e a b r i e f report of c e l l f u s i o n i n d u c t i o n w i t h Sendai v i r u s , between i n s e c t c e l l s and human H e l a c e l l s (Zepp et aL, 1 9 7 1 ) . It has been r e c o g n i z e d s i n c e , t h a t t h e i n s e c t c e l l s were l e p i d o p t e r a n , and not d i p teran in o r i g i n (Greene et aL 1972). C o n f i r m a t i o n of t h i s c e l l f u s i o n w o r k is l a c k ing. Other a t t e m p t s by u s , w i t h some r e p u t e d l y f u s i n g v i r u s e s ( a t l e a s t for mammalian c e l l s ) , s u c h a s t h e H e r p e s - t y p e A u j e s k i v i r u s or t h e Simian p a r a i n f l u e n z a SV5 v i r u s , w e r e a l s o u n s u c c e s s f u l ( E c h a l i e r , 1971). A paper, by E. Suitor and Paul ( 1 9 6 9 ) , r e c o r d i n g the c y t o p a t h i c e f f e c t s of D e n g u e 2 v i r u s on S i n g h ' s Aedes c e l l s and the f o r m a t i o n of large s y n c y t i a prompted us to i n v e s t i g a t e the p o s s i b l e f u s i n g a c t i v i t y of t h i s v i r u s and many other A r b o v i r u s e s . Most of the 18 Sero-group Β A r b o v i r u s e s t h a t we t e s t e d (Hannoun and E c h a l i e r , 1971) m u l t i p l i e d in Drosophila c e l l s for very long p e r i o d s , but no c e l l f u s i o n w a s ever o b s e r v e d . S t i l l w i t h the same purpose in m i n d , w e became i n t e r e s t e d in s e v e r a l s u b s t a n c e s w h i c h are known t o m o d i f y t h e c e l l s u r f a c e . F i n a l l y , Becker, from our g r o u p , was able t o e s t a b l i s h t h a t t h e p h y t o h e m a g g l u t i n i n C o n c a n a v a l i n A not o n l y a g g l u t i n a t e s Drosophila c e l l s - as it is e x t e n s i v e l y s h o w n f o r mammalian c e l l s — b u t does i n d u c e a r a p i d c e l l f u s i o n and the f o r m a t i o n of a p p a r e n t l y v i a b l e s y n k a r y o n s (Becker 1972). The t e c h n i q u e d e v i s e d by Becker is rather s i m p l e : c e l l s are m a i n t a i n e d in a dense s u s p e n s i o n (3X106 c e l l s per ml) by a s l o w l y r o t a t i n g magnet and t r e a t e d , d u r i n g 20 m i n u t e s at room t e m p e r a t u r e , w i t h a c o n c e n t r a t i o n o f 100 μg of Con A ( C a l b i o c h e m , Los A n g e l e s ) per m l . We knew t h a t Con A a c t i v i t y is s t r e n g t h e n e d by t h e a d d i t i o n of Ca and Mn c a t i o n s ( 1 0 - 4 M) to the standard medium D 2 2 . T h e n , t h e c e l l s are seeded into c u l t u r e f l a s k s a n d , as soon as t h e y a d h e r e , w a s h e d c a r e f u l l y w i t h f r e s h medium. Shortly a f t e r t h e b e g i n n i n g of t h e t r e a t m e n t , c e l l s gather i n p a i r s or larger g r o u p s . A t l e a s t f o r our l i n e Κ c e l l s , t h e e f f i c i e n c y is v e r y h i g h : a l m o s t 50% of t h e popup u l a t i o n may be a f f e c t e d by t h e p h e n o m e n o n . S p e c i a l c o n n e c t i o n s are c r e a t e d b e t w e e n t h e p a r t n e r s , a s o b s e r v e d by p h a s e - c o n t r a s t m i c r o s c o p y ( F i g . 3 a ) . With an e l e c t r o n m i c r o s c o p e ( F i g . 3 b ) , a c o m p l e x w e b of m i c r o v i l l i c a n be seen in t h i s c o n -
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CELLS
Fig. 3. C o n c a n a v a l i n A - i n d u c e d f u s i o n o f Drosophi/a c e l l s , o n e hour a f t e r t h e b e g i n n i n g of t h e t r e a t m e n t , (a) w i t h p h a s e c o n t r a s t (X 2 5 0 0 ) (b) w i t h E.M. (X 8 0 0 0 ) O b s e r v e t h e c o m p l e x i n t r i c a t i o n o f m i c r o v i l l i i n t h e c o n t a c t area. ( C o u r t e s y o f J . L . Becker a n d P. G r e l l e t . The E l e c t r o n m i c r o g r a p h y i s s t i l l u n p u b l i s h e d ) . t a c t area. They f u s e d u r i n g t h e n e x t f e w h o u r s , w i t h - t h e f o r m a t i o n of d i - or p o l y k a r y o n s , t h e n , some 24 hours l a t e r , of s y n k a r y o n s . A u t o r a d i o g r a p h i c p i c t u r e s were o b t a i n e d of d i k a r y o n s f o r m e d b e t w e e n one s u b l i n e w h o s e n u c l e i had been l a b e l l e d w i t h t r i t i a t e d t h y m i d i n e and another u n l a b e l l e d c e l l l i n e .
141
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U n f o r t u n a t e l y , in t h e a b s e n c e of an o p e r a t i v e s e l e c t i v e d e v i c e , w e have not y e t been a b l e to s t u d y t h e fate of s u c h C o n A induced h y b r i d c e l l s . It must not be f o r g o t t e n t h a t , in mammalian c e l l s , even w i t h t h e c l a s s i c a l Sendai m e t h o d , v i a b l e h y b r i d c e l l s never e x c e e d more than 5% of t h e f u s e d c e l l s . L e t us n o t e t h a t , among t r e a t e d c e l l s of our very s t a b l e l i n e K, an u n u s u a l l y high p e r c e n t a g e of t e t r a p l o i d c e l l s w a s o b s e r v e d d u r i n g the f i r s t f e w d a y s . It r a p i d l y dropped to t h e normal l e v e l ( a b o u t 10%), w h i c h might very w e l l mean t h a t d i p l o i d c e l l s outnumbered h y b r i d o n e s . A t t e m p t s of i s o l a t i n g h y b r i d s by c l o n i n g h a v e been u n s u c c e s s f u l so f a r . The r e a l i t y and e f f i c i e n c y of Drosophila c e l l f u s i o n by C o n c a n a v a l i n A were c o n f i r m e d by G v o s d e v et aL, in M o s c o w , and G e h r i n g et aL, in Basel (personal commun i c a t i o n s ) . The rate of c e l l f u s i o n s seems t o v a r y , n e v e r t h e l e s s , from one l i n e t o another. - More r e c e n t l y , t w o q u i t e d i f f e r e n t t y p e s of compounds w e r e reported to induce c e l l f u s i o n in Drosophila c e l l s : L y s o l e c i t h i n s ( B a r i g o z z i et aL; Lane DeCamp; pers o n a l c o m m u n i c a t i o n s ) and p o l y e t h y l e n e g l y c o l ( G e h r i n g et aLt p e r s o n a l c o m m u n i c a t i o n ) . N o t h i n g is known a b o u t the s u r v i v a l of t h e observed d i k a r y o n s . So, a l t h o u g h a d e c i s i v e step may have been t a k e n , it s t i l l remains c r u c i a l t o c o n t r i v e an e f f i c a c i o u s s e l e c t i v e method to sort out s o m a t i c h y b r i d c e l l s . c) Selective
system
for hybrid
cells
— The u n i v e r s a l l y used t e c h n i q u e for s e l e c t i n g mammalian s o m a t i c h y b r i d c e l l s w a s d e v i s e d by L i t t l e f i e l d ( 1 9 6 4 ) . It c o n s i s t s in " c r o s s i n g " a c e l l l i n e w h i c h i s d e f i c i e n t for an enzyme of t h e p u r i n e " s a l v a g e " p a t h w a y ( h y p o x a n t h i n e guanine p h o s p h o r i b o s y l t r a n s f e r a s e , H GpT) w i t h a n o t h e r l i n e w h i c h l a c k s one enzyme of the p y r i m i d i n e " s a l v a g e " p a t h w a y ( t h y m i d i n e k i n a s e ) . In t h e p r e s e n c e of a m i n o p t e r i n e (a f o l i c a c i d a n a l o g u e w h i c h b l o c k s t h e n e o s y n t h e s i s routes t o n u c l e o t i d e s ) , and p r o v i d e d t h a t t h e c u l t u r e medium is s u p p l e m e n t e d w i t h h y p o x a n t h i n e and t h y m i dineO), o n l y h y b r i d c e l l s , by c o m p l e m e n t a t i o n , w i l l be able to g r o w , but not t h e p a r e n t a l o n e s . Such e n z y m e - d e f i c i e n t l i n e s a r e , in p r a c t i c e , rather e a s y t o i s o l a t e , b e c a u s e t h e y prove to be r e s p e c t i v e l y r e s i s t a n t to v a r i o u s g u a n i n e and t h y m i d i n e a n a l o g u e s . Several v a r i a n t s of t h i s s e l e c t i v e method were d e s c r i b e d . F i r s t a t t e m p t s t o adapt t h e s y s t e m to Drosophila c e l l s encountered unexpected d i f f i c u l t i e s : A l l our c e l l l i n e s were f o u n d t o be r e s i s t a n t t o h i g h d o s e s of azaguan i n e . F i n a l l y , i t was e s t a b l i s h e d , by d i r e c t e n z y m a t i c a s s a y a s w e l l as by a u t o r a d i o g r a p h i c s t u d i e s , t h a t t h e enzyme HGPRT does not e x i s t in Drosophila established c e l l l i n e s , nor in any Drosophila normal t i s s u e (Becker, 1 9 7 4 a ) . T h i s s u r p r i s i n g s i t u a t i o n had been p r e v i o u s l y s u s p e c t e d , but not d i r e c t l y p r o v e d , in o v a r i a n t i s s u e s o f a f l y from another g e n u s , Musca. A c o m p l e t e s u r v e y o f the i n t e r c o n v e r s i o n routes of p u r i n e m e t a b o l i s m in Drosophila c e l l s w a s n e c e s s a r y (Becker 1 9 7 4 b , 1 9 7 5 ) . It w a s c o n c l u d e d t h a t , in Drosophila, the only working " s a l v a g e " pathway for purine n u c l e o t i d e s is through a d e n i n e p h o s p h o - r i b o s y l t r a n s f e r a s e (APRT is d i s t i n c t from HGPRT). ( s e e f i g . 4 ) . Spontaneous d e f i c i e n t mutants w e r e , t h e r e f o r e , s e l e c t e d a g a i n s t a z a a d e n i n e . Their f r e q u e n c y , in our main l i n e s , is about 10*6, They w e r e f o u n d in one s e l e c t i o n _ s t e p , to be r e s i s t a n t t o r e l a t i v e l y high doses (10 j j g / m l , i.e. 1 0 6 M) and t h e i r r e s i s t a n c e may indeed be a c c o u n t e d f o r by an absence of APRT a c t i v i t y (Becker 1 9 7 4 a ) .
(1)
This
selective
medium
is called
HAT, for hypoxanthine,
142
aminopterine and thymidine.
LINES OF DROSOPHILA
2 ar. kinase 3 hgprt 4 gr.kinase
7 ar.désaminase 8 a.désaminase 9 g.désaminase
D 5'nucleotidase
10 pji.p.ase
CELLS
/ / xanthine oxydase
Fig. 4. I n t e r c o n v e r s i o n r o u t e s o f p u r i n e m e t a b o l i s m i n Drosophila e x i s t in Drosophila c e l l s ) (Becker 1 9 7 4 b ) .
(dotted pathways donot
In order to c o m p l e t e the s e l e c t i v e s y s t e m , T K - d e f i c i e n t mutants had t o be l o o k e d f o r . U n f o r t u n a t e l y , new d i f f i c u l t i e s a r o s e . T h o s e are n o t , a s a matter of f a c t , spec i f i c to Drosophila c e l l s a n d , even in mammalian c e l l s , BUdR r e s i s t a n c e i s n o t a l w a y s c o r r e l a t e d w i t h TK d e f i c i e n c y . In Drosophila c e l l l i n e s , a f e w v a r i a n t s res i s t a n t t o 1 0 - 4 M BUdR were i s o l a t e d , but t h e i r measured TK a c t i v i t y w a s never f o u n d to be l o w e r t h a n about 3 0 % of t h e normal v a l u e ( w h i c h e x c e e d s v a l u e s due t o t h e m i t o c h o n d r i a l e n z y m e ) . P r o c e s s e s of BUdR p e n e t r a t i o n and u t i l i z a t i o n seem t o be c o m p l e x : w h e n u s i n g l a b e l l e d a n a l o g u e s , o n l y t r a c e s of t h e r a d i o a c t i v i t y w e r e r e c o v e r e d in normal c e l l D N A ( B e c k e r , p e r s o n a l c o m m u n i c a t i o n ) . B e s i d e s , Drosophila c e l l s w e r e o b s e r v e d to be h i g h l y s e n s i t i v e t o FUdR, and no s p o n t a n e o u s res i s t a n c e c o u l d be f o u n d . A d i s t i n c t s e l e c t i v e s y s t e m , b a s e d on the use w a s r e c e n t l y s u g g e s t e d for murine c e l l s by Baker et t o i n h i b i t the plasma membrane N a / K A T P a s e . The b e h a v e s a s a c o d o m i n a n t t r a i t m i g h t be of some use o u a b a i n r e s i s t a n t and o u a b a i n s e n s i t i v e c e l l l i n e s .
of o u a b a i n - r e s i s t a n t v a r i a n t s , al. ( 1 9 7 4 ) . The drug is known fact that ouabain resistance for selecting hybrids between
The method p r o v e d d i s a p p o i n t i n g in Drosophila c e l l s : a few spontaneously resist a n t c e l l s w e r e i s o l a t e d , but t h e y w e r e u s e l e s s b e c a u s e t h e y no longer f u s e d w i t h c o n c a n a v a l i n A . T h e i r N a / K ATPase a c t i v i t y w a s not measured and s u c h a r e s i s t a n t to 1 0 * ^ M o u a b a i n m i g h t very w e l l be due t o c o m p l e x m o d i f i c a t i o n s o f t h e p l a s ma membrane. — To c o n c l u d e , f u r t h e r s t u d i e s are r e q u i r e d of s e l e c t i n g s o m a t i c h y b r i d Drosophila c e l l s . H o w e v e r , a s e m i - s e l e c t i v e m e t h o d , u s i n g APRT c e l l s , i s a l r e a d y a v a i l a b l e .
143
G. E C H A L I E R
s i m i l a r t o t h a t c o n t r i v e d by D a v i d s o n and Ephrussi (1965) for mammalian c e l l s . It may be a p p l i e d t o i n v e s t i g a t i o n s on f u s i o n of Drosophila established cell lines with various freshly explanted tissue c e l l s . 3- Analysis
of functional
states
of in vitro
cultured
cells:
isoenzymatic
patterns
Under in vitro c o n d i t i o n s , at l e a s t in l i q u i d m e d i a , most c e l l l i n e s a d o p t a s i m p l e m o r p h o l o g y , w i t h o u t apparent d i f f e r e n t i a t e d s t r u c t u r e s , w h i c h is u s u a l l y d e s c r i b e d as " f i b r o b l a s t - l i k e " . T h i s t e r m , h o w e v e r , does not t e l l a n y t h i n g a b o u t p h y s i o l o g i c a l f u n c t i o n s . It i s now o b v i o u s , from e x p e r i m e n t s w i t h mammalian c e l l c u l t u r e s , t h a t , even a f t e r s e v e r a l y e a r s of g r o w t h in vitro c e l l l i n e s may h a v e k e p t open one or a f e w of t h e s p e c i a l i z e d m e t a b o l i c p a t h w a y s w h i c h w e r e s p e c i f i c f o r t h e d i f f e r e n t i a t ed t i s s u e s they w e r e d e r i v e d f r o m . It w a s t h o u g h t t h a t an a n a l y s i s of t h e i s o e n z y m a t i c p a t t e r n s of our c e l l l i n e s c o u l d reveal some degree of f u n c t i o n a l d i f f e r e n t i a t i o n .
Drosophila
I t i s indeed w e l l known t h a t , in h i g h e r o r g a n i s m s , the same e n z y m a t i c a c t i v i t y may c o r r e s p o n d to several d i s t i n c t m o l e c u l a r f o r m s and t h a t t h e d i s t r i b u t i o n of such " i s o z y m e s " seems to be t i s s u e s p e c i f i c and to vary w i t h the d e v e l o p m e n t a l s t a g e s of the a n i m a l . D u r i n g t h e l a s t ten y e a r s , such i s o z y m i c v a r i a t i o n s h a v e been e x t e n s i v e l y s t u d i e d in t h e f l y Drosophila, by w o r k e r s in many l a b o r a t o r i e s a l l o v e r t h e w o r l d ( s e e rec e n t r e v i e w s : U r s p r u n g , 1 9 7 1 ; Fox et al., 1 9 7 1 ; D i c k i n s o n and S u l l i v a n , 1 9 7 5 ) . A large number of e n z y m a t i c s y s t e m s (about 40) h a v e been e x p l o r e d , w i t h t h e usual e l e c t r o p h o r e t i c i d e n t i f i c a t i o n m e t h o d s . In many c a s e s , t h e i r r e p a r t i t i o n w a s s y s t e m a t i c a l l y a n a l y s e d in the v a r i o u s o r g a n s and d u r i n g t h e s u c c e s s i v e s t a g e s , from embryo to i m a g o . There i s now a v a i l a b l e a v a s t s y n o p t i c t a b l e g i v i n g t h e s p e c i f i c i s o e n z y m a t i c e q u i p m e n t of any p a r t i c u l a r t i s s u e (for i n s t a n c e , of n e r v o u s g a n g l i a at t h e b e g i n n i n g o f the t h i r d l a r v a l i n s t a r ) . Moreover, i t w a s p o s s i b l e to map t h e genes c o r r e s p o n d i n g to some 30 of t h o s e e n z y m a t i c s y s t e m s , w i t h t h e u s u a l p r e c i s i o n of Drosophila genetics. Debec (1974; p e r s o n a l c o m m u n i c a t i o n ) , in our l a b o r a t o r y , s t u d i e d t h e f u n c t i o n a l s t a t e s of our c e l l l i n e s , l o o k i n g for t w o k i n d s of i n f o r m a t i o n . - F i r s t an a t t e m p t w a s made to c h a r a c t e r i z e s i g n i f i c a n t d i f f e r e n c e s in t h e i s o z y m i c p a t t e r n s of v a r i o u s s t r a i n s or c l o n e s , in order to u s e them as r e l i a b l e m a r k e r s . — S e c o n d l y , an a t t e m p t w a s made to compare such i s o z y m i c p r o f i l e s w i t h r e s p e c t i v e p a t t e r n s of the d i f f e r e n t t i s s u e s of Drosophila, to p r o v i d e i n f o r m a t i o n about t h e s t a t e s of f u n c t i o n a l d i f f e r e n t i a t i o n , at t h e m o l e c u l a r l e v e l , p o s s i b l y r e t a i n e d by these established cell lines. In a f i r s t s e r i e s , 13 e n z y m a t i c s y s t e m s w e r e e x p l o r e d in some 20 c e l l s t r a i n s or s u b l i n e s and more r e c e n t l y , 12 o t h e r e n z y m e s h a v e been s t u d i e d . The diagram ( F i g . 5) sums up D e b e c ' s r e s u l t s c o n c e r n i n g our l i n e K. It can be s e e n , at a g l a n c e , on melanogaster, which the g e n e t i c map of t h e c o r r e s p o n d i n g enzymes o f Drosophila s t r u c t u r a l genes are c u r r e n t l y e x p r e s s e d in t h e in vitro c e l l s , under s t a n d a r d c o n d i t i o n s . I n t e r e s t i n g remarks may be made on t h e p o s s i b l e c l u s t e r i n g of a c t i v e or i n a c t i v e g e n e s . T h i s p a t t e r n i s t y p i c a l a n d , w i t h but f e w e x c e p t i o n s , s i m i l a r to general p r o f i l e s o b s e r v e d in a l l s t u d i e d c e l l s t r a i n s . Three main e x p l a n a t i o n s may be put f o r w a r d to a c c o u n t f o r such a r e l a t i v e homog e n e i t y of t h e o b s e r v e d i s o e n z y m a t i c p r o f i l e s :
144
LINES OF DROSOPHILA
CELLS G6PD
,G0T 2 PGK
G ΡΟΗ
MOH
ADH
Fructok
Amylase
2
το..
AcPh
IDH
Aldox
LapOv
3 Ac.
4
Chr. 2
Fig. 5. I s o z y m i c p a t t e r n of t h e c e l l l i n e Κ, on t h e g e n e t i c m a p of Drosophila melanogaster: D a r k c i r c l e s c o r r e s p o n d t o l o c i w h i c h a r e a c t i v e u n d e r t h e s t a n d a r d c o n d i t i o n s o f in vitro culture. ADH Aldox AcPh Aph Est Fructok. GOT G6PD GPDH IDH
Alcohol Dehydrogenase Aldehyde Oxydase A c i d Phosphatase A l k a l i n e Phosphatase Esterases Fructokinase Glutamate Oxalate Transaminase Glucose 6 Phosphate Dehydrogenase Glycerophpsphate Dehydrogenase Isocitrate Dehydrogenase
Lap LeucineAmino Peptidases Malate Dehydrogenase MDH ME "Malic Enzyme" ODH Octanol Dehydrogenase 6PGD Phospho G l u c o n a t e D e h y d r o g e n a s e PGK Phospho G l y c e r a t e K i n a s e PGM Phospho G l u c o m u t a s e TO Tetrazolium Oxidase XDH Xanthin Dehydrogenase ( C o u r t e s y of A . DEBEC 1 9 7 5 , t o b e p u b l i s h e d ) .
- The f i r s t one i s t h a t our c e l l s t r a i n s ( m o s t l y s u b l i n e s from our t w o o r i g i n a l C and K, d e r i v e from f l i e s w i t h a common g e n e t i c b a c k g r o u n d . A s a m a t t e r of the most c l e a r - c u t d i f f e r e n c e in s e v e r a l e n z y m a t i c s y s t e m s c o n c e r n e d a f e w r e c e n t l y grown by Richard-Mo lard ( 1 9 7 5 ) , w h i c h h a v e a d i s t i n c t g e n o t y p e . l i k e l y t h a t in t h e f u t u r e when a large number of new l i n e s w i t h very d i f f e r e n t t y p e s w i l l become a v a i l a b l e , t h e i r i s o z y m i c p r o f i l e s w i l l d i f f e r c o n s i d e r a b l y .
lines fact, lines It is geno-
— The s e c o n d p o s s i b i l i t y i s t h a t a l l Drosophila c e l l l i n e s , d e r i v e d from f r a g m e n t ed embryos m i g h t c o n s i s t of c l o s e l y r e l a t e d t i s s u e s : o n l y a f e w c e l l t y p e s m i g h t be a b l e to m u l t i p l y under our p r e s e n t in vitro c o n d i t i o n s . In regard t o t h i s i n t e r e s t i n g q u e s t i o n i s o z y m i c p a t t e r n s can p e r h a p s p r o v i d e e v i d e n c e c o n c e r n i n g t h e p r e c i s e t i s s u e o r i g i n of our c e l l l i n e s . By c o m p a r i s o n w i t h t h e k n o w n p r o f i l e s of t h e t i s s u e s of e m b r y o s , l a r v a e , and imagos of Drosophila, i t i s t e m p t i n g to p o i n t o u t some r e s e m b l a n c e s , f o r i n s t a n c e , b e t w e e n our l i n e C a n d n e r v o u s t i s s u e s o f t h e larva. The p a t t e r n of our l i n e Κ w o u l d be more a k i n to t h e p r o f i l e of i m a g i n a i d i s k s . Supporting d a t a , h o w e v e r , are s t i l l too f e w and w e have t o be e x t r e m e l y c a u t i o u s w i t h such a s s u m p t i o n s .
145
G. ECHALIER - The t h i r d h y p o t h e s i s - and the most a t t r a c t i v e one - is to suppose t h a t c e l l s of any o r i g i n , under the very p e c u l i a r c o n d i t i o n s of in vitro c u l t u r e s , w o u l d a l l carry on a sort of " b a s i c p r o g r a m " w h i c h a l l o w s them o n l y to grow and d i v i d e ( w h a t B. Ephrussi c a l l s " h o u s e k e e p i n g a c t i v i t i e s " ) . Even t h e n , as i t w a s p o i n t e d o u t at t h e b e g i n n i n g of t h i s c h a p t e r , it is now w e l l k n o w n , from r e s u l t s o b t a i n e d w i t h mammalian c e l l l i n e s , t h a t , beyond t h i s common program, c e l l s in vitro can keep open (or o n l y p a r t i a l l y open) a few s p e c i a l i z e d m e t a b o l i c p a t h w a y s ( " l u x u r y a c t i v i t i e s " , according to Ephrussi's t e r m i n o l o g y ) . In t h i s r e s p e c t , the r e l a t i v e l y r a p i d and w i d e l y spread s c r e e n i n g t h a t can be done, in Drosophila c e l l l i n e s , w i t h an e l e c t r o p h o r e t i c a n a l y s i s of i s o z y m i c p a t t e r n s may reveal some p o s s i b l e degree of f u n c t i o n a l d i f f e r e n t i a t i o n . It must be u n d e r s t o o d , h o w e v e r , t h a t in s p i t e of the c o n s i d e r a b l e work a l r e a d y a c c o m p l i s h e d , more date are needed, e s p e c i a l l y about c e l l l i n e s w i t h d i s t i n c t g e n o t y p e s . When t h e f u n c t i o n a l s t a t e s of many c e l l l i n e s become better k n o w n , i t m i g h t be p o s s i b l e to c o r r e l a t e some m o d i f i c a t i o n s of t h e i r s p e c i a l i z e d programs w i t h s p e c i f i c v a r i a t i o n s of t h e i r k a r y o t y p e . It w i l l be w o r t h w h i l e to study the b i o s y n t h e s i s of a p a r t i c u l a r enzyme and the r e s u l t i n g monosomy or p o l y s o m y of the chromosome t h a t c a r r i e s the s t r u c t u r a l gene of t h i s e n z y m e . P r e l i m i n a r y r e s u l t s , in t h i s f i e l d , w e r e reported about X - l i n k e d G6PD and gene dosage in Drosophila cultured cells ( G v o s d e v e f al., 1 9 7 1 ) . Moreover, i t w i l l be t e m p t i n g to m a n i p u l a t e t h o s e c e l l l i n e s t o e x c e e d t h e i r u s u a l and monotonous program - e i t h e r by c h a n g i n g c o m p l e t e l y t h e i r c u l t u r e c o n d i t i o n s or by a p p l y i n g s p e c i f i c hormonal s t i m u l a t i o n s . 4-
Action
of insect
hormones
at the cellular
level
A p p l i c a t i o n s of i n v e r t e b r a t e c e l l c u l t u r e f o r the study of i n s e c t hormones have been d i s c u s s e d by Marks ( 1 9 7 5 ) . Our own w o r k c o n c e r n i n g the r e s p o n s e s of Drosophila e s t a b l i s h e d c e l l l i n e s is p r e s e n t e d here. C o u r g e o n , from our g r o u p , was a b l e to d e m o n s t r a t e t h a t some c e l l s t r a i n s , and m a i n l y our l i n e K, seem to respond s p e c i f i c a l l y to the s y n t h e t i c i n s e c t m o u l t i n g hormones, cx and /3-ecdysones, and to several of t h e i r a n a l o g u e s . The e f f e c t i v e dos e s may i n d e e d be c o n s i d e r e d a s very c l o s e to p h y s i o l o g i c a l v a l u e s , as l o w as 0.006 ,ug/ml f o r / î - e c d y s o n e . Moreover, in order to make sure t h a t the o b s e r v e d resp o n s e s are n o t u n s p e c i f i c p h a r m a c o l o g i c a l e f f e c t s of any s t e r o i d c o m p o u n d , many other s t e r o i d s were c h e c k e d , such as c h o l e s t e r o l and mammalian h o r m o n e s , but none were f o u n d to have any d i s c e r n a b l e a c t i v i t y , even at much h i g h e r d o s e s . - It c o u l d be e s t a b l i s h e d t h a t , in a d d i t i o n to t h e i r s t r i k i n g l y d i f f e r e n t t h r e s h o l d c o n c e n t r a t i o n s (/9-ecdysone i s 500 t i m e s more a c t i v e than o c - e c d y s o n e ) , t h e t w o main e c d y s o n e s have rather d i s t i n c t t y p e s of a c t i o n : - / 3 - e c d y s o n e m o s t l y i n d u c e s a c h a r a c t e r i s t i c s e r i e s of m o r p h o l o g i c a l m o d i f i c a t i o n s (Courgeon 1 9 7 2 a ) , w h i c h c o i n c i d e s w i t h an a r r e s t of m u l t i p l i c a t i o n : hormone t r e a t e d c e l l s of l i n e Κ c o n s i s t ing u s u a l l y of small round c e l l s , l o o s e l y a t t a c h e d t o the f l a s k bottom - are s e e n , after 16 to 24 h o u r s , to f l a t t e n a n d e l o n g a t e . Then, they t e n d to aggregate and bebecome f i b r o b l a s t i c or very f l a t t e n e d , and the membrane s h o w s an apparent exp a n s i o n ; t h e i r c y t o p l a s m i s f u l l of i n c l u s i o n s . The r a p i d i t y of the phenomena i s d o s e dependant. A s for cx-ecdysone, i t s t i m u l a t e s c e l l m u l t i p l i c a t i o n (Courgeon 1972b). These o b s e r v e d e f f e c t s on c u l t u r e d c e l l s are in good agreement w i t h c u r r e n t i d e a s about the d i s t i n c t r o l e s of the t w o e c d y s o n e s in the p h y s i o l o g i c a l r e g u l a t i o n of i n 146
L I N E S OF DROSOPHILA
CELLS
s e c t growth and m o u l t i n g . It w a s f o u n d t h a t the p r e s e n c e of f e t a l c a l f serum in t h e c u l t u r e medium i s nec e s s a r y for hormone a c t i o n . T h i s f a c t i s r e m i n i s c e n t of t h e s y n e r g i s t i c " M a c r o m o l e c u l a r F a c t o r " d e s c r i b e d by K a m b y s e l l i s and W i l l i a m s (1971) f o r t h e in vitro spermat o g e n e s i s of an i n s e c t . - Beyond such m o r p h o l o g i c a l v a r i a t i o n s i n d u c e d by β - e c d y s o n e in Drosophila c e l l s , i t s h o u l d be p o i n t e d out t h a t a f t e r hormonal t r e a t m e n t a f e w m o d i f i c a t i o n s of p r o t e i n p a t t e r n s of t h e c e l l s have been r e v e a l e d by i m m u n o l o g i c a l m e t h o d s . T h i s w o r k , in c o l l a b o r a t i o n w i t h Robert's group ( p e r s o n a l c o m m u n i c a t i o n ) , i s in a p h a s e . - B e s i d e s , s e v e r a l c l o n e s h a v e been s e l e c t e d from l i n e K, w h i c h d i s p l a y a w i d e range of r e c e p t i v i t y to e c d y s o n e , from the u t m o s t s e n s i t i v i t y to a t r u e r e s i s t a n c e ( i . e . no e f f e c t w i t h c o n c e n t r a t i o n s of e c d y s o n e 10,000 t i m e s e x c e e d i n g t h e u s u a l a c t i v e d o s e s ) . T h i s t r a i t w a s f o u n d t o be very s t a b l e in c l o n a l p o p u l a t i o n s (Courg e o n , 1 9 7 5 ) . T h i s d i f f e r e n t i a l c e l l u l a r r e s p o n s e t o e c d y s o n e may g i v e us a good t o o l f o r a n a l y s i n g the c o m p l e x m e c h a n i s m s of hormone a c t i o n . In t h i s c o n n e c t i o n , r e c e n t p r e l i m i n a r y r e s u l t s w i l l be m e n t i o n e d : Best-Belpomme and Courgeon ( 1 9 7 5 ) , by u s i n g l a b e l l e d / ^ - e c d y s o n e , have j u s t been a b l e t o c h a r a c t e r i z e s a t u r a b l e " r e c e p t o r s " in a " m e m b r a n e f r a c t i o n " from s e n s i t i v e c e l l s , w h i l e none c o u l d be f o u n d in r e s i s t a n t c l o n e s . IV.
P r o s p e c t s in Drosophila
cell culture
- In recent y e a r s , s l o w but s i g n i f i c a n t p r o g r e s s has been made, in s e t t i n g up long term as w e l l as primary c u l t u r e s of Drosophila c e l l s . A major f a c t o r , in t h i s p r o g r e s s , i s t h e r e c e n t i n v o l v e m e n t of an i n c r e a s i n g number o f r e s e a r c h g r o u p s . One can e x p e c t t h a t many new c e l l l i n e s w i t h i n t e r e s t i n g g e n o t y p e s w i l l soon become a v a i l a b l e . E s p e c i a l l y m u t a n t s of Drosophila w h i c h are b e i n g e x t e n s i v e l y s t u d i e d a t t h e o r g a n i s m l e v e l : such a s , f o r i n s t a n c e , " b o b b e d " m u t a n t s ( r e d u c t i o n of t h e number of r i b o s o m a l genes) or " r u d i m e n t a r y " m u t a n t s ( d e f i c i e n c y of one o f t h e f i r s t e n z y m e s of t h e p y r i m i d i n e b i o s y n t h e t i c p a t h w a y ) and many o t h e r s m i g h t t h u s p r o v i d e a d e c i s i v e a d v a n t a g e and i n c o m p a r a b l e p o s s i b i l i t i e s in p h y s i o l o g i c a l melanogaster. g e n e t i c s Drosophila - So f a r , a l l recorded s u c c e s s e s , at l e a s t in long term c u l t u r e s , c o n c e r n e d emb r y o n i c c e l l s . It w o u l d be e x t r e m e l y i m p o r t a n t to grow in vitro, f o r p r o l o n g e d per i o d s , t i s s u e s of l a r v a e or i m a g o s . N o n e of t h e p r e s e n t l y a v a i l a b l e media seems to be s u i t a b l e f o r t h i s p u r p o s e , even i f i t i s c l a i m e d t h a t they i m i t a t e t h e c o m p o s i t i o n of the body f l u i d of 3rd i n s t a r l a r v a e . I t w o u l d seem w o r t h w h i l e to a t t a c k t h i s p r o b l e m a g a i n , w i t h o u t any p r e j u d i c e , and to d e v i s e p a t i e n t l y new f o r m u l a e . It i s l i k e l y , i n d e e d , t h a t d i f f e r e n t i a t e d t i s s u e c e l l s are l i v i n g , in vitro, in a m i c r o - e n v i r o n m e n t w h i c h m i g h t d i f f e r c o n s i d e r a b l y from t h e e n v i r o n m e n t p r o v i d e d by c i r c u l a t i n g h e m o l y m p h . Moreover, much i m m a g i n a t i o n w o u l d h a v e to be d e v o t e d t o m o d i f y , perhaps in a r a d i c a l w a y , the s t a n d a r d c o n d i t i o n s of t h e c u l t u r e in l i q u i d media - in order to p r e s e r v e t h e s u p p o s e d l y imp o r t a n t c e l l t o c e l l c o n n e c t i o n s of d i f f e r e n t i a t e d t i s s u e s . Let us n o t e t h a t t h e a b o v e remarks c o r r e s p o n d to a new g e n e r a l t r e n d , in v e r t e b r a t e c e l l c u l t u r e as w e l l . Ten y e a r s ago (and d u r i n g t h e f i f t i e s f o r m a m m a l i a n c e l l s ) , methods w e r e d e v i s e d to f a v o r c e l l d i v i s i o n s and to grow l a r g e c e l l p o p u l a t i o n s . A t t h a t t i m e , m o n o l a y e r s of f i b r o b l a s t c e l l s w e r e m o s t l y u s e d as s u b s t r a t e f o r v i r a l m u l t i p l i c a t i o n . The p o i n t of v i e w c h a n g e d e n t i r e l y when t h e main c u r r e n t of b i o l o -
147
G. E C H A L I E R
g i c a l research s h i f t e d t o p h y s i o l o g i c a l g e n e t i c s of e u k a r y o t i c c e l l s of h i g h e r org a n i s m s . N o w , l e s s e m p h a s i s i s put on c e l l m u l t i p l i c a t i o n and greater c a r e is t a k e n in t r y i n g t o grow t h e c e l l s under c o n d i t i o n s w h i c h w o u l d a l l o w them t o m a i n t a i n , a t l e a s t p a r t l y , t h e i r d i f f e r e n t i a t e d s t r u c t u r e s and m e t a b o l i c p r o g r a m s . - U n d o u b t e d l y , among t h e v a r i o u s Drosophila t i s s u e s t o be grown and p r o p a g a t e d in vitro, i m a g i n a i d i s c c e l l s have r e c e i v e d the g r e a t e s t a t t e n t i o n . Imaginai d i s c c e l l s indeed c o n s t i t u t e one of t h e m o s t c o n v e n i e n t m a t e r i a l s f o r a n a l y s i n g t h e c o m p l e x m e c h a n i s m s of c e l l d e t e r m i n a t i o n . T h i s can be d e d u c e d from t h e r e m a r k a b l e s e r i e s of i n v e s t i g a t i o n s c a r r i e d o u t in vitro by Hadorn a n d h i s c o w o r k e r s in Z u r i c h ( U r s p r u n g , 1 9 7 2 ) . I t must be r e c a l l e d t h a t i m a g i n a i d i s c s are s m a l l groups of c e l l s w h i c h are k e p t apart d u r i n g t h e w h o l e l a r v a l p e r i o d of t h e i n s e c t a n d , at t h e t i m e of the metamorp h o s i s , w i l l permit t h e b u i l d i n g of a n e n t i r e l y new o r g a n i s m , t h e imago. The determ i n a t i o n of t h o s e c e l l s , t h a t i s t h e s e l e c t i o n of t h e program they w i l l h a v e to acc o m p l i s h , goes back to e a r l y e m b r y o n i c l i f e , even though t h e e x p r e s s i o n o f t h i s d i f f e r e n t i a t e d program i s p o s t p o n e d f o r s e v e r a l d a y s and w i l l t a k e p l a c e o n l y under t h e hormonal s t i m u l a t i o n s of m e t a m o r p h o s i s . The b a s i c s t a b i l i t y of t h i s d e t e r m i n e d s t a t e has been c l e a r l y e s t a b l i s h e d . There are, in t h e l i t t é r a t u r e , many r e p o r t s on more or l e s s s u c c e s s f u l d e v e l o p m e n t of i m a g i n a i d i s c s in vitro. Among t h e most r e c e n t r e p o r t s , l e t us m e n t i o n t h e p a r t i a l s u c c e s s o b t a i n e d by Schneider ( 1 9 6 4 , 1 9 6 6 ) , in her l i q u i d m e d i u m , w i t h e y e - a n t e n nal d i s c s a n d , m o s t l y , the o u t s t a n d i n g r e s u l t s of Mandaron ( 1 9 7 0 , 1 9 7 1 ) : in a s y n t h e t i c c u l t u r e medium and under the s t i m u l a t i o n of e c d y s o n e , leg or w i n g d i s c s from l a t e 3rd i n s t a r l a r v a e can e v a g i n a t e and d i f f e r e n t i a t e c o m p l e t e l y in a f e w d a y s . The v a r i o u s s e g m e n t s of t h e appendage, w i t h t h e i r s p e c i f i c c u t i c u l a r s t r u c t u r e s , are p e r f e c t l y r e c o g n i z a b l e . It must be p o i n t e d o u t , h o w e v e r , t h a t o n e i s d e a l i n g w i t h i n t a c t i m a g i n a i d i s c s and not w i t h i n d i v i d u a l c e l l s ; t h e r e i s no or v e r y l i t t l e , c e l l m u l t i p l i c a t i o n . Therefore d e s p i t e the a t t r a c t i v e n e s s of t h i s e x p e r i m e n t a l model f o r an a n a l y s i s of l a t e s t a g e s of d i f f e r e n t i a t i o n , a b i o c h e m i c a l approach r e m a i n s d i f f i c u l t and r e s t r i c t e d , even though i t o f f e r s the p o s s i b i l i t y of i s o l a t i n g r e l a t i v e l y large amounts of d i s c s , a c c o r d i n g t o t h e method of F r i s t r o m and M i t c h e l l ( 1 9 6 5 ) . A p r o m i s i n g step w a s t a k e n when Schneider (1972) o b s e r v e d t h e g r o w t h of f l o a t i n g v e s i c l e s in primary c u l t u r e s from l a t e e m b r y o s . They c o n t i n u e d t o s w e l l and bud d u r i n g a f e w p a s s a g e s a n d , when f i n a l l y t r a n s p l a n t e d i n t o l a r v a e , t h e y g a v e r i s e , a t the t i m e of t h e i r h o s t m e t a m o r p h o s i s , t o i d e n t i f i a b l e c u t i c l e and b r i s t l e s . T h i s means t h a t they w e r e d i s c c e l l s . U n f o r t u n a t e l y , t h e e s t a b l i s h e d l i n e s w h i c h w e r e d e r i v e d from t h e same c u l t u r e s a p p a r e n t l y d i f f e r e d from t h i s i m a g i n a i m a t e r i a l . Q u i t e r e c e n t l y , Dubendorfer, in c o l l a b o r a t i o n w i t h t h e group of Sang ( p e r s o n a l comm u n i c a t i o n ) e s t a b l i s h e d t h a t s i m i l a r v e s i c l e s , d e v e l o p e d in p r i m a r y c y l t u r e s from d i s s o c i a t e d e a r l y e m b r y o s , can d i f f e r e n t i a t e and s e c r e t e c u t i c u l a r s t r u c t u r e s , not o n l y when t r a n s p l a n t e d in vivo, but a l s o in vitro w h e n t r e a t e d w i t h e c d y s o n e . U n f o r t u n a t e l y , such v e s i c l e s , in the p r e s e n t c o n d i t i o n s of c u l t u r e , do not s u r v i v e longer than a f e w w e e k s . A new f r o n t i e r w i l l open w h e n v e r i t a b l e permanent l i n e s of such d e t e r m i n e d i m a g i nai d i s c c e l l s became a v a i l a b l e . In c o n c l u s i o n , in recent y e a r s Drosophila c e l l c u l t i v a t i o n has g i v e n r i s e to a s p e c t a c u l a r r e v i v a l of i n t e r e s t in d e v e l o p m e n t a l b i o l o g y . It i s q u i t e p r o b a b l e t h a t a l m o s t
148
L I N E S OF DROSOPHILA
CELLS
a l l p r e d i c t a b l e e x p e r i m e n t a l a p p r o a c h e s e m p l o y i n g Drosophila y i e l d s i g n i f i c a n t and p e r h a p s d e c i s i v e r e s u l t s . V.
m a t e r i a l in vitro
will
References
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C h a p t e r 10
METAMORPHOSIS OF IMAGINAL DISC TISSUE GROWN FROM DISSOCIATED EMBRYOS OF
IN
VITRO
DROSOPHILA
A . DÜBENDORFER
I.
Introduction
151
II.
M a t e r i a l s and M e t h o d s
152
III.
R e s u l t s and C o n c l u s i o n s
152
1 . M e t a m o r p h o s i s o f i m a g i n a i d i s c c e l l s IN VITRO
152
2. T h e d i f f e r e n t i a t e c a p a c i t y a f t e r m e t a m o r p h o s i s IN VIVO IV.
Discussion 1 . The s i g n i f i c a n c e o f t i s s u e v e s i c u f a r i s a t i o n a n d c e l l f l a t t e n i n g 2. H o r m o n e a c t i o n
V.
References
I.
Introduction
154 ·
156 155 157 6 gms on t h e t h i r d day o f t h e i n s t a r i s s t i l l c o m m i t t e d to l a r v a l d i f f e r e n t i a t i o n . Yet by t h i s t i m e the J H t i t e r in the hemolymph h a s begun t o d e c l i n e ( N i j h o u t a n d W i l l i a m s , 1 9 7 4 b ) . T h e r e f o r e , t h i s e p i d e r m i s w a s i n c u b a t e d i n G r a c e ' s medium w i t h d i f f e r e n t a m o u n t s of /3-ecdysone f o r 2 0 - 2 3 hours to d e t e r m i n e t h e c o n c e n t r a t i o n n e c e s s a r y t o e f f e c t a c o m p l e t e s w i t c h o v e r in c o m mitment to p u p a l d e v e l o p m e n t . The c i r c l e s ( · ) in f i g u r e 2 show the % w h i c h f o r m e d c o m p l e t e l y pupal c u t i c l e in the in vivo a s s a y as a f u n c t i o n of t h e l o g a r i t h m of t h e d o s e of /^-ecdysone. With 1 j j g / m l / 3 - e c d y s o n e 9 3 % o f t h e e p i d e r m i s c o m p l e t e d t h e 215
L M. R I D D I F O R D
c h a n g e b e f o r e i m p l a n t a t i o n into the l a r v a l h o s t Recovery of i m p l a n t e d c y s t s ranged b e t w e e n 50 and 8 0 % , and the c u t i c l e formed w a s hard and b r i t t l e and u s u a l l y tan or b e g i n n i n g to t a n . When the c o n c e n t r a t i o n of fi-e cd y s one w a s i n c r e a s e d to 5 j u g / m l , 100% o f the c y s t s formed p u p a l c u t i c l e . But t h e p e r c e n t a g e o f e p i d e r m a l c y s t s r e c o v e r e d from the m o l t e d 5th instar host w a s low ( 9 5 % t h e natural trans, trans, eis isomer. A l t h o u g h the m o r p h o g e n e t i c a c t i o n of J H in vitro in t h e p r e v e n t i o n of i m a g i n a i d i s c d e v e l o p m e n t has p r e v i o u s l y been r e p o r t e d ( C h i h a r a a n d F r i s t r o m , 1973; Oberl a n d e r a n d T o m b l i n , 1972; Sanburg et al, 1 9 7 5 ) , t h e e f f e c t i v e d o s e s have a l w a y s been about 10-4 M or h i g h e r . A l s o , in t h e s e s y s t e m s t h e r e s p o n s e s h a v e been a c o m p l e t e i n h i b i t i o n of d i f f e r e n t i a t i o n and c u t i c l e f o r m a t i o n , a f a c t n o t s u r p r i s i n g s i n c e i m a g i n a i d i s c s o n l y g r o w and do not make c u t i c l e in t h e p r e s e n c e of J H in t h e l a r v a . Yet in s u c h a s y s t e m o n e c a n n o t be s u r e t h a t the i n h i b i t i o n seen at t h e s e h i g h d o s e s is n o t j u s t p h a r m a c o l o g i c a l , a l t h o u g h C h i h a r a and Fristrom (1973) report immediate r e v e r s i b i l i t y of the i n h i b i t i o n upon removal of the J H . In the Manduca e p i d e r m a l s y s t e m d e s c r i b e d in t h i s paper the e f f e c t o f J H is a l s o i n h i b i t i o n a s i t p r e v e n t s the e c d y s o n e - i n d u c e d o n s e t of p u p a l c o m m i t m e n t . But t h e f a c t t h a t the e p i d e r m i s is not a d v e r s e l y a f f e c t e d b y t h e J H is shown b y i t s a b i l i t y t o m a k e larval c u t i c l e when exposed t o an in vivo m o l t . Furthermore, p r e l i m i n a r y data i n d i c a t e t h a t w h e n the e p i d e r m i s i s e x p o s e d to e c d y s o n e in the p r e s e n c e o f J H f o r 2 0 h o u r s , then removed to hormone-free m e d i a , i t w i l l form a d e f i n i t i v e l a r v a l c u t i c l e i n a b o u t 2 d a y s ( R i d d i f o r d , u n p u b l i s h e d ) . But w h e n e x p o s e d o n l y to e c d y s o n e f o r 2 0 h o u r s , then to hormone f r e e m e d i a , no c u t i c l e i s f o r m e d . Rather a s e c o n d e x p o s u r e t o e c d y s o n e i s n e c e s s a r y and r e s u l t s i n p u p a l c u t i c l e f o r m a t i o n ( M i t s u i and R i d d i f o r d , m a n u s c r i p t in p r e p a r a t i o n ) .
218
IN VITRO A C T I O N OF E C D Y S O N E A N D J U V E N I L E H O R M O N E
Sanburg et al., (1975) h a v e s u g g e s t e d t h a t the J H - b i n d i n g p r o t e i n i s o l a t e d f r o m t h e hemolymph of f i f t h i n s t a r Manduca l a r v a e p l a y s an i m p o r t a n t r o l e not o n l y in p r o t e c t i n g J H from d e g r a d a t i o n b y t h e g e n e r a l e s t e r a s e s b y t a l s o in t h e a c t i o n of J H on i t s t a r g e t t i s s u e . Their e x p e r i m e n t s w h i c h show the e n h a n c e m e n t of t h e J H i n h i b i t i o n of c u t i c l e d e p o s i t i o n in P/odia w i n g d i s c s upon a d d i t i o n of t h e b i n d i n g p r o t e i n (Sanburg et al., 1975) tend to s u p p o r t t h i s c o n c l u s i o n . H o w e v e r , even w i t h t h e b i n d i n g p r o t e i n the d o s e of C 1 8 J H n e c e s s a r y f o r 50% i n h i b i t i o n w a s > 1 0-4 M. In the e x p e r i m e n t s reported here t h e r e w a s no p r o t e i n in the medium a n d no b i n d i n g p r o t e i n a d d e d . A l t h o u g h the c o m p l e t e a b s e n c e o f b l o o d c e l l s i s u n l i k e l y b e c a u s e of t h e i r a d h e r e n c e t o i n s e c t t i s s u e , t h e i r number was m i n i m a l due t o t h e e x t e n s i v e w a s h i n g . The a b s e n c e of f a t body i n t h e c u l t u r e s i n s u r e s t h e v i r t u a l a b s e n c e o f b i n d i n g p r o t e i n s i n c e N o w o c k and G i l b e r t ( t h i s c o n f e r e n c e ) h a v e n o w r e p o r t e d t h e p r o d u c t i o n of the b i n d i n g p r o t e i n b y t h e f a t b o d y in vitro. T h e r e f o r e , one m u s t c o n c l u d e t h a t J H can a l s o a c t on t h e e p i d e r m i s w i t h o u t t h e i n t e r m e d i a r y of the b i n d i n g p r o t e i n . It r e m a i n s to be seen if the a d d i t i o n of the J H - b i n d i n g p r o t e i n w o u l d s u b s t a n t i a l l y l o w e r the e f f e c t i v e d o s e of J H required f o r i t s status quo e f f e c t . VI.
T i m i n g of D N A s y n t h e s i s in t h e e p i d e r m i s d u r i n g t h e l a r v a l - p u p a l t r a n s f o r m a tion.
In t h e c l a s s i c a l v i e w of t h e e n d o c r i n e c o n t r o l of m e t a m o r p h o s i s , e c d y s o n e i s t h o u g h t t o cause a p o l y s i s from t h e o v e r l y i n g c u t i c l e , D N A s y n t h e s i s , a n d then s e c r e t i o n of a n e w c u t i c l e ( f o r r e v i e w s see W y a t t , 1972; G i l b e r t and K i n g , 1973; W i l l i s , 1974) S i n c e in many d e v e l o p i n g s y s t e m s D N A s y n t h e s i s i s t h o u g h t t o be n e c e s s a r y t o c o m m i t t h e c e l l s t o a p a r t i c u l a r path of d i f f e r e n t i a t i o n ( H o l t z e r et al, 1 9 7 2 ) , t h e p r e v a l e n t idea in t h e i n s e c t l i t e r a t u r e has been t h a t e c d y s o n e i n d u c e s t h i s c r i t i c a l round of D N A s y n t h e s i s and the l e v e l of JH d e t e r m i n e s w h e t h e r t h e l a r v a l , p u p a l , or a d u l t g e n e - s e t w i l l be r e a d t o s y n t h e s i z e the c u t i c l e ( S c h n e i d e r men, 1972; W i l l i a m s and K a f a t o s , 1972). C o n s e q u e n t l y , t h e m o r p h o g e n e t i c a c t i o n o f J H has been assumed to be somehow i n v o l v e d w i t h t h i s D N A s y n t h e s i s , b u t W i l l i s ( 1 9 7 4 ) argues p e r s u a s i v e l y a g a i n s t t h i s n a r r o w v i e w . S i n c e t h e e c d y s o n e - i n d u c e d c h a n g e in c o m m i t m e n t from l a r v a l to p u p a l d i f f e r e n t i a t i o n o c c u r s at a very s p e c i f i c t i m e in Manduca, i t seemed to be an i d e a l s y s t e m in w h i c h t o look a t t h i s p r o b l e m . C o n c u r r e n t l y w i t h the d e v e l o p m e n t of t h e in vitro s y s t e m for t h e study o f the hormonal c o n t r o l o f e p i d e r m a l c o m m i t m e n t , D N A synthesis in the i n t a c t animal was monitored by autoradiography. Fifth i n s t a r l a r v a e of k n o w n age were i n j e c t e d w i t h 5 ,uc/gm 3 H - t h y m i d i n e ( s p e c i f i c a c t i v i t y 2 0 C i / m m o l e , New England N u c l e a r C o r p . ) a n d then s a c r i f i c e d 2 hours l a t e r . The i n t e g u m e n t f r o m v a r i o u s r e g i o n s w a s f i x e d , s e c t i o n e d , c o a t e d w i t h NTB-3 N u c l e a r t r a c k e m u l s i o n (Eastman K o d a k ) , and e x p o s e d f o r 4 w e e k s a t - 2 0 ° C . Table 2 compares the t i m i n g of t h e s w i t c h o v e r of c o m m i t m e n t o f t h e e p i d e r m a l c e l l s as a s s a y e d by i m p l a n t a t i o n into 4th i n s t a r l a r v a e to t h a t for DNA s y n t h e s i s . A s i s r e a d i l y apparent, no D N A s y n t h e s i s o c c u r s d u r i n g the d a y t h a t t h e s w i t c h o v e r to pupal c o m m i t m e n t o c c u r s . Even t h e f o l l o w i n g d a y there is o n l y D N A s y n t h e s i s i n t h e p r o l e g region b u t none in the d o r s a l e p i d e r m i s . But on d a y 1 of t h e w a n d e r i n g p h a s e when the s e c o n d e c d y s o n e s e c r e t i o n i s b e g i n n i n g , all o f the e p i d e r m i s is h e a v i l y l a b e l e d . By the f o l l o w i n g d a y t h i s b u r s t o f D N A s y n t h e s i s i s completeand c u t i c l e secretion begins. These data thus i n d i c a t e that the e c d y s o n e - i n d u c e d c h a n g e in c o m m i t m e n t o f t h e e p i d e r m a l c e l l s o c c u r s n e a r l y 2 days b e f o r e D N A s y n t h e s i s is s e e n . T h u s , it i s
219
L. M. R I D D I F O R D TABLE 1 Commitment of epidermis from 6.5, ± 0.3 gm fifth exposure to ecdysone and juvenile hormone. Hormone
instar
MANDUCA
after
Type of cuticle
(μg/m/)
C18JH
larvae
Ecdysone
Number
Larval
IN
produced
VITRO
(%)
Larval & Pupal
Pupal
0
0
59
100
0
0
0
1
39
0
7
93
0.005
1
8
12
38
50
0.05
1
21
10
52
38
0.5
1
15
60
33
7
5
1
6
100
0
0
TABLE 2 DNA
synthesis
in MANDUCA
epidermis
Epidermal
during
Commitment
the larval-pupal
(%}*
transformation.
Epidermal
DNA
Synthesis**
Dorsal
Ventral
Larval Stage Feeding
Larva/ 5th
& Pupal
Pupal
Pro/eg
instar
3rd day p.m. 4th day a.m. p.m. Wandering
100
0
0
0
0
0
30 9
40 16
30 78
0 0
0 0
0 0
stage
Day 0 a.m.
0
0
100
0
+/ -
p.m.
—
- -
—
0
0
Day 1 a.m. p.m.
- - -
- - -
- - -
++++ ++++
++ ++
Day 2 a.m.
_
0
0
*
_
_
_
_
+++ ++ 0
E p i d e r m i s w a s r e m o v e d at t h e i n d i c a t e d t i m e a n d i t s c o m m i t m e n t w a s d i r e c t l y a s s a y e d by the in vivo
**
_
++ +
l a r v a l a s s a y d e s c r i b e d in s e c t i o n I I I .
A u t o r a d i o g r a p h s o f e p i d e r m i s f r o m t w o l a r v a e at e a c h t i m e p o i n t w e r e s c a n n e d , and t h e a m o u n t o f l a b e l i n g w a s s c o r e d as f o l l o w s : 0: no d e t e c t a b l e label + / - : l a b e l in a f e w c e l l s in o n l y o n e o f t h e t w o a n i m a l s + : < 10 c e l l s l a b e l e d per s e c t i o n + + : 10-30 c e l l s l a b e l e d per s e c t i o n + + + : 3 0 - 5 0 c e l l s l a b e l e d per s e c t i o n + + + + : > 50 c e l l s l a b e l e d per s e c t i o n
220
IN VITRO A C T I O N OF ECDYSONE A N D J U V E N I L E H O R M O N E n o t D N A s y n t h e s i s i t s e l f t h a t e f f e c t s t h e i r r e v e r s i b l e s w i t c h o v e r . Rather i t i s some c e l l u l a r e v e n t , perhaps t h e s y n t h e s i s of a r e g u l a t o r y p r o t e i n , t h a t o c c u r s w e l l b e f o r e t h i s s y n t h e s i s . Later the f i n a l round of D N A s y n t h e s i s o c c u r s j u s t p r i o r to t h e e x p r e s s i o n of the d i f f e r e n t i a t e d s t a t e , i . e . , c u t i c l e f o r m a t i o n . VII.
Conclusion.
Manduca l a r v a l e p i d e r m i s t h u s p r o v i d e s an e x c e l l e n t s y s t e m in w h i c h t o s t u d y t h e mode of a c t i o n of both e c d y s o n e and j u v e n i l e hormone in vitro. I t can b e e a s i l y c u l t u r e d i n G r a c e ' s medium and upon exposure t o / ^ - e c d y s o n e in vitro w i l l change i t s c o m m i t m e n t f o r d i f f e r e n t i a t i o n f r o m l a r v a l to p u p a l . If, h o w e v e r , J H i s a l s o p r e s e n t d u r i n g t h i s in vitro e x p o s u r e t o e c d y s o n e , i t s l a r v a l c o m m i t m e n t w i l l b e m a i n t a i n e d . The b e a u t y of t h i s s y s t e m i s t h a t the c o m m i t m e n t to d i f f e r e n t i a t i o n and the e x p r e s s i o n of t h i s c o m m i t m e n t , i . e . c u t i c l e s y n t h e s i s , o c c u r a t t w o d i s t i n c t l y separate t i m e s , t h u s r e q u i r i n g t w o p u l s e s of e c d y s o n e . The c e l l u l a r e v e n t i n v o l v e d in t h e s w i t c h o v e r of c o m m i t m e n t in t h i s s y s t e m i s not D N A s y n t h e s i s b u t some p r i o r h a p p e n i n g . I n v e s t i g a t i o n s of t h e n a t u r e o f t h i s e v e n t are c o n t i n u i n g w i t h t h i s in vitro s y s t e m . Acknowledgements I thank M s . J a n i c e Moore f o r t e c h n i c a l a s s i s t a n c e , D r s . C . Gunthart a n d W. V o g e l o f R. Maag f o r t h e g i f t of Ro6-9550, D r . Margery Fain f o r a d v i c e on c u l t u r e t e c h n i q u e s , and D r . James W. Truman f o r a c r i t i c a l r e a d i n g o f the m a n u s c r i p t . Supported by grants BMS74-02781-A02 from NSF and RF7301 9 from the R o c k e f e l l e r F o u n d a t i o n .
VIII.
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385-
C h a p t e r 1a
METABOLISM OF MOLTING HORMONE ANALOGS BY CULTURED COCKROACH TISSUES
E.P. Marks
ι.
Introduction
223
II.
Methods
223
III.
Results
224
IV.
Discussion
225
V.
References
226
I.
Introduction
Treatment of l e g regenerates o f c u l t u r e d c o c k r o a c h e s , Leucophaea maderae ( F , ) , w i t h / ^ - e c d y s o n e w i l l i n d u c e the d e p o s i t i o n o f a c h i t i n - c o n t a i n i n g c u t i c l e (Marks a n d L e o p o l d , 1971). Treatment w i t h oc-ecdysone w i l l a l s o i n d u c e c u t i c l e d e p o s i t i o n , b u t o n l y when t h e e c d y s o n e is i n c u b a t e d w i t h t h e leg r e g e n e r a t e f o r s e v e r a l d a y s ( M a r k s , 1973a). R e c e n t l y , King and Marks (1974) d e m o n s t r a t e d t h a t c u l t u r e d l e g reg e n e r a t e s s l o w l y c o n v e r t oc-ecdysone t o / ^ - e c d y s o n e . In a d d i t i o n , Kaplanis eta/. ( 1 9 6 9 ) r e p o r t e d t h a t 2 2 , 2 5 - d i d e o x y e c d y s o n e was c o n v e r t e d to o 3 0 0 0 0 ) in general p r o b a b l y r e s u l t f r o m t h e a g g r e g a t i o n of smaller units. From t h i s b r i e f d i s c u s s i o n i t i s apparent t h a t t h e quaternary s t r u c t u r e of Hbs var i e s c o n s i d e r a b l y b e t w e e n s p e c i e s . In a d d i t i o n , s e v e r a l t y p e s of Hb c a n c o - e x i s t in t h e same o r g a n i s m , as is the c a s e for Chironomus and some A n n e l i d s ( B r a u n i t z e r et al., 1968; M a n w e l l , 1966; T h o m p s o n , 1968; V i n o g r a d o v et al., 1 9 7 0 ; Seamonds et al., 1971). In t h e l a t t e r c a s e , there are a t l e a s t t w o H b s in t h e c o e l o m i c c o r p u s c l e s in a d d i t i o n to t h o s e in t h e c o e l o m i c c a v i t y of Glycera. A s many as 10-14 d i f f e r e n t H b s are known f o r Chironomus, i n c l u d i n g monomers and d i m e r s . H b s in Chironomus r e p r e s e n t up to 90% of the hemolymph p r o t e i n s ( E n g l i s h , 1 9 6 9 ) . The t e r t i a r y s t r u c t u r e o f monomeric H b s f r o m A n n e l i d s , Chironomus and vertebrat e s have been compared by x-ray c r y s t a l l o g r a p h y (Padlan et al., 1 9 7 4 ) . The r e s u l t s d e m o n s t r a t e a s t r i k i n g s i m i l a r i t y in t h e c a r b o n b a c k b o n e s b e t w e e n a l l s p e c i e s e x a m i n e d . A l l of t h e m o l e c u l e s have t h e m y o g l o b i n - l i k e f o l d i n w h i c h t h e heme group is i n s e r t e d . In a l l c a s e s , h y d r o p h o b i c amino a c i d r e s i d u e s surround t h e heme m o i e t y . Such r e s i d u e s are r e q u i r e d f o r O2 e x c h a n g e . W i t h o u t t h e m , O 2 w o u l d be r a p i d l y d i s c h a r g e d t o t h e s u r r o u n d i n g medium. In s p i t e of t h e o v e r a l l s i m i l a r i t y of some monomeric Hbs f r o m w i d e l y d i v e r g e n t s p e c i e s , t h e s e c o n d a r y and p a r t i c u l a r l y t h e p r i m a r y s t r u c t u r e s can v a r y c o n s i d e r a b l y . Thus t h e a l p h a - h e l i c a l c o n t e n t o f V e r t e b r a t e H b , sperm w h a l e m y o g l o b i n , Chironomus Hb and Glycera H b are not i d e n t i c a l (Huber et al., 1 9 6 8 ; Padlan et al., 1 9 7 4 ) . A m i n o a c i d a n a l y s e s and s e q u e n c i n g (where a v a i l a b l e ) s h o w t h a t t h e r e i s up to 93% non-homology of amino a c i d r e s i d u e s and o n l y t w o r e s i d u e s are in homol o g o u s p o s i t i o n s in a l l Hbs sequenced (Padlan et al., 1974). Even among t h e numethummi h e m o l y m p h , t h e r e are many d i s s i m i l a r i rous Hbs i s o l a t e d from Chironomus t i e s in amino a c i d c o n t e n t and N- and C - t e r m i n i (Braun et al., 1 9 6 8 ) . A p p a r e n t l y c e r t a i n Key p o s i t i o n s in t h e d i f f e r e n t H b m o l e c u l e s are o c c u p i e d by s i m i l a r amino a c i d r e s i d u e s w h i c h s e r v e to break or a l t e r t h e h e l i c a l s t r u c t u r e o f t h e p o l y p e p t i d e c h a i n s in j u s t t h e r i g h t p l a c e s , b i n d i n g t h e m o l e c u l e t o a s s u m e t h e o v e r a l l s i m i l a r i t y of t e r t i a r y s t r u c t u r e . D i f f e r e n c e s in t h e h y d r o p h o b i c groups w h i c h s e c l u d e t h e heme f r o m an o t h e r w i s e h y d r o p h i l l i c e n v i r o n m e n t p r o b a b l y r e s u l t in t h e d i f f e r e n c e s in O2 (and other l i g a n d ) b i n d i n g a f f i n i t i e s o b s e r v e d f o r t h e s e H b s . Other s u b t l e d i f f e r e n c e s in p r i m a r y , secondary and t e r t i a r y s t r u c t u r e may d e 229
H. L A U F ER et al.
t e r m i n e t h e a b i l i t y o f H b s to d i m e r i z e or a g g r e g a t e and t h e r e l a t e d c o o p e r a t i v i t y o r n o n - c o o p e r a t i v i t y of O2 b i n d i n g ( i . e . , t h e b i n d i n g of t h e f i r s t O2 m o l e c u l e gener a l l y f a c i l i t a t e s b i n d i n g of t h e s u c c e e d i n g m o l e c u l e s in p o l y m e r i c H b s ( H a u r o w i t z , 1963). 3.
Location
and source
of hemoglobins
in
non-vertebrates
The s y n t h e s i s and o r i g i n o f Hbs has been d e m o n s t r a t e d in t h e root n o d u l e s of l e g u m e s . S t u d i e s o f i s o t o p i c p r e c u r s o r i n c o r p o r a t i o n i n t o H b s show t h a t protoporp h y r i n IX is s y n t h e s i z e d by t h e b a c t e r o i d s y m b i o n t , w h i l e t h e g l o b i n is d e t e r m i n e d b y t h e p l a n t genome ( D i l w o r t h , 1969; C u t t i n g et al., 1 9 7 1 ; 1 9 7 2 ; Broughton et al., 1 9 7 1 ) . In y e a s t s and p r o t o z o a , H b is a component of t h e c y t o p l a s m . G e n e r a l l y , Hb i s f o u n d f r e e in t h e body f l u i d s o f i n v e r t e b r a t e s ( L a n k e s t e r , 1 8 7 2 ; Shlom et al., 1973). The c o e l o m i c c e l l s o f some p o l y c h a e t e a n n e l i d s a l s o c o n t a i n H b ( V i n o g r a d o v et al., 1970). C e l l s in t h e body w a l l of f l a t w o r m s appear to c o n t a i n Hb ( F o x , 1 9 5 5 ; Fox et al., 1955). H b - c o n t a i n i n g c e l l s ( e . g . , pore c e l l s ) are f o u n d in M o l l u s c s (Sminia et al., 1972) u s i n g H b - s p e c i f i c s t a i n i n g p r o c e d u r e s . A m o n g c r u s t a c e a n s ( e . g . , Daphnia) Hb is f o u n d in c e l l s of t h e nervous s y s t e m , in e g g s and in f a t c e l l s ( F o x , 1955b). I n s e c t H b s are s o m e t i m e s f o u n d in groups of c e l l s r i c h l y p e n e t r a t e d as w e l l as in c e l l s in t h e gut and nervous by t r a c h e a e ( e . g . , Buenoa, Gastrophilus) personal o b s e r v a t i o n ) . These " t r a c h e a l c e l l s " seem o n t i s s u e ( e . g . , Chironomus, t o g e n i c a l l y to be d e r i v e d from c e l l s of t h e f a t body ( D i n o l e s c u , 1932; Bare, 1 9 2 8 ) . In a d d i t i o n to t h e s e H b s , M y o g l o b i n has been f o u n d a s s o c i a t e d p r i m a r i l y w i t h M o l l u s c and A n n e l i d m u s c l e s (Fox 1955a; F a n e l l i et al., 1958; T e n t o r i et al., 1 9 7 2 ) . The c y t o l o g i c a l or h i s t o l o g i c a l l o c a l i z a t i o n o f Hbs in c e l l s and o r g a n s of i n v e r t e b r a t e s has led to c o n s i d e r a b l e s p e c u l a t i o n r e g a r d i n g t h e source of t h e s e p i g m e n t s i n t h e o r g a n i s m . The main l i n e s o f e v i d e n c e s u p p o r t i n g such s p e c u l a t i o n s stem f r o m o b s e r v e d d i f f e r e n c e s in t h e p h y s i o c h e m i c a l p r o p e r t i e s of v a r i o u s i n v e r t e b r a t e a n d v e r t e b r a t e H b s . D i f f e r e n c e s in a b s o r p t i o n s p e c t r a , m o l e c u l a r w e i g h t s , l i g a n d b i n d i n g a f f i n i t i e s , etc. suggested that different organisms elaborated t h e i r own H b s , pres u m a b l y in t h e t i s s u e s where t h e y were l o c a l i z e d . H o w e v e r , H b s a n d p o r p h y r i n s are c e r t a i n l y a v a i l a b l e to a l l i n v e r t e b r a t e s from d i e t a r y s o u r c e s . C l e a r l y , in Rhodnius, a b l o o d s u c k i n g i n s e c t , t h e l o c a l i z a t i o n o f Hb in t h e eggs and m i d g u t is not i n d i c a t i v e o f Hb s y n t h e s i s b y t h e s e c e l l s ( W i g g l e s w o r t h , 1 9 4 3 ) . The p r e s e n c e o f Hbs w i t h i n c e l l s in an i n v e r t e b r a t e may s u g g e s t a s i t e o f s y n t h e s i s , s t o r a g e , b r e a k d o w n , or u t i l i z a t i o n . It i s t h e p u r p o s e o f t h i s s t u d y t o e x p a n d a p r e l i m i n a r y report (Bergtrom et al., 1975b) on t h e s i t e o f h e m o g l o b i n s y n t h e s i s , a s s e m b l y and s e c r e t i o n , a n d t h e s y n t h e s i s of s t a g e - s p e c i f i c H b s in Chironomus b y t h e use of s h o r t - t e r m t i s s u e and organ c u l t u r e .
III.
S y n t h e s i s of i n s e c t h e m o g l o b i n s - s i t e o f s y n t h e s i s
A n organ may manufacture a m a c r o m o l e c u l e f r o m s m a l l e r p r e c u r s o r m o l e c u l e s , or i t c a n s e q u e s t e r it in w h o l e or in p a r t f r o m d i e t a r y s o u r c e s . Organ c u l t u r e e x p e r i ments w e r e d e s i g n e d to d i s t i n g u i s h b e t w e e n t h e s e t w o a l t e r n a t i v e s in t h e c a s e of Chironomus Hb. 7.
Organs and culture
procedures
T i s s u e s from l a r v a e in t h e f o u r t h i n s t a r of C. thummi were d i s s e c t e d and c u l t u r e d i n C a n n o n ' s m o d i f i e d medium (Ringborg & R y d l a n d e r , 1 9 7 1 ) , s u p p l e m e n t e d w i t h e i t h e r ^ H - < / - a m i n o l e v u l i n i c a c i d (cf-ALA, a p o r p h y r i n p r e c u r s o r ) or a m i x t u r e o f
230
H E M O G L O B I N S Y N T H E S I S IN A N I N V E R T E B R A T E
t r i t i a t e d L-amino a c i d s (5.050 C i / m M and 1.3 C i / m M , r e s p e c t i v e l y ) . In d o u b l e l a b e l 3 4 e x p e r i m e n t s , H - c f - A L A and 1 C - L - a m i n o a c i d s ( 5 . 0 5 0 C i / m M a n d 4 9 C i / m M , r e s p e c t i v e l y ) were u s e d . S a l i v a r y g l a n d s from 3 5 l a r v a e , o r t h e g u t c o m p l e x ( i n c l u d i n g t h e m a l p h i g i a n t u b u l e s ) or f a t body f r o m 12 l a r v a e w e r e c u l t u r e d i n 50-75 jul o f medium containing the isotopes. The m o r p h o l o g y of t h e g u t c o m p l e x and s a l i v a r y g l a n d s has been d e s c r i b e d ( M i a l l and H a m m o n d , 1900; K l o e t z e l and L a u f e r , 1 9 6 9 ) , F a t body is an i r r e g u l a r s h e e t o f c e l l s s e v e r a l l a y e r s t h i c k s u s p e n d e d in t h e b o d y c a v i t y o f Chironomus. Another c o m p o n e n t o f t h e l a r v a l f a t b o d y , l y i n g b e t w e e n t h e e p i d e r m i s and t h e m u s c u l a t u r e o f t h e body w a l l , c o u l d not be i s o l a t e d w i t h o u t d i f f i c u l t y and w a s t h e r e f o r e , n o t i n c l u d e d in t h e c u l t u r e s . Fat body c e l l s adhere l o o s e l y to each o t h e r , and in phase c o n t r a s t m i c r o s c o p y , are h i g h l y v a c u o l a t e d . Some o f t h e s e v a c u o l e s are p i n k w h i l e o t h e r are y e l l o w , o r c o n t a i n i r r e g u l a r g r e e n i s h i n c l u s i o n s . T h i s w a s t h e major c e l l t y p e f o u n d in t h e c u r s o r y e x a m i n a t i o n o f t h e f a t body used in organ c u l t u r e s . Fat b o d y - l i k e c e l l s were a l s o t h e major c e l l component f o u n d f l o a t i n g in t h e hemolymph o f C . thummi, based o n p h a s e c o n t r a s t m i c r o s c o p y . The v a r i o u s t i s s u e s were each c u l t u r e d w i t h i s o t o p e s f o r 24 h o u r s . The a b i l i t y o f t h e f a t b o d y , gut c o m p l e x and s a l i v a r y g l a n d t o s y n t h e s i z e , a s s e m b l e and s e c r e t e H b s w a s d e t e r m i n e d by e x a m i n i n g t h e p r o t e i n r e l e a s e d i n t o t h e c u l t u r e medium d u r i n g the incubation period. 2.
Detection
of hemoglobin
synthesis
and
secretion
The m e d i u m , f r o m w h i c h t h e t i s s u e s w e r e removed b y low speed (121 χ g) c e n t r i f u g a t i o n , w a s d i a l y z e d a g a i n s t s e v e r a l c h a n g e s o f 0.009M PO4 (Na) b u f f e r ( 0 . 0 4 M N a C I , 0.04% K C N , p H 8.1) o v e r n i g h t to remove u n i n c o r p o r a t e d c o u n t s . In some c a s e s , t h e same r e s u l t w a s a c h i e v e d by Sephadex G-25 g e l f i l t r a t i o n . The r a d i o a c t i v i t y o f an a l i q u o t of e a c h s a m p l e w a s d e t e r m i n e d and a k n o w n amount ( 2 0 , 0 0 0 5 0 , 0 0 0 cpm) w a s p r e p a r e d f o r gel e l e c t r o p h o r e s i s . P r e p a r a t i o n i n v o l v e d t h e a d d i t i o n of 10 jul f r e s h u n l a b e l l e d f o u r t h i n s t a r hemolymph as marker, s u c r o s e t o i n c r e a se s a m p l e d e n s i t y , and bromophenol b l u e . D i s c o n t i n u o u s a c r y l a m i d e g e l s w e r e p r e p a r e d and run a t 125-200V for 2-4 hours a c c o r d i n g t o p r o c e d u r e s d e s c r i b e d e l s e w h e r e (Bergtrom et al., 1 9 7 5 a ) . N i n e h e m o g l o b i n s were v i s i b l e as red bands after e l e c t r o p h o r e s i s (Bergtrom et al., 1 9 7 5 a , b ) . The Hb p a t t e r n is c o m p a r a b l e t o t h a t o b t a i n e d by B r a u n i t z e r et al. ( 1 9 6 8 ) , e x c e p t t h a t t h e s e w o r k e r s d e t e c t an a d d i t i o n a l H b b a n d . The band p o s i t i o n s w e r e r e c o r d e d a n d t h e g e l s w e r e s l i c e d , d i g e s t e d in H2O2 at 9 0 ° C a n d c o u n t e d in A q u a s o l (or a f l u o r made from T o l u e n e , PPO & POPOP, and T r i t o n X-100) (Bergtrom et al., 1 9 7 5 a ) . The p o s i t i o n s o f t h e H b b a n d s and t h e d e t a i l s o f t h e e x p e r i m e n t are p r e s e n t e d in t h e f i g u r e s * 3.
Isotope
controls 3
3
Hbs w e r e l a b e l l e d e i t h e r w i t h H ° c T - A L A or H - a m i n o a c i d s . A f t e r a c i d - a c e t o n e t r e a t m e n t o f t h e sample (Bergtrom er al., 1975a) a p p r o x i m a t e l y 86% of t h e l a b e l 3 f r o m t h e H - a m i n o a c i d s is f o u n d in t h e p r o t e i n f r a c t i o n , w h i l e 87% o f t h e l a b e l 3 from t h e H - t / - A L A is r e m o v e d b y t h e a c i d - a c e t o n e . T h i s i n d i c a t e s t h a t t h e r e i s l i t t l e or no s i g n i f i c a n t i n t e r c o n v e r s i o n of p o r p h y r i n and p o l y p e p t i d e p r e c u r s o r s . When l a b e l l e d Hb bands are i n d i v i d u a l l y e l u t e d and r e - e l e c t r o p h o r e s e d a l o n g w i t h c o l d hemolymph m a r k e r s , r a d i o a c t i v i t y i s found m i g r a t i n g o n l y w i t h t h e band o r i g i n a l l y e l u t e d , a n d , in s m a l l e r a m o u n t s , i t s a d j a c e n t b a n d s (Bergtrom et al., 1 9 7 5 a ) . 231
H. L A U F E R
etal.
T h i s s u g g e s t s t h a t t h e r e is no heme e x c h a n g e d e t e c t a b l e b e t w e e n l a b e l l e d and unlabelled Hbs. 4.
Results
demonstrating
the site of hemoglobin
synthesis
in the fourth
larval
instar
1 4
Fat b o d y w a s c u l t u r e d w i t h ^ H - Z - A L A a n d C - a m i n o a c i d s f o r 2 4 h o u r s . A n e l e c t r o p h e r o g r a m o f t h e medium i s s h o w n in F i g u r e 1 . The ^ C - a m i n o a c i d incorp o r a t i o n p r o f i l e d e m o n s t r a t e s t h a t the n i n e H b bands present in f o u r t h i n s t a r hemolymph c o m i g r a t e w i t h t h e l a b e l . In a d d i t i o n , t h e r e are s e v e r a l a m i n o a c i d p e a k s b e t w e e n bands 1 and 2 . These are p r e s u m a b l y non-heme p r o t e i n s o r g l o b i n s , s i n c e t h e ^ H - c f - A L A p r o f i l e s h o w s no i n c o r p o r a t i o n in t h i s r e g i o n . T r i t i a t e d c f - A L A is i n c o r p o r a t e d into t h e n i n e Hb b a n d s , h o w e v e r , i n d i c a t i n g t h a t t h e amino a c i d incorp o r a t i o n i n t o t h e bands r e p r e s e n t s g l o b i n s y n t h e s i s . T h u s , a l l n i n e v i s i b l e H b s are s y n t h e s i z e d and s e c r e t e d by f a t body in organ c u l t u r e . A l t h o u g h t h e s e r e s u l t s can be e x p l a i n e d by a r t i f a c t s s u c h as t h e l o s s of t e r m i n a l amino a c i d s d u r i n g H b p u r i f i c a t i o n or i n c o m p l e t e p u r i f i c a t i o n , the l a b e l l i n g t e c h n i que used here does reveal t h a t t h e r e may be a t l e a s t 2 and 3 c o m p o n e n t s in Hb bands 6 and 4 , r e s p e c t i v e l y . In some e x p e r i m e n t s , as many as 1 4 / - A L A - l a b e l l e d heme p r o t e i n s m i g r a t i n g w i t h v i s i b l e red r e g i o n s of t h e gel can be d e t e c t e d . Braunitzer et al. (1971) s u g g e s t e d t h a t there may be more Hbs than t h o s e r e s o l v e d by e l e c t r o p h o r e s i s or ion e x c h a n g e c h r o m a t o g r a p h y . A l t h o u g h s a l i v a r y g l a n d s do i n c o r p o r a t e amino a c i d s in 24 hours i n t o p r o t e i n s on g e l e l e c t r o p h o r e s i s , none of t h e s e p r o t e i n s i n c o r p o r a t e ^ H - ^ - A L A ( F i g u r e s 2 a , 2 b , o n n e x t p a g e ) . Thus t h e s a l i v a r y g l a n d does not s y n t h e s i z e and secrete H b s . Based o n a 24-hour i n c o r p o r a t i o n of both ^ H - c f - A L A a n d ^ H - a m i n o a c i d s into gut t i s s u e ( i n c l u d i n g t h e M a l p h i g i a n t u b u l e s ) t h e r e i s a l o w l e v e l o f Hb s y n t h e s i s and s e c r e t i o n . T h i s s y n t h e s i s amounted t o about 5% of t h e t o t a l Hb s y n t h e s i s by f a t body and gut c o m p l e x from t h e same l a r v a e . If t h e f a t body b e t w e e n t h e body w a l l m u s c u l a t u r e and t h e e p i d e r m i s a l s o s y n t h e s i z e d H b , t h e Hb s y n t h e s i z e d by t h e gut may be s t i l l l e s s t h a n t h i s f i g u r e . We have f o u n d t h a t t h e body w a l l in t h e absence of t h e gut c o m p l e x , s a l i v a r y g l a n d s , and d i s s e c t a b l e f a t b o d y , is s t i l l c a p a b l e o f H b s y n thesis. These e x p e r i m e n t s d e m o n s t r a t e t h a t a) i s o l a t e d l a r v a l t i s s u e s in o r g a n c u l t u r e s y n t h e s i z e heme and g l o b i n from s m a l l p r e c u r s o r m o l e c u l e s , b) t h e heme and g l o b i n a r e a s s e m b l e d and s e c r e t e d by t h e s e t i s s u e s , c ) t h e f a t b o d y i s t h e major s i t e of s y n t h e s i s o f Hbs in t h e f o u r t h l a r v a l i n s t a r and d) t h e r e may be more t h a n 9 (or 10) H b s in C . thummi hemolymph s e p a r a b l e b y a c r y l a m i d e gel e l e c t r o p h o r e s i s . IV.
H e m o g l o b i n s in d e v e l o p m e n t
Changing patterns of hemoglobin synthesis during vertebrate development have been r e c o g n i z e d . Mouse embryonic h e m o g l o b i n s , d i s t i n c t from a d u l t h e m o g l o b i n s , h a v e been c h a r a c t e r i z e d by e l e c t r o p h o r e s i s and c h r o m a t o g r a p h y ( F a n t o n i et al., 1 9 6 7 ) . Fantoni et al., (1968) d e m o n s t r a t e d t h a t t h e s y n t h e t i c rates o f t h e t h r e e d i s t i n c t embryonic mouse h e m o g l o b i n s c h a n g e d u r i n g f e t a l d e v e l o p m e n t l e a d i n g t o a l t e r a t i o n s in hemoglobin p a t t e r n s . The a d u l t a l p h a - g l o b i n c h a i n i s a l s o p r e s e n t d u r i n g f e t a l l i f e , a n d c o n t r i b u t e s to f e t a l t e t r a m e r i c h e m o g l o b i n s ( d e s i g n a t e d E|, E|| and E | | | ) . The changeover f r o m embryonic t o a d u l t h e m o g l o b i n s r e f l e c t s an a l t e r a t i o n in t h e s i t e of hemoglobin s y n t h e s i s , l i v e r - d e r i v e d e r y t h r o i d c e l l s p r o d u c i n g adult hemoglobins w h i c h replace the y o l k sac derived erythroid c e l l s w h i c h were r e s p o n s i b l e f o r t h e e l a b o r a t i o n of f e t a l h e m o g l o b i n s . T h i s changeover o c c u r s a b o u t 232
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Fig. /. , L a b e l l e d p r e c u r s o r i n c o r p o r a t i o n i n t o p r o d u c t s o f f a t body s y n t h e s i s a n d s e c r e t i o n i n organ c u l t u r e f o r 2 4 h o u r s . I n c o r p o r a t i o n of ^ H - J - A L A (1 m C i / m l ) a n d ^ C - a m i n o a c i d s (0.5 m C i / m l ) . Fifteen per cent o f t h e d i a l y z e d sample were co-electrophoresed w i t h c o l d f o u r t h i n s t a r h e m o l y m p h m a r k e r s . P o s i t i o n s o f t h e H b m a r k e r s a r e i n d i c a t e d by a r r o w s . ( A d a p t e d from Bergtrom et al., 1975). m i d w a y t h r o u g h e m b r y o n i c d e v e l o p m e n t ( R i f k i n d et al., 1 9 6 9 ; Moore et al., 1 9 7 0 ; Marks et al., 1 9 7 2 ; I n g r a m , 1 9 7 2 ) 0 In f r o g s , t h e s h i f t from l a r v a l to a d u l t hemoglob i n s a l s o o c c u r s d u r i n g d e v e l o p m e n t (Weber, 1 9 6 7 ) , a p r o c e s s mediated by t h y r o i d g l a n d hormones. C h i r o n o m i d s , w h o s e l i f e c y c l e i n c l u d e a s e r i e s of a q u a t i c l a r v a l s t a g e s , c o n t a i n s e v e r a l e l e c t r o p h o r e t i c a l l y d i s t i n g u i s h a b l e h e m o g l o b i n s in t h e hemolymph (Braunitzer et al., 1968); Laufer and P o l u h o w i c h , 1 9 7 1 ; T i c h y , 1970; P l a g e n s et al., 1 9 7 0 ) . The e l e c t r o p h o r e t i c pattern a n d number o f h e m o g l o b i n s p r e s e n t i n a n y g i v e n i n s t a r i s s p e c i f i c and c h a n g e s w i t h d e v e l o p m e n t (Wulker et al., 1 9 6 9 ; M a n w e l l , 1966; Schin et al., 1974; Bergtrom e i al. , 1 9 7 5 b ) . /.
Procedures
for detecting
in vivo hemoglobin
synthesis
Hemolymph w a s c o l l e c t e d from l a r v a e o f C . thummi in t h e m i d - t h i r d i n s t a r and e a r l y and m i d - f o u r t h i n s t a r and d i l u t e d 1:1 w i t h KCN-PO4 buffer ( a b o v e ) . T w e n t y ,ul o f t h i s s o l u t i o n w e r e el e c t r o p h o r e s e d on a c r y l a m i d e g e l s prepared in a s l a b g e l a p p a r a t u s w i t h i n g r e d i e n t s i d e n t i c a l t o t h o s e u s e d in t h e t u b e g e l s in t h e p r e v i o u s e x p e r i m e n t s , in order to c o n f i r m t h e stage s p e c i f i c i t y o f Hbs o b s e r v e d by o t h e r w o r k ers and d e t e r m i n e t h e e a r l i e s t s t a g e at w h i c h c h a n g e s are d e t e c t a b l e .
233
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Fig. 2. L a b e l l e d p r e c u r s o r i n c o r p o r a t i o n i n t o p r o d u c t s o f s a l i v a r y g l a n d s y n t h e s i s a n d s y n t h e s i s and s e c r e t i o n i n organ c u l t u r e f o r 2 4 h o u r s . a) i n c o r p o r a t i o n o f ^ Η - a m i n o a c i d s ( 1 . 5 m C i / m l ) 3 b) i n c o r p o r a t i o n o f H - c f - A L A ( 1 . 0 m C i / m l ) In each e x p e r i m e n t t h e t o t a l s a m p l e , a f t e r d i a l y s i s , w a s c o - e l e c t r o p h o r e s e d w i t h c o l d f o u r t h i n s t a r h e m o l y m p h m a r k e r s . ( A d a p t e d f r o m Bergtrom et al., 1 9 7 5 ) .
The s y n t h e s i s o f H b s b y t h i r d a n d f o u r t h i n s t a r l a r v a e w a s e x a m i n e d in vivo. T h i r t y a n d 2 0 l a r v a e ( r e s p e c t i v e l y ) were p l a c e d in a g l a s s v i a l ( 1 " in d i a m e t e r ) c o n t a i n i n g 0.75 JJI o f tap w a t e r s u p p l e m e n t e d w i t h ^ H - a m i n o a c i d s o r ^ H - Z - A L A and mg o f f o o d (Laufer a n d W i l s o n , 1960) a n d a e r a t e d . The l a r v a e w e r e r i n s e d t h o roughly in i s o t o p e - f r e e r u n n i n g w a t e r , b l o t t e d a n d b l e d . The hemolymph c o l l e c t e d w a s c e n t r i f u g e d (12,000 g , 1 0 ' ) t o remove c e l l p a r t i c u l a t e s and d e b r i s , a n d t h e s u p e r n a t a n t d i a l y z e d i n t h e same manner a s t h e organ c u l t u r e medium s a m p l e s . A n a l i q u o t o f s a m p l e w i t h 20,000-50,000 c p m ( s u f f i c i e n t f o r e l e c t r o p h o r e t i c r e s o l u t i o n ) w a s made d e n s e w i t h a s u c r o s e c r y s t a l a n d e l e c t r o p h o r e s e d . T h e g e l s w e r e f i x e d and s l i c e d for s c i n t i l l a t i o n c o u n t i n g . To d e t e r m i n e if H b s l a b e l l e d o n l y i n t h e f o u r t h a n d n o t i n t h e t h i r d i n s t a r w e r e s y n t h e s i z e d de novo, t h e larvae s t i l l i n t h e t h i r d i n s t a r a f t e r a 2 4 - h o u r e x p o s u r e t o ^ H - a m i n o a c i d s were c o l l e c t e d a n d w a s h a b l e r a d i o a c t i v i t y w a s removed by t h o rough r i n s i n g 0 The i n c u b a t i o n w a s t h e n c o n t i n u e d f o r 10 d a y s w i t h a e r a t i o n in a p p r o x i m a t e l y 100 ml o f i s o t o p e - f r e e w a t e r c o n t a i n i n g f o o d . A t t h e e n d o f t h i s " c h a s e " p e r i o d , l a r v a e h a d a d v a n c e d 4-7 d a y s into t h e f o u r t h i n s t a r . T h e s e w e r e b l e d . The hemolymph c o l l e c t e d w a s c e n t r i f u g e d , d i a l y z e d a n d e l e c t r o p h o r e s e d a s d e s c r i b e d . Fourth i n s t a r larvae were s u b j e c t e d t o s i m i l a r " p u l s e - c h a s e " t r e a t m e n t s . In t h i s c a s e t h e c h a s e p e r i o d w a s s h o r t e n e d to f i v e d a y s in order t o a v o i d t h e o n s e t of metamorphosis.
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H E M O G L O B I N S Y N T H E S I S IN A N I N V E R T E B R A T E 2.
Results
demonstrating
stage-specific
hemoglobin
synthesis
The e l e c t r o p h o r e t i c p a t t e r n s o f H b s c o l l e c t e d f r o m l a r v a e in d i f f e r e n t d e v e l o p m e n t a l s t a g e s are compared in Figure 3 ( s c h e m a t i c e l e c t r o p h e r o g r a m ) . Hb in b a n d s 2 and 3 appear in t h e m i d - f o u r t h i n s t a r a n d are not p r e s e n t in s u f f i c i e n t q u a n t i t y t o be d e t e c t a b l e in e i t h e r t h i r d i n s t a r or " r e d - h e a d " f o u r t h i n s t a r l a r v a e . H b s in bands 1 and 4-9 are common t o a l l t h r e e s t a g e s . 11 η g e l s s t a i n e d w i t h C o o m a s s i e b r i l l i a n t b l u e , a f a i n t sharp band f r o m t h i r d a n d e a r l y f o u r t h i n s t a r hemolymph m i g r a t e s w i t h an Rf near band 2 Hb from m i d - f o u r t h i n s t a r h e m o l y m p h . To d e t e r m i n e i f d i f f e r e n t stage l a r v a e c o u l d s y n t h e s i z e t h e H b s f o u n d in t h e i r h e m o l y m p h in vivo, l a r v a e were a l l o w e d t o i n c o r p o r a t e ^ Η - a m i n o a c i d s f o r 2 4 hours in vivo d u r i n g t h e m i d - t h i r d and f o u r t h i n s t a r s . Larvae in t h e f o u r t h i n s t a r i n c o r p o r a t e d l a b e l into m a t e r i a l m i g r a t i n g w i t h Hb bands 1-9 o n e l e c t r o p h o r e t i c g e l s ( F i gure 4 ) . R e s u l t s of s i m i l a r e x p e r i m e n t s u s i n g ^ H - c f - A L A i n s t e a d of amino a c i d s s u g g e s t t h a t f o u r t h i n s t a r l a r v a e s y n t h e s i z e a l l of t h e v i s i b l e H b s f o u n d in t h e i r heF i g.
Θ
3
A
Β
c 2 . 5%
I
I O%
2 m MI
M
3
M
5
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7 8 9
m NN
mm
Θ Fig. 3. Slab g e l e l e c t r o p h o r e s i s o f w h o l e h e m o l y m p h from t h i r d ( A ) , e a r l y f o u r t h ( B , " r e d h e a d " ) , and mid-fourth (C) instar larvae. Unstained g e l s s h o w i n g resolution of red hemoglobin bands. Note boundary between 2.5% s t a c k i n g and 1 0 % running acrylamide g e l s . Hb b a n d s are n u m b e r e d c o n s e c u t i v e l y from 1 t o 9 . m o l y m p h . In a d d i t i o n , amino a c i d l a b e l l e d p r o t e i n s m i g r a t i n g b e t w e e n H b bands 1 and 2 are p r o b a b l y not heme p r o t e i n s . When m i d - t h i r d i n s t a r l a r v a e i n c o r p o r a t e 3|-|-amino a c i d s in vivo, b a n d s 1 and 4-9 are most h e a v i l y l a b e l l e d (Figure 5 ) . L a b e l is a l s o i n c o r p o r a t e d into m a t e r i a l m i g r a t i n g b e t w e e n bands 1 and 4 , w h i c h on t h e b a s i s of e x p e r i m e n t s w i t h ^ H - ^ - A L A , are non-heme proteins^ To d e t e r m i n e if H b bands 2 a n d 3 are s y n t h e s i z e d de novo in t h e f o u r t h i n s t a r , t h i r d i n s t a r l a r v a e were f e d ^ H - a m i n o a c i d s f o r 24 h o u r s , and t h e n " c h a s e d " , i n t h e a b s e n c e of i s o t o p e , into t h e f o u r t h i n s t a r . The r e s u l t s ( F i g u r e 6) s h o w t h a t o n l y bands 1 and 4 - 8 (and p o s s i b l y 9 ) remain h e a v i l y l a b e l l e d a f t e r t h e c h a s e . Since t h e l a r v a e w e r e b l e d i n t h e f o u r t h i n s t a r a l l n i n e H b s are p r e s e n t o n t h e g e l . In a d d i t i o n , t h e l a b e l in non-heme p r o t e i n s b e t w e e n b a n d s 1 a n d 4 i s near b a c k ground l e v e l s , s u g g e s t i n g t h a t non-heme p r o t e i n s are degraded d u r i n g t h e c h a s e
235
H. L A U F ER et al.
Fig. 4. D i s c g e l s o f l a b e l l e d h e m o l y m p h p r o t e i n s from f o u r t h i n s t a r l a r v a e i n c u b a t e d in vivo 3 w i t h H - a m i n o a c i d s (0.1 m C i / m l ) for 24 hours. Incorporation p r o f i l e of s t a c k i n g gel i s n o t s h o w n . A p p r o x i m a t e l y 3 0 % o f t h e s a m p l e w a s e l e c t r o p h o r e s e d . A r r o w s i n d i c a t e H b band positions. Fig. 5. D i s c g e l s o f l a b e l l e d h e m o l y m p h p r o t e i n s from t h i r d i n s t a r l a r v a e i n c u b a t e d in vivo 3 w i t h H - a m i n o a c i d s (0.1 m C i / m l ) for 24 hours. A p p r o x i m a t e l y 30% of t h e sample w a s electrophoresed. p e r i o d . Two minor peaks in t h i s r e g i o n (2X b a c k g r o u n d ) may r e f l e c t t h e r e u t i l i z a t i o n of the p r o d u c t s of d e g r a d a t i o n o f non-heme p r o t e i n s in t h e s y n t h e s i s of bands 2 and 3 . The p o s s i b i l i t y e x i s t s , h o w e v e r , t h a t H b s 2 and 3 are s y n t h e s i z e d in s m a l l q u a n t i t i e s d u r i n g t h e t h i r d instar. Thus f a r , t h e r e s u l t s i n d i c a t e t h a t p a r t i c u l a r Hbs are s y n t h e s i z e d at s p e c i f i c dev e l o p m e n t a l s t a g e s of Chironomus, and s u g g e s t t h a t Hbs 2 and 3 , p r e s e n t in s u f f i c i e n t q u a n t i t y t o be d e t e c t e d w i t h o u t s t a i n i n g o n l y in f o u r t h i n s t a r l a r v a e , are o n l y s y n t h e s i z e d in s i g n i f i c a n t amounts in t h e f o u r t h i n s t a r and are p r o b a b l y " n e w " p r o t e i n s , not a g g r e g a t e s or a l t e r a t i o n s of H b s p r e s e n t a l r e a d y in t h e t h i r d i n s t a r . Further, " p u l s e - c h a s e " e x p e r i m e n t s s u g g e s t t h a t H b s s y n t h e s i z e d in t h e t h i r d i n s t a r are not turned over as r a p i d l y as non-heme p r o t e i n s s y n t h e s i z e d d u r i n g t h e same 24-hour p u l s e . The l a t t e r o b s e r v a t i o n w a s c o n f i r m e d in p u l s e - c h a s e e x p e r i ments c o n d u c t e d on l a r v a e e n t i r e l y w i t h i n t h e f o u r t h i n s t a r (Figure 7 ) . A f t e r t h e 24 hour l a b e l l i n g p e r i o d in e i t h e r t h i r d or f o u r t h i n s t a r l a r v a e , t h e " c h a s e " p e r i o d s i n c l u d e d at l e a s t 5-7 d a y s in t h e f o u r t h i n s t a r . Thus H b s s y n t h e s i z e d d u r i n g t h e l a b e l i n c u b a t i o n in both i n s t a r s are e x p o s e d t o f o u r t h i n s t a r hemolymph f o r s i m i l a r l e n g t h s o f t i m e . T h i s s u p p o r t s t h e c o n c l u s i o n t h a t H b s 2 a n d 3 are s y n t h e s i z e d de novo in t h e f o u r t h i n s t a r . Further, a m i n o a c i d a n a l y s e s of H b s 1-9 s u g g e s t t h a t each i s a u n i q u e p r o t e i n (Braun et al., 1 9 6 8 ) . H o w e v e r , v a r i a t i o n s in the hormonal m i l i e u in d i f f e r e n t p e r i o d s of t h e f o u r t h i n s t a r a f f e c t i n g t h e s e l e c t i v e or s p e c i f i c a g g r e g a t i o n of Hbs 4-9 to r e s u l t in t h e a p p e a r a n c e o f Hbs 2 and 3 c a n n o t be r u l e d o u t .
236
H E M O G L O B I N S Y N T H E S I S IN A N I N V E R T E B R A T E The o b s e r v a t i o n t h a t f a t body s y n t h e s i z e s H b is not u n r e a s o n a b l e in l i g h t of t h e f a c t t h a t i n s e c t f a t body s e c r e t e s many b l o o d p r o t e i n s ( S h i g m a t s u , 1 9 5 8 ; L a u f e r , 1960). Evidence, though i n c o n c l u s i v e , suggest that fat c e l l s of crustaceae ( e . g . , Daphnia) may s y n t h e s i z e t h e heme o f Hb ( S m a r i d g e , 1956). A t l e a s t t w o H b s ( e l e c t r o p h o r e t i c bands 2 and 3 ) are not s y n t h e s i z e d in t h e m i d - t h i r d i n s t a r , or are s y n t h e s i z e d in i n s i g n i f i c a n t a m o u n t s . The s y n t h e s i s of t h e s e t w o H b s , p r e s e n t in t h e f o u r t h i n s t a r , is p r e s u m a b l y induced or s t i m u l a t e d by a f a c t o r ( s ) a c t i n g on t h e s i t e o f s y n t h e s i s . S i n c e t h e i r appearance i s c o r r e l a t e d w i t h t h e l a s t l a r v a l m o l t , i t i s p o s s i b l e t h a t e c d y s o n e s p l a y a r o l e , d i r e c t or i n d i r e c t , i n t h i s p r o c e s s . I t is of i n t e r e s t t h a t H b s are d e g r a d e d , and Hb s y n t h e s i s is p r e s u m a b l y t u r n e d o f f a t m e t a m o r p h o s i s (Laufer and P o l u h o w i c h , 1 9 7 1 ; Schin et aL, 1974). These p r o c e s s e s may a l s o be m e d i a t e d by e c d y s o n e s .
Fig.
7
i .
Top
Slice #
Bottom
Top
Slice #
i
Bottom
Fig. 6. D i s c g e l s o f l a b e l l e d h e m o l y m p h p r o t e i n s f r o m f o u r t h i n s t a r l a r v a e a f t e r a 2 4 - h o u r pulse exposure to ^H-amino a c i d s (0.1 m C i / m l ) d u r i n g t h e t h i r d instar a n d a 240-hour chase in t h e a b s e n c e o f i s o t o p e . Fig. 7. L a b e l l e d h e m o l y m p h p r o t e i n s f r o m f o u r t h i n s t a r l a r v a e a f t e r a 2 4 - h o u r p u l s e e x p o s u r e t o 3|-|-amino a c i d s ( 0 . 1 m C i / m l ) d u r i n g t h e f o u r t h i n s t a r a n d a 120-hour c h a s e in t h e a b s e n c e of i s o t o p e . V.
D i s c u s s i o n and c o n c l u s i o n s
The f i r s t c a s e of h e m o g l o b i n s y n t h e s i s b y an i n v e r t e b r a t e is d e m o n s t r a t e d b o t h and by t h e in vivo i n c o r p o r a t i o n of l a b e l l e d amino a c i d s a n d / - A L A by Chironomus t h e i r i n c o r p o r a t i o n into H b s in organ c u l t u r e s . Fat body is t h e major s i t e o f Hb s y n t h e s i s in f o u r t h i n s t a r l a r v a e and is c a p a b l e of s y n t h e s i z i n g and s e c r e t i n g a l l of t h e e l e c t r o p h o r e t i c a l l y d i s t i n g u i s h a b l e H b s n o r m a l l y s y n t h e s i z e d by f o u r t h i n s t a r larv a e in vivo. The l a b e l l i n g t e c h n i q u e i n d i c a t e s t h a t s e v e r a l of t h e v i s i b l e H b s on a c r y l a m i d e g e l s may be made up o f 2-3 c o m p o n e n t s . It is a l l e g e d ( B r a u n i t z e r et aL, 1971) on t h e b a s i s of t e r m i n a l a m i n o a c i d a n a l y s e s , t h a t t h e r e may be as many as 4 0 d i f f e r e n t H b s in Chironomus ( s e e a b o v e ) . A l s o , a f t e r 2 4 hours of i s o t o p e incor-
237
H. L A U F E R et al. p o r a t i o n , t h e l e v e l of l a b e l l i n g d i f f e r s among the h e m o g l o b i n s . T h e s e d i f f e r e n c e s a l s o v a r y b e t w e e n e x p e r i m e n t s , p e r h a p s d u e to d i f f e r e n c e s in t h e p h y s i o l o g i c a l or d e v e l o p m e n t a l s t a t e of t h e l a r v a e . D i f f e r e n c e s in p e a k h e i g h t , may be t h e r e s u l t o f d i f f e r e n t i a l r a t e s of s y n t h e s i s of t h e Hbs or may be due to d i f f e r e n c e s in t h e s p e c i f i c a c t i v i t i e s of the amino a c i d s in the i s o t o p e m i x t u r e , or in t h e g l o b i n p o l y peptides. The c h a n g e in t h e e l e c t r o p h o r e t i c p a t t e r n o f H b s in Chironomus during development o r i g i n a l l y d e m o n s t r a t e d by Wulker et a / . , (1969) and M a n w e l l (1966) has been c o n f i r m e d . H e m o g l o b i n bands 2 and 3 are p r e s e n t o n l y in m i d - f o u r t h i n s t a r l a r v a e , and are a p p a r e n t l y not s y n t h e s i z e d in t h e t h i r d i n s t a r . " P u l s e - c h a s e " e x p e r i m e n t s s u g g e s t t h a t Hb bands 2 and 3 are s y n t h e s i z e d de novo in t h e f o u r t h i n s t a r . The t e m p o r a l c o r r e l a t i o n b e t w e e n t h e s y n t h e s i s a n d a p p e a r a n c e of t h e t w o a d d i t i o n a l H b s and m o l t i n g f r o m t h e t h i r d t o t h e f o u r t h i n s t a r s t r o n g l y s u g g e s t s t h a t c e r t a i n a s p e c t s of Hb s y n t h e s i s may be under hormonal ( e . g . , e c d y s o n e ) r e g u l a t i o n . Low l e v e l s of Hb s y n t h e s i s and s e c r e t i o n b y g u t t i s s u e s c a n be e x p l a i n e d in t w o w a y s . The g u t may have been a c t i v e in H b s y n t h e s i s a t an e a r l i e r l a r v a l s t a g e , e x h i b i t i n g o n l y r e s i d u a l Hb s y n t h e s i s in t h e f o u r t h i n s t a r . A l t e r n a t i v e l y , t h e r e may b e a p o p u l a t i o n of c e l l s common to both g u t and f a t b o d y , b u t p r e s e n t i n g r e a t e r q u a n t i t y in t h e l a t t e r . These c e l l s might s y n t h e s i z e H b . The p r e s e n c e of f r e e f l o a t i n g c e l l s in t h e hemolymph r e s e m b l i n g f a t body c e l l s have been noted in Chironomus. These or other c e l l t y p e s ( e . g . , o e n o c y t e s ) may adhere to v a r i o u s organs in t h e b o d y c a v i t y . Fat body c e l l s have in f a c t been f o u n d a s s o c i a t e d w i t h t h e g u t (K. J u d y and E. M a r k s , personal c o m m u n i c a t i o n ) . In v i e w of the p r e s e n c e o f Hbs in v a r i o u s i n v e r t e b r a t e t i s s u e s and c e l l s , it w o u l d be u s e f u l t o a p p l y r a d i o i s o t o p e t e c h n i q u e s to d e t e r m i n e if such c e l l s are s i t e s of H b s y n t h e s i s . Of p a r t i c u l a r i n t e r e s t are t h e s p e c i a l i z e d c e l l s d e r i v e d f r o m f a t body in Castrophilus ( t r a c h e a l c e l l s ) w h i c h c o n t a i n H b ( D i n o l e s c u , 1 9 3 2 ) . S i m i l a r Hbc o n t a i n i n g c e l l s are f o u n d in t h e n o n - p a r a s i t i c n o t o n e c t i d Buenoa (Bare, 1 9 2 8 ) . Our f i n d i n g s in Chironomus s u g g e s t t h a t Hb in t h e s e o t h e r s p e c i e s may be s y n t h e s i z e d by f a t b o d y - d e r i v e d c e l l s . VI.
Acknowledgment
The a u t h o r s g r a t e f u l l y a c k n o w l e d g e the h o s p i t a l i t y of P r o f e s s o r s J . E . Edstrom and H.O. H a l v o r s o n of t h e K a r o l i n s k a I n s t i t u t e and Brandeis U n i v e r s i t y , r e s p e c t i v e l y , where the e a r l y p h a s e s of t h i s w o r k were i n i t i a t e d . We a l s o a c k n o w l e d g e t h e t e c h n i c a l a s s i s t a n c e of Thomas G o r a l s k i and Henry D a n z i g e r . T h i s research w a s supported in p a r t by g r a n t s from the NSF, the U n i v e r s i t y of C o n n e c t i c u t Research F o u n d a t i o n , an N I H s p e c i a l f e l l o w s h i p and a NATO s e n i o r fellowship. VI.
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C h a p t e r 20
DISSOCIATION AND REAGGREGATION OF FAT BODY CELLS DURING INSECT METAMORPHOSIS H . OBERLANDER
I.
Introduction
II.
Influence of beta-ecdysone
........
.....
241 242
III.
Effect of larval age
242
IV.
I n f l u e n c e o f j u v e n i l e hormone
244
V.
S p e c i f i c i t y o f f a t body a d h e s i o n t o i m a g i n a i d i s k s
244
VI.
Conclusions
,
245
VII.
References
I.
246
Introduction
Insect f a t body undergoes a d r a m a t i c t r a n s f o r m a t i o n d u r i n g m e t a m o r p h o s i s . The c o n n e c t i v e t i s s u e sheath s u p p o r t i n g t h e f a t b o d y i s d e s t r o y e d , and t h e c o m p a c t s t r a n d s of l a r v a l f a t body become l o o s e l y c o h e s i v e . Many of t h e d i s s o c i a t e d c e l l s t h e n r e a s s e m b l e t o form new compact a d u l t t i s s u e ( W a l k e r , 1966; W a l t e r s , 1 9 6 9 , 1972). Reaggregation of d i s s o c i a t e d c e l l s has been t h e s u b j e c t of s c i e n t i f i c i n q u i r y s i n c e W i l s o n p u b l i s h e d h i s o b s e r v a t i o n s o f sponges in 1907. S p e c i f i c c e l l a g g r e g a t i o n in s p o n g e s c o n t i n u e s t o be s t u d i e d w i t h in vitro t e c h n o l o g y ( H u m p h r e y s , 1 9 7 0 ) . S i m i l a r l y , t h e in vitro r e a g g r e g a t i o n of d i s s o c i a t e d v e r t e b r a t e e m b r y o n i c c e l l s has been i n v e s t i g a t e d i n t e n s i v e l y (See r e v i e w s b y Z w i l l i n g , 1968; and R o t h , 1 9 7 3 ) . H e n c e , it seemed l i k e l y t h a t d i s s o c i a t i o n and r e a g g r e g a t i o n of i n s e c t f a t b o d y c e l l s s h o u l d a l s o be amenable t o s t u d y in vitro. Some p r o g r e s s has been made in t h i s d i r e c t i o n . Walters and W i l l i a m s (1966) d e m o n s t r a t e d t h a t d i s s o c i a t e d p u p a l f a t body c e l l s of s a t u r n i i d moths reaggregate when c u l t u r e d in p u p a l b l o o d . F u r t h e r m o r e , J u d y and Marks (1971) o b s e r v e d t h a t f a t body a d h e r i n g t o h i n d g u t o f Manduca sexta d i s p e r s e d in vitro. In t h e c o u r s e of i n v e s t i g a t i n g t h e a c t i o n o f b e t a - e c d y s o n e on i m a g i n a i d i s k s o f Plodia interpunctella (Hübner) in vitro, w e n o t e d t h a t " t h e f a t body c e l l s d i s s o c i a t e d after s e v e r a l d a y s in vitro and surrounded t h e d i s k s . . . " ( D u t k o w s k i and O b e r l a n d e r , 1973). T h u s , there w a s an o p p o r t u n i t y t o s t u d y f o r t h e f i r s t t i m e t h e e f f e c t s o f hormones on f a t body d i s s o c i a t i o n in vitro. T h i s paper reports on t h e e f f e c t s o f betae c d y s o n e and j u v e n i l e hormone on t h e d i s s o c i a t i o n o f P. interpunctella larval fat b o d y . In a d d i t i o n , t h e s p e c i f i c i t y o f t h e r e a g g r e g a t i o n of f a t b o d y c e l l s o n t h e c u l t u r e d imaginai d i s k s was examined.
241
H. II.
OBERLANDER
I n f l u e n c e of b e t a - e c d y s o n e
The s e n s i t i v i t y of t h e f a t body to hormone w a s s t u d i e d by e x a m i n i n g t h e e f f e c t s of d i f f e r e n t c o n c e n t r a t i o n s of b e t a - e c d y s o n e . Fat body w a s d i s s e c t e d f r o m f i n a l i n s t a r l a r v a e ( w e i g h e d 15 t o 18 mg) and c u l t u r e d (5 s h e a t h s / d i s h ) in 1 ml of m o d i f i e d G r a c e ' s medium as p r e v i o u s l y d e s c r i b e d (Oberlander et aL, 1 9 7 3 ) . In a l l e x p e r i m e n t s t h e f a t body w a s c u l t u r e d 24 hours b e f o r e t h e a d d i t i o n of hormone. A l s o , e a c h expedient i n c l u d e d matched c o n t r o l s to w h i c h s o l v e n t (10% e t h a n o l ) w a s a d d e d . In no c a s e d i d t h e c o n t r o l f a t body d i s s o c i a t e . The degree o f d i s s o c i a t i o n i s based on e s t i m a t e s from e x a m i n a t i o n w i t h a d i s s e c t i n g m i c r o s c o p e . A s reported in T a b l e 1 , c o n c e n t r a t i o n s of hormone as l o w as 0.005 p g / m l w e r e eff e c t i v e in s t i m u l a t i n g f a t body d i s s o c i a t i o n , a l t h o u g h c o n c e n t r a t i o n s of 0.05 p g / m I or greater had a more p r o n o u n c e d e f f e c t (See F i g . 1 ) . E x t e n s i v e d i s s o c i a t i o n w a s not o b s e r v e d f o r f a t body i n c u b a t e d w i t h 0.05 Ajg/ml (or greater) b e t a - e c d y s o n e u n t i l t h e second w e e k of c u l t u r e ; a f u l l r e s p o n s e t o 0.005 p g / m l t o o k t h r e e w e e k s .
TABLE 1 Concentration
of Beta-ecdysone
and Fat Body (15-18 mg larvae)
Concentration beta-ecdysone ( u g / m l )
dissociation.
Degree o f d i s s o c i a t i o n * 1 week
2 weeks
0
0 +
0 +
0
0.005
++ +
++
3 weeks
+++
0.05 0.2
+
+++
+++
0.5
+
+++
+++
5.0
+
++
+++
+
d e n o t e s 10-25%,
++
d e n o t e s 25-50%,
+++ d e n o t e s 50-75%. Each t r e a t m e n t w a s r e p l i c a t e d 3 t i m e s .
The p r e c e d i n g e x p e r i m e n t s d e m o n s t r a t e d t h a t t h e f a t body is s e n s i t i v e t o l o w c o n c e n t r a t i o n s of b e t a - e c d y s o n e (0.005 - p g / m l ) , a l t h o u g h t h e f u l l r e s p o n s e t o o k a c o n s i d e r a b l e t i m e t o d e v e l o p . In v i e w o f t h i s l a t e n t p e r i o d , t h e e f f e c t s o f hormone t r e a t m e n t s of l i m i t e d d u r a t i o n w e r e e x a m i n e d . T h e m i n i m u m e f f e c t i v e d u r a t i o n of hormone t r e a t m e n t (0.5 p g / m l ) w a s 15 h o u r s . A 48-hour e x p o s u r e w a s r e q u i r e d to o b t a i n a pronounced e f f e c t , a l t h o u g h even 48 hours w a s l e s s e f f e c t i v e t h a n c o n t i nuous e x p o s u r e to hormone (See Table 2 ) . When t h e f a t body w a s c u l t u r e d w i t h b e t a - e c d y s o n e longer t h a n 48 h o u r s , it became f r a g i l e and b r o k e up d u r i n g h a n d l i n g .
III.
E f f e c t o f l a r v a l age
A c o m p a r i s o n of t h e degree of d i s s o c i a t i o n of f a t body from f i n a l - i n s t a r l a r v a e o f d i f f e r e n t a g e s i s shown in T a b l e 3 . The f a t body w a s f u l l y c o m p e t e n t t o d i s s o c i a t e in r e s p o n s e t o b e t a - e c d y s o n e at any t i m e d u r i n g t h e f i n a l l a r v a l i n s t a r . F u r t h e r m o r e ,
242
INSECT M E T A M O R P H O S I S
Fig.
1A.
A s h e a t h of f a t b o d y c u l t u r e d f o r t w o w e e k s in m o d i f i e d G r a c e ' s m e d i u m .
Fig.
1B.
Fat body c u l t u r e d t w o w e e k s in m e d i u m c o n t a i n i n g 0.5 u g / m l b e t a - e c d y s o n e .
TABLE 2 Duration
of Exposure
to Beta-ecdysone
(0.5%jg/ml)
and Fat Body (15-18 mg larvae)
Dissociation.
D e g r e e of d i s s o c i a t i o n *
Exposure to
2 weeks
3 weeks
0
0
0
2
0
0
0
6
0
0
0
beta-ecdysone (hours)
1 week
0
15
+
+
+
24
+
+
+
48
+
+
++
+
+++
+++
Continuous +
denotes 10-25%
++
denotes 25-50%
+++
denotes 50-75%
Each t r e a t m e n t w a s r e p l i c a t e d 3 t i m e s .
243
H.OBERLANDER TABLE 3 Effect
of Fat Body Age on Beta-ecdysone
(0.5 pg/m/)-/nduced
Dissociation.
Degree o f d i s s o c i a t i o n *
Larval Age: 1 week
2 weeks
11 ( 8 - 11 mg)
+
++
+++
12 ( 1 2 - 15 mg)
+
++
+++
13 ( 1 5 - 18 mg)
+
+++
+++
1 4 (wandering)
+
++
+++
D a y s a f t e r egg l a y i n g
3 weeks
+ d e n o t e s 10-25% ++ d e n o t e s 2 5 - 5 0 % +++ d e n o t e s 50-75% Each t r e a t m e n t w a s r e p l i c a t e d 3 t i m e s . M a t c h e d c o n t r o l c u l t u r e s o f f a t body a t each a g e d i d n o t d i s s o c i a t e in t h e a b s e n c e o f b e t a - e c d y s o n e .
f a t body from w a n d e r i n g - s t a g e larvae had not r e c e i v e d a s u f f i c i e n t hormonal s t i m u l u s in vivo b e c a u s e it d i d not d i s s o c i a t e in vitro u n l e s s a d d i t i o n a l b e t a - e c d y s o n e w a s p r e s e n t . T h i s is c o n s i s t e n t w i t h t h e t i m i n g of f a t body d i s s o c i a t i o n d u r i n g metamorphosis. IV.
I n f l u e n c e of j u v e n i l e hormone
Larval f a t body does not d i s s o c i a t e d u r i n g t h e l a r v a l m o l t c y c l e , t h o u g h f a t body from n e w l y m o l t e d l a s t - i n s t a r l a r v a e is r e s p o n s i v e t o b e t a - e c d y s o n e in vitro. E x p e r i j u v e n i l e hormone ( m i x e d isomers of ments were t h e r e f o r e c o n d u c t e d w i t h Cecropia m e t h y l - 1 0 p1 1 - e p o x y - 7 - e t h y l - 3 , 1 1 - d i m e t h y l - 2 , 6 - t r i d e c a d i e n o a t e ) t o d e t e r m i n e w h e t h e r j u v e n i l e hormone is d i r e c t l y r e s p o n s i b l e for p r e v e n t i n g h i s t o l y s i s of l a r v a l f a t body. Larvae t h a t w e i g h e d 12-15 mg w e r e used as a source of f a t body b e c a u s e a t t h i s s t a g e the i m a g i n a i d i s k s are s e n s i t i v e to j u v e n i l e hormone (Oberlander and T o m b l i n , 1 9 7 2 ) . In a c o n t r o l e x p e r i m e n t f a t body w a s i n c u b a t e d in c u l t u r e medium w i t h e i t h e r j u v e n i l e hormone (200 jug/mI ) or an e q u i v a l e n t amount of s o l v e n t (DMSO). N e i t h e r t r e a t m e n t w a s t o x i c , nor w a s d i s s o c i a t i o n i n d u c e d . N e x t , f a t body a n d i m a g i n a i w i n g d i s k s were c u l t u r e d in a g l a s s m i c r o d i s h c o n t a i n i n g 200 p\ of m o d i f i e d G r a c e ' s m e d i u m , 12-jjg j u v e n i l e hormone and I 6 0 p g of b i n d i n g p r o t e i n ( i s o l a t e d from Manduca sexta; c o u r t e s y of Prof. J o h n L a w , U n i v e r s i t y o f C h i c a g o ) . [ The b i n d i n g p r o t e i n e n h a n c e s t h e e f f e c t of j u v e n i l e hormone, p r o b a b l y b y r e t a r d i n g i t s d e g r a d a t i o n (Sanburg et ai., 1 9 7 5 ) J . A f t e r 24 h o u r s , 0.1 p g b e t a - e c d y s o n e w a s added to t h e c u l t u r e s . By three w e e k s t h e f a t b o d y c u l t u r e d w i t h b e t a - e c d y s o n e a l o n e had d i s s o c i a t e d ; t h e r e w a s l i t t l e or no d i s s o c i a t i o n in t h e c u l t u r e s t r e a t e d w i t h j u v e n i l e hormone. The imaginai d i s k s s e r v e d as an i n t e r n a l c o n t r o l f o r t h e e f f e c t i v e n e s s of j u v e n i l e hormone; 80% p r o d u c e d c u t i c l e in r e s p o n s e to b e t a - e c d y s o n e a l o n e , but none made c u t i c l e in t h e p r e s e n c e of b e t a - e c d y s o n e and j u v e n i l e hormone.
V.
S p e c i f i c i t y of f a t body a d h e s i o n t o i m a g i n a i d i s k s
Larval f a t body t h a t d i s s o c i a t e s in vitro reaggregates s p o n t a n e o u s l y a t t h e s u r f a c e o f t h e l i q u i d a l o n g t h e edge of t h e c u l t u r e d i s h . Ilf i m a g i n a i d i s k s are p r e s e n t , t h e y
244
INSECT M E T A M O R P H O S I S
become a f o c u s for f a t body r e a g g r e g a t i o n . The s p e c i f i c i t y of a d h e s i o n o f f a t body (15-18 mg larvae) t o t h e i m a g i n a i d i s k s w a s t h e r e f o r e a s s e s s e d by i n c u b a t i n g w i t h b e t a - e c d y s o n e (0.5 J i g / m l ) and (a) w i n g d i s k s ; (b) w i n g d i s k s t h a t w e r e f i x e d f o r 5 m i n u t e s in 70% e t h a n o l ; ( c ) p i e c e s of m i d g u t ; or (d) c h i p s of agar. In t h e s e e x p e r i ments t h e f a t b o d y d i s s o c i a t e d b y t h e s e c o n d w e e k a n d b y t h e t h i r d w e e k w e r e a d h e r i n g t o t h e midgut or normal w i n g d i s k s . There w a s no a d h e s i o n t o t h e agar or the f i x e d w i n g t i s s u e . Hence, d i s s o c i a t e d fat body c e l l s w i l l eventually adhere to e a c h other and t o other l i v e t i s s u e t h a t may be p r e s e n t as a f o c u s f o r r e a g g r e g a t i o n .
VI.
Conclusions
The e x p e r i m e n t s reported in t h i s paper d e m o n s t r a t e t h a t f a t body of P. interpunctella l a r v a e d i s s o c i a t e s in r e s p o n s e t o b e t a - e c d y s o n e , b u t is i n h i b i t e d f r o m d o i n g s o in t h e p r e s e n c e of j u v e n i l e hormone. The d e v e l o p m e n t a l b e h a v i o r o f P. interpunctella f a t body in vitro i s d i s t i n g u i s h e d from t h a t of w i n g i m a g i n a i d i s k s b e c a u s e f u l l c o m p e t e n c e t o r e s p o n d t o b e t a - e c d y s o n e is p r e s e n t t h r o u g h o u t t h e l a s t l a r v a l i n s t a r ; i n t h e c a s e of w i n g d i s k s it appears o n l y a t m i d - i n s t a r ( O b e r l a n d e r and T o m b l i n , 1972). T h e r e f o r e , w e may c o n c l u d e t h a t l a r v a l f a t b o d y d o e s not d i s s o c i a t e u n t i l metamorp h o s i s b e g i n s b e c a u s e j u v e n i l e hormone p r e v e n t s t h e f a t body f r o m r e s p o n d i n g t o b e t a - e c d y s o n e , even t h o u g h it i s c o m p e t e n t to do s o . The l o w c o n c e n t r a t i o n (0.005 x j g / m l ) o f b e t a - e c d y s o n e t o w h i c h f a t b o d y r e s p o n d s makes t h i s one of t h e most s e n s i t i v e e c d y s o n e r e s p o n s e s e x a m i n e d in vitro. H o w e v e r , t h e long e x p o s u r e r e q u i r e d t o a c h i e v e a f u l l e f f e c t i n d i c a t e s t h a t t h e a c t i o n o f b e t a - e c d y s o n e on t h i s t i s s u e is c o n s i s t e n t w i t h t h e t h e o r y of O h t a k i et al. ( 1 9 6 8 ) : " c o v e r t " e f f e c t s of t h e hormone a c c u m u l a t e w i t h i n t h e t a r g e t t i s s u e u n t i l t h r e s h o l d i s reached f o r an " o v e r t " r e s p o n s e . T h u s , t h e f a c t t h a t d u r i n g m e t a m o r p h o s i s w i n g d i s k d e v e l o p m e n t p r o c e e d s in a d v a n c e of f a t b o d y h i s t o l y s i s c a n be e x p l a i n e d b y experit h e d i f f e r e n t s e n s i t i v i t i e s to b e t a - e c d y s o n e d e m o n s t r a t e d in the in vitro than m e n t s . Imaginai d i s k s r e q u i r e a s h o r t e r e x p o s u r e to b e t a - e c d y s o n e in vitro d o e s f a t body. A l s o , d i s k s from w a n d e r i n g l a r v a e begin to m e t a m o r p h o s e in vitro w i t h o u t a d d i t i o n a l hormone, but f a t body from w a n d e r i n g l a r v a e does n o t d i s s o c i a t e without ecdysone. The u l t r a s t r u c t u r a l c h a n g e s t h a t accompany f a t body h i s t o l y s i s in vivo h a v e b e e n d e s c r i b e d (Walker, 1 9 6 6 ) , but b i o c h e m i c a l c h a n g e s have not been s t u d i e d . W a l t e r s (1974) s h o w e d t h a t f a t body c o u l d be d i s s o c i a t e d by t r e a t m e n t w i t h t r y p s i n , p r o n a s e or c o l l a g e n a s e . H o w e v e r , t r y p s i n d i g e s t i o n w a s i n c o m p l e t e , and p r o n a s e damaged t h e c e l l s . Collagenase d i s s o c i a t e d the fat body completely w i t h o u t damaging the c e l l s . W a l t e r s s u g g e s t e d that a t i s s u e c o l l a g e n a s e may be r e s p o n s i b l e f o r f a t body h i s t o l y s i s during metamorphosis. Manifestly, the action of beta-ecdysone on t h e a c t i v i t y o f s u c h e n z y m e s s h o u l d be i n v e s t i g a t e d . Further, i t is p o s s i b l e t h a t t h e i n h i b i t o r y a c t i o n o f j u v e n i l e hormone on f a t body d i s s o c i a t i o n c a n be e x p l a i n e d in t e r m s of s u p p r e s s i n g s u c h enzyme a c t i v i t i e s . Walters (1969) a l s o f o u n d t h a t r e a g g r e g a t i o n o f f a t body c e l l s o c c u r e d e i t h e r in t h e p r e s e n c e of hemocytes or w i t h g e n t l e a g i t a t i o n . In t h e P. interpunctella cultures the f a t body w a s w a s h e d s e v e r a l t i m e s and c u l t u r e d w i t h o u t b l o o d . P r e s u m a b l y , f e w hemocytes a c c o m p a n i e d t h e f a t b o d y into c u l t u r e . The c u l t u r e s w e r e not a g i t a t e d , e x c e p t as n e c e s s a r y f o r p e r i o d i c e x a m i n a t i o n . T h i s p r o b a b l y a c c o u n t s f o r t h e s l o w p a c e o f d i s s o c i a t i o n and r e a g g r e g a t i o n o b s e r v e d in t h e P. interpunctella cultures.
245
H. O B E R L A N D E R A n i n s e c t p l a s m a f a c t o r w a s r e q u i r e d f o r r e a g g r e g a t i o n o f p u p a l f a t body c e l l s in W a l t e r ' s (1969) e x p e r i m e n t s . The f a c t o r had some p r o p e r t i e s of p r o t e i n s , b u t v a r i o u s s o u r c e s o f v e r t e b r a t e p r o t e i n s d i d not s e r v e as s u b s t i t u t e s . In t h e P. interpunctella c u l t u r e s i n s e c t plasma p r o t e i n w a s not r e q u i r e d . S i n c e it is w e l l k n o w n t h a t t h e f a t body i t s e l f adds p r o t e i n s to t i s s u e c u l t u r e medium ( e . g . Pan et aL, 1 9 6 9 ) , t h i s may a c c o u n t f o r t h e d i f f e r e n c e in o b s e r v a t i o n s , s i n c e t h e P. interpunctella c u l t u r e s were i n i t i a t e d w i t h i n t a c t l a r v a l f a t body rather t h a n d i s s o c i a t e d p u p a l c e l l s . The a d h e s i o n of P. interpunctella f a t body c e l l s to e a c h o t h e r or t o i m a g i n a i d i s k s and midgut in vitro in t h e a b s e n c e o f h e m o c y t e s or i n s e c t p l a s m a p r o t e i n m a k e s it p o s s i b l e to s t u d y t h e r e c o n s t i t u t i o n o f a d u l t f a t body under c o n t r o l l e d c o n d i t i o n s . C l e a r l y , f a t body d i s s o c i a t i o n and r e a g g r e g a t i o n c a n be d u p l i c a t e d in vitro a n d prov i d e s a f a v o r a b l e s y s t e m f o r s t u d y i n g both hormone a c t i o n a n d c e l l a d h e s i o n i n insects. VII.
References
D u t k o w s k i , Α . , and O b e r l a n d e r , Η . ( 1 9 7 3 ) . J. Insect H u m p h r e y s , T . D . ( 1 9 7 0 ) . Transpl.
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O b e r l a n d e r , Η . , L e a c h , C . E., a n d T o m b l i n , C . ( 1 9 7 3 ) . J. Insect O b e r l a n d e r , H . , a n d T o m b l i n , C . ( 1 9 7 2 ) . Science
O h t a k i , T . , M i l k m a n , R., a n d W i l l i a m s , C . M . (1968) Biol. Bull. P a n , M . L., B e l l , W. J . , a n d T e l f e r , W. H . ( 1 9 6 9 ) . Science R o t h , S . ( 1 9 7 3 ) . Quart.
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177, 4 4 1 . W o o d s H o l e 135, 3 2 2 .
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S a n b u r g , L. L., Kramer, K. J . , K e z d y , F. J . , L a w , J . H . , a n d O b e r l a n d e r , Η . ( 1 9 7 5 ) . 253, 2 6 6 . W a l k e r , P. A . ( 1 9 6 6 ) . J. Insect
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W a l t e r s , D. R. ( 1 9 6 9 ) . Biol. Bull.,
12, 1 0 0 9 .
Woods H o l e 137, 2 1 7 .
W a l t e r s , D. R. ( 1 9 7 2 ) . Am. Zoo/. 12, 1 0 2 . W a l t e r s , D. R. ( 1 9 7 4 ) . J. Insect
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20, 4 9 .
W a l t e r s , D. R., a n d W i l l i a m s , C. M. ( 1 9 6 6 ) . Science W i l s o n , Η. V . ( 1 9 0 7 ) . J. Exp. Zoo/. 5, 2 4 5 . Z w i l l i n g , Ε. ( 1 9 6 8 ) . Deve/op.
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Nature,
C h a p t e r 21
THE DEVELOPMENT OF AN INSECT TISSUE CULTURE MEDIUM
G.R. Wyatt and S.S. Wyatt
I.
Introduction
,
249
II.
T h e e s t a b l i s h m e n t of an c u l t u r e m e d i u m
249
III.
References
254
I.
Introduction
From t h e r e m a r k a b l y d i v e r s e and s u c c e s s f u l a p p l i c a t i o n s of i n s e c t c e l l and t i s s u e c u l t u r e reported a t t h i s m e e t i n g , it is q u i t e c l e a r t h a t t h i s f i e l d of endeavour c a n now be p r o p e r l y d e s c r i b e d as h a v i n g come of a g e . So i t may perhaps be e x c u s a b l e t o t a k e t h i s o p p o r t u n i t y to d e s c r i b e some e x p e r i m e n t s done j u s t 21 y e a r s ago w h i c h , as e v e n t s have t u r n e d o u t , p l a y e d a s i g n i f i c a n t r o l e in t h e embryonic d e v e l o p m e n t of insect tissue c u l t u r e . II.
The e s t a b l i s h m e n t o f an c u l t u r e m e d i u m .
In the e a r l y 1 9 5 0 ' s , w e were both e m p l o y e d at t h e r e c e n t l y e s t a b l i s h e d Laboratory of I n s e c t P a t h o l o g y of t h e C a n a d i a n D e p a r t m e n t of A g r i c u l t u r e a t Sault S t e - M a r i e , O n t a r i o , where one of the major p r o j e c t s w a s r e s e a r c h on i n s e c t - p a t h o g e n i c v i r u s e s , w i t h a v i e w t o p o s s i b l e a p p l i c a t i o n s in s e l e c t i v e i n s e c t c o n t r o l . S.S.W., as a graduat e s t u d e n t , undertook t h e p r o j e c t of a t t e m p t i n g t o produce i n s e c t t i s s u e c u l t u r e s f o r u s e in v i r u s s t u d i e s . The s t a t e of i n s e c t t i s s u e c u l t u r e a t t h e t i m e w a s q u i t e p r i m i t i v e . Since t h e p i o n e e r i n g w o r k of G o l d s c h m i d t (1916) d e s c r i b e d by W i l l i a m s at t h i s m e e t i n g , t h e r e had been o n l y a f e w o t h e r c o n t r i b u t i o n s , m o s t l y not very s u c c e s s f u l . A r e m a r k a b l e paper f o r i t s t i m e , h o w e v e r , w a s t h a t of Trager ( 1 9 3 5 ) . U s i n g fragments of l a r v a l s i l k w o r m (Bombyx mori) o v a r i e s e x p l a n t e d into a s i m p l e s y n t h e t i c m e d i u m , Trager had o b t a i n e d c o n s i d e r a b l e c e l l o u t g r o w t h , i n c l u d i n g some m i t o s e s . A f t e r a d d i n g to t h e s e c u l t u r e s some hemolymph from v i r u s - d i s e a s e d s i l k w o r m s he o b s e r v e d t h e i n f e c t i v e p r o c e s s , l e a d i n g to p r o d u c t i o n of large numbers of the p o l y h e d r a l i n c l u s i o n b o d i e s t y p i c a l of t h i s c l a s s of v i r u s . By 1951 t h i s h i g h l y p r o m i s i n g report had never been f o l l o w e d up. S.S.W. s u c c e e d e d in r e p e a t i n g T r a g e r ' s c u l t u r e m e t h o d , but t h e o u t g r o w i n g c e l l s never appeared r e a l l y h e a l t h y . A l t h o u g h t h e i r numbers i n c r e a s e d f o r s e v e r a l d a y s , a l r e a d y by 24 hours t h e c e l l s were g r a n u lar (by phase o p t i c s ) and t h e i r a c t i v i t y w a s d e c l i n i n g . Many a t t e m p t s w e r e made t o improve t h e s e c u l t u r e s by a d j u s t m e n t s in t h e medium and a d d i t i o n s of t i s s u e ext r a c t s and p r o t e i n d i g e s t s , but i n i t i a l l y w i t h q u i t e l i m i t e d s u c c e s s . G.R.W. became i n t e r e s t e d in t h e p r o j e c t , and we c o l l a b o r a t e d on t h e p r o b l e m of i n s e c t t i s s u e c u l t u r e m e d i u m . We assumed t h a t an a p p r o p r i a t e medium s h o u l d , in main f e a t u r e s , r e s e m b l e the b l o o d or body f l u i d s of the s p e c i e s b e i n g u s e d , s o began t o c o l l e c t i n f o r m a t i o n on s i l k w o r m h e m o l y m p h . To s u p p l e m e n t w h a t w a s in 249
an its we the
G. R. W Y A T T A N D S. S. W Y A T T l i t e r a t u r e , w e p e r f o r m e d , w i t h t h e aid of Dr. C r o s s l e y L o u g h h e e d , some new a n a l y s e s (Wyatt, Loughheed and W y a t t , 1956). Then u s i n g t h e a v a i l a b l e data together w i t h a generous a d d i t i o n of g u e s s w o r k and t r i a l and error, we f o r m u l a t e d a new p h y s i o l o g i c a l s o l u t i o n for s i l k w o r m t i s s u e c u l t u r e (Table 1 ; S.S. W y a t t , 1956). TABLE I Physiological
Component
solution for culture of tissue from the f B o m b y x m o r i ; (S.S. Wyatt, 1956).
m g / 1 0 0 ml
mM
Component
silkworm
m g / 1 0 0 ml
mM
Inorganic salts N a H 2P 0 . 4H 20
110
8
L-Arginine HCl
70
M g C I 2. 6 H 20
304
15
DL-Lysine HCl
125
3.3 6.9
M g S 0 4. 7 H 20
370
250
15.7
298
15 40
L-H i s t i n e
KCL CaCI2
Amino acids
81
7.2
Sugars
L-Aspartic acid
35
L-Asparagine
35
2,65
L-Glutamic acid
60
4.08
2.63
L-Glutamine
60
4.11
Glucose
70
3.9
Glycine
65
8.66
Fructose
40
2.2
DL-Serine
Sucrose
40
1.1
DL-Alanine
Organic acids
110
10.5
/^-Alanine
45 20
2.25
L-Prol ine
35
3.0
Malic
67
5
L-Tyrosine
(x-Ketogluaric
37
2.5
DL-Threonine
5.05
5
0.27
35
2.94
Succinic
6
0.5
DL-Methionine
10
0.67
Fumaric
5.5
0.5
L-Phenylalanine
0.9
DL-Valine
15 20
DL-lsoleucine
10
0.77
DL-Leucine
15 10
0.49
L-Tryptophan L-Cystine Cysteine HCl
2.5 8
1.7 1.14 0.1 0.5
L-amino a c i d s were used i f a v a i l a b l e ; otherwise, DL-forms were u s e d . The s o l u t i o n w a s a d j u s t e d t o pH 6 . 3 5 and u s e d w i t h a d d i t i o n of 10% of h e a t - t r e a t e d s i l k w o r m h e m o l y m p h . (Some a r i t h m e t i c a l e r r o r s in t h e o r i g i n a l t a b l e h a v e been c o r r e c t e d . ) . The i n o r g a n i c c o m p o n e n t s were based on the e x c e l l e n t s y s t e m a t i c a n a l y s e s of B i a l a s c e w i c z and Landau ( 1 9 3 8 ) . We used t h e i r d a t a o n h e m o l y m p h of f u l l y g r o w n f e e d i n g l a r v a e , t h e s t a g e used f o r t i s s u e c u l t u r i n g , but lowered t h e c a l c i u m c o n t e n t s o m e w h a t t o a l l o w f o r t h e p r o b a b i l i t y t h a t a p r o p o r t i o n of t h i s ion in hemolymph w a s p r o t e i n - b o u n d . The b a l a n c i n g a n i o n s p o s e d a p r o b l e m , s i n c e t h e p u b l i s h e d a n a l y s e s s h o w e d c h l o r i d e e q u i v a l e n t to l e s s t h a n 2 0 % of t h e t o t a l c a t i o n t i t e r and most of t h e remainder w a s u n i d e n t i f i e d . We i n c l u d e d a low l e v e l of i n o r g a n i c p h o s p h a t e , b a s e d on a n a l y s e s , . a n d some s u l f a t e , on t h e s u p p o s i t i o n t h a t v a r i e t y w o u l d be a g o o d t h i n g , and made up most of the b a l a n c e w i t h c h l o r i d e , f o r l a c k of better i d e a s . S i n c e t h e n it has become k n o w n t h a t major a n i o n i c c o m p o n e n t s of the hemolymph o f higher
250
D E V E L O P M E N T OF A N INSECT TISSUE C U L T U R E M E D I U M
i n s e c t s i n c l u d e a number of organic p h o s p h a t e s , b i c a r b o n a t e and s o m e t i m e s c i t r a t e and other organic a c i d s ( W y a t t , 1961). We had an i n d i c a t i o n f r o m t h e e a r l y w o r k of T s u j i ( 1 9 0 9 ) , as c i t e d in the useful t a b u l a t i o n s of Y a m a f u j i ( 1 9 3 7 ) , t h a t s i l k w o r m hemolymph c o n t a i n e d s u b s t a n t i a l l e v e l s of s e v e r a l d i - a n d t r i - c a r b o x y l i c a c i d s rel a t e d to t h e c i t r i c a c i d c y c l e . F r o m e m p i r i c a l t e s t i n g in s i l k w o r m o v a r i a n c u l t u r e s , i t appeared t h a t a c o m b i n a t i o n of f o u r of t h e s e g a v e some s t i m u l a t i o n o f g r o w t h , and a c c o r d i n g l y t h i s m i x t u r e w a s i n c l u d e d in the c u l t u r e m e d i u m . The sugars p r e s e n t e d a problem s i n c e there w e r e no p u b l i s h e d data on sugars in s i l k w o r m hemolymph t h a t appeared to be r e l i a b l e , a n d , i n d e e d , v e r y few on t h e hemolymph of any other i n s e c t . W h i l e some a u t h o r s had r e p o r t e d g l u c o s e from t e s t s t h a t measured r e d u c i n g power, more c r i t i c a l w o r k by o t h e r s had shown t h a t t h e comp o u n d s r e s p o n s i b l e w e r e in f a c t l a r g e l y not s u g a r s . We c a r r i e d out a n a l y s e s of s i l k w o r m b l o o d sugars by paper c h r o m a t o g r a p h y and f o u n d e v i d e n c e for g l u c o s e , f r u c t o s e and s u c r o s e , but o n l y a t t r a c e l e v e l s (Wyatt et a / . , 1956). [ ^ H o w e v e r , determ i n a t i o n of t o t a l c a r b o h y d r a t e in hemolymph e x t r a c t by means of t h e anthrone react i o n s h o w e d f a r higher l e v e l s , of a g l u c o s e d e r i v a t i v e w h i c h w a s later i d e n t i f i e d as t r e h a l o s e (Wyatt and K a l f , 1958)]]· For t h e f o r m u l a t i o n of a c u l t u r e m e d i u m , i t seemed t h a t some c a r b o h y d r a t e w a s d e s i r a b l e as a s u s t a i n e d e n e r g y s o u r c e , so w e p r o v i d e d w h a t seemed a r e a s o n a b l e level by s i m p l y e l e v a t i n g t h e l e v e l s of t h e t h r e e sugars d e t e c t e d on c h r o m a t o g r a m s t o a t o t a l o f 150 m g / 1 0 0 m l . For t h e a m i n o a c i d s , w e o b t a i n e d f a i r l y s a t i s f a c t o r y a n a l y t i c a l data by q u a n t i t a t i v e paper c h r o m a t o g r a p h y (Wyatt et a / . , 1 9 5 6 ) , a n d a l s o d u r i n g t h e c o u r s e o f t h e w o r k r e c e i v e d the paper of S a r l e t , D u c h a t e a u and F l o r k i n ( 1 9 5 2 ) , r e p o r t i n g d e t e r m i n a t i o n s of 14 a m i n o a c i d s in s i l k w o r m hemolymph b y m i c r o b i o l o g i c a l a s s a y . We f o r m u l a t e d a m i x t u r e based on our a n a l y s e s from the late l a r v a l s t a g e , w i t h q u a n t i t i e s rounded o f f for c o n v e n i e n t w e i g h i n g . Knowing l i t t l e a b o u t t h e p a t h w a y s of p r o t e i n s y n t h e s i s and w i s h i n g t o o m i t n o t h i n g t h a t might b e e s s e n t i a l , w e i n c l u d e d both c y s t i n e and c y s t e i n e , as w e l l as t r y p t o p h a n , w h i c h t h e chromatograms from hemolymph d i d not show. The chromatograms d i d , h o w e v e r , r e g u l a r l y r e v e a l / 3 - a l a n i n e , so w e i n c l u d e d t h i s in the m e d i u m , h a v i n g no idea of its f u n c t i o n . The m i x t u r e was a d j u s t e d t o pH 6 . 3 5 , c o r r e s p o n d i n g t o our b e s t e s t i m a t e f o r s i l k w o r m h e m o l y m p h , and had an o s m o t i c p r e s s u r e (measured as f r e e z i n g p o i n t d e p r e s s i o n of 0 . 5 3 ° C ) c l o s e t o reported v a l u e s for s i l k w o r m b l o o d . To p r o v i d e f o r u n k n o w n needs of t h e c e l l s , it seemed a p p r o p r i a t e t o add a proport i o n of s i l k w o r m hemolymph i t s e l f , as Trager (1935) had d o n e . T h i s c o n f r o n t e d us w i t h t h e problem of t h e phenol o x i d a s e a c t i v i t y , w e l l k n o w n in i n s e c t b l o o d , w h i c h , upon e x p o s u r e t o a i r , l e a d s to t h e p r o d u c t i o n o f m e l a n i n and v a r i o u s t o x i c intermed i a t e s . Of s e v e r a l compounds t e s t e d to i n h i b i t p h e n o l o x i d a s e , o n l y p h e n y l t h i o u r e a gave permanent i n h i b i t i o n , but t h e r e w a s an i n d i c a t i o n t h a t t h i s s u b s t a n c e m i g h t i t s e l f be harmful t o t h e c e l l s . E v e n t u a l l y , t a k i n g a cue f r o m t h e e x p e r i m e n t s of Levenbook (1950) on Gastrophilus h e m o l y m p h , we f o u n d t h a t h e a t i n g of t h e hemolymph t o 6 0 ° C for 5 m i n u t e s c a u s e d c o a g u l a t i o n of some p r o t e i n i n c l u d i n g t h e p h e n o l o x i d a s e , w h i c h c o u l d t h e n be removed b y c e n t r i f u g i n g . H e a t - t r e a t e d s i l k w o r m hemolymph w a s added t o t h e m e d i u m , u s u a l l y at a c o n c e n t r a t i o n of 10%. Of course a new c u l t u r e medium w a s not f o r m u l a t e d in a s i n g l e s t e p . But i t i s our r e c o l l e c t i o n t h a t t h e f i r s t e x p e r i m e n t s w i t h a medium d e s i g n e d on t h e b a s i s of t h e c o m p o s i t i o n of hemolymph s h o w e d marked and o b v i o u s improvement over t h o s e done p r e v i o u s l y . From then o n , m o d i f i c a t i o n s were r e a d i l y r e f l e c t e d in the appearance and number of the c e l l s , so t h a t p r o g r e s s i v e improvement became p o s s i b l e . Under t h e 251
G. R. W Y A T T A N D S. S. W Y A T T
Fig. 7-3. C e l l g r o w t h in h a n g i n g - d r o p c u l t u r e s o f l a r v a l s i l k w o r m o v a r i a n t i s s u e , p r i n t e d f r o m t h e n e g a t i v e s o f S.S. W y a t t ( 1 9 5 6 ) . Phase c o n t r a s t . 1. T y p i c a l g r o w t h o f d i s p e r s e d c e l l s , 4 d a y s a f t e r e x p l a n a t i o n o f o v a r i a n f r a g m e n t s Fig. x168. Fig. 2. L e s s u s u a l t y p e o f o u t - g r o w t h of c e l l s in c o n t a c t ; a p o r t i o n o f a n o v a r i o l e c a n b e seen. 5 days. x175. Fig. 3. M i t o s i s i n a 4 - d a y o l d c u l t u r e . x 7 3 5 . a, 0 m i n ; b, 9 m i n ; c, 2 0 m i n .
c o n d i t i o n s f i n a l l y a c h i e v e d in t h i s p r o j e c t , o u t g r o w t h c o n t i n u e d in hanging drop c u l t u r e s for as long a s a w e e k a n d in s m a l l r o l l e r t u b e c u l t u r e s , in w h i c h t h e medium c o u l d be c h a n g e d , for more than 2 w e e k s . The c e l l s appeared h e a l t h y , w i t h c l e a r c y t o p l a s m a n d f r e q u e n t m i t o s i s . Figure 1 s h o w s a h a n g i n g drop c u l t u r e a t 4 d a y s of d e v e l o p m e n t , w i t h t h e m i g r a t i n g , s e p a r a t e d , f i b r o b l a s t - l i k e c e l l s that w e r e regul a r l y o b t a i n e d . F i g u r e 2 shows a s l o w e r - g r o w i n g c o n t i n u o u s o u t g r o w t h from t h e e x p l a n t w h i c h w a s o b s e r v e d in o c c a s i o n a l c u l t u r e s . Figure 3 s h o w s three s t a g e s of m i t o s i s in a 4 d a y c u l t u r e .
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D E V E L O P M E N T OF A N INSECT TISSUE C U L T U R E M E D I U M
In 1954, q u i t e soon a f t e r t h e s e p r o m i s i n g r e s u l t s w e r e o b t a i n e d , w e b o t h l e f t t h e l a b o r a t o r y w h e r e t h i s w o r k w a s d o n e . S.S.W. t u r n e d to r a i s i n g a f a m i l y , and G. R.W. to s t u d i e s on i n s e c t b i o c h e m i s t r y , b e g i n n i n g w i t h some p r o b l e m s o f h e m o l y m p h c h e m i s t r y t h a t arose f r o m the w o r k j u s t d e s c r i b e d . There w a s no o p p o r t u n i t y f o r f u r t h e r r e f i n e m e n t of t h e t i s s u e c u l t u r e m e d i u m , or s e r i o u s a t t e m p t s at long-term c u l t u r i n g or e s t a b l i s h m e n t of a c e l l l i n e . A s is w e l l k n o w n , t h e s e l a t t e r p r o b l e m s were soon t a k e n up by o t h e r s . Tom G r a c e , in A u s t r a l i a , improved t h e m e d i u m , b y t h e a d d i t i o n of group of w a t e r - s o l u b l e v i t a m i n s and some a d j u s t m e n t of ionic r a t i o s , p H and o s m o t i c p r e s s u r e ( G r a c e , 1 9 6 2 ; 1 9 6 7 ) . In 1962 he r e p o r t e d t h e landmark s t e p o f t h e e s t a b l i s h m e n t of t h e f i r s t c o n t i n u o u s l y eucalypti. g r o w i n g i n s e c t c e l l l i n e , from t h e A u s t r a l i a n w i l d s i l k m o t h Antheraea soon to be f o l l o w e d by o t h e r s . Imogene Schneider ( 1 9 6 4 , 1966) m o d i f i e d t h e medium by a l t e r a t i o n of the s o d i u m : p o t a s s i u m r a t i o to make s o d i u m f o r use w i t h Drosophila t h e d o m i n a n t c a t i o n , as in the hemolymph of f l i e s . Marks and R e i n i c k e ( 1 9 6 5 ; M a r k s , 1973) f r o m the same b a s i s d e v e l o p e d media s u i t a b l e for c u l t u r i n g t i s s u e s of c o c k r o a c h e s and other O r t h o p t e r a . Many o t h e r s have s i n c e c o n t r i b u t e d to the d e v e l o p ment and m o d i f i c a t i o n of c u l t u r e media s u i t e d t o a w i d e v a r i e t y of i n s e c t c e l l s ( V a g o , 1971). R e v i e w i n g t h e v a r i o u s i n s e c t t i s s u e c u l t u r e m e d i a t h a t have been d e s c r i b e d , i t i s i n t e r e s t i n g to see how t h e i n h e r i t a n c e from our e a r l y e x p e r i m e n t s has been p a s s e d d o w n . A m o n g t h e amino a c i d s , for e x a m p l e , w e i n c l u d e d / ^ - a l a n i n e in t h e medium s i m p l y b e c a u s e w e f o u n d i t in s i l k w o r m h e m o l y m p h . / 9 - A l a n i n e i s , o f c o u r s e , n o t o n e of t h e amino a c i d s of p r o t e i n s and i s u n l i k e l y to a s s i s t t h e g r o w t h of i n s e c t c e l l s ; more r e c e n t l y , i t has been shown to h a v e a r o l e in t h e t a n n i n g of i n s e c t c u t i c l e ( B o d n a r y k , 1 9 7 1 ) . Yet i t w a s i n c l u d e d in t h e media of G r a c e ( 1 9 6 2 ) , S c h n e i d e r ( 1 9 6 6 ) , and S h i e l d s and Sang ( 1 9 7 0 ) , among o t h e r s Landureau ( 1 9 6 6 ) , i n c l u d e d / ^ - a l a n i n e in h i s f i r s t c u l t u r e medium f o r c o c k r o a c h t i s s u e s , b u t , a f t e r f a i l i n g to f i n d e v i d e n c e f o r u t i l i z a t i o n of i t , o m i t t e d i t from s u b s e q u e n t m o d i f i c a t i o n s ( L a n d u reau and Jolies, 1969; Landureau and G r e l l e t , 1 9 7 2 ) . N o w , at t h i s m e e t i n g , M i t s u h a s h i (1976) has shown t h a t / ô - a l a n i n e may even h a v e some d e t r i m e n t a l e f f e c t on the g r o w t h of c e l l c u l t u r e s from b u t t e r f l y p u p a l o v a r i e s . It w o u l d seem t h a t / ' - a l a n i n e s h o u l d h e n c e f o r t h be o m i t t e d from i n s e c t t i s s u e c u l t u r e m e d i a . The p r o p o r t i o n s of the p r o t e i n amino a c i d s t h a t w e u s e d , w h i c h are c o n t i n u e d in G r a c e ' s (1962) m e d i u m , w e r e based on t h e a n a l y s i s o f s i l k w o r m h e m o l y m p h , b u t t h e y may not be n e c e s s a r y or o p t i m a l f o r i n s e c t c e l l g r o w t h . These p r o p o r t i o n s have been v a r i e d by recent w o r k e r s ; Marks and R e i n e c k e (1965) and Landureau and G r e l l e t (1972) d e v e l o p e d f o r m u l a t i o n s for c o c k r o a c h t i s s u e c u l t u r e w i t h more n e a r l y e q u i m o l a r q u a n t i t i e s of the d i f f e r e n t a m i n o a c i d s . That t h e p r o p o r t i o n s of the amino a c i d s are not at a l l c r i t i c a l is a l s o s u g g e s t e d by t h e s u c c e s s w i t h w h i c h l a c t a l bumin h y d r o l y s a t e has been used in many c u l t u r e m e d i a . O n e m u s t , h o w e v e r , be on guard f o r the e f f e c t s of i n t e r a c t i o n s b e t w e e n medium c o m p o n e n t s . H i s t i d i n e , f o r e x a m p l e , t h e most abundant a m i n o a c i d in our o r i g i n a l f o r m u l a t i o n , is an e f f e c t i v e c h e l a t o r for d i v a l e n t c a t i o n s , and its p r e s e n c e in large a m o u n t s may h e l p t o p r e v e n t the p r e c i p i t a t i o n of the rather i n s o l u b l e p h o s p h a t e s of c a l c i u m and m a g n e s i u m . L o w e r i n g of h i s t i d i n e l e v e l s s h o u l d , t h e r e f o r e , perhaps be a c c o m p a n i e d by r e d u c t i o n of the l e v e l s of t h e s e i o n s ; a l t e r n a t i v e l y , t h e i n c l u s i o n of c i t r a t e , w h i c h is o f t e n abundant in i n s e c t hemolymph ( L e v e n b o o k a n d H o l l i s , 1961) and a l s o f o r m s c o m p l e x e s w i t h t h e m , m i g h t be v a l u a b l e . T h e m i x t u r e of Krebs c y c l e a c i d s w h i c h w e i n t r o d u c e d , or some v a r i a n t of t h e m , has been i n c l u d e d in many i n s e c t t i s s u e c u l t u r e m e d i a , and as t h e r e is some e v i d e n c e t h a t t h e y improve c e l l g r o w t h ( W y a t t , 1956)
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G. R. W Y A T T A N D S. S. W Y A T T c o n t i n u a t i o n of t h i s p r a c t i c e may be j u s t i f i e d . The three s u g a r s , g l u c o s e , f r u c t o s e and s u c r o s e , added t o our o r i g i n a l medium on t h e s l e n d e r grounds t h a t have been d e s c r i b e d , are s t i l l i n c l u d e d in the medium of Grace (1972) and some o t h e r s . A f t e r t r e h a l o s e w a s d i s c o v e r e d as t h e major blood sugar of most i n s e c t s , it was i n c l u d e d in some i n s e c t t i s s u e c u l t u r e m e d i a , f o r e x a m p l e , t h a t of Schneider (1966) and Landureau ( 1 9 6 6 ) . W h i l e t h i s seemed reasonab l e , and t r e h a l o s e can support t h e growth o f i n s e c t c e l l c u l t u r e s ( S t o c k d a l e and G a r d i n e r , 1 9 7 6 ) , it has never been shown to have any s u p e r i o r i t y over g l u c o s e in t h i s r o l e . Sucrose is p r o b a b l y g e n e r a l l y not u t i l i z e d by i n s e c t c e l l s ( S t o c k d a l e and G a r d i n e r , 1976); i t s c o n s u m p t i o n by Antheraea eucalypti c e l l s reported b y C l e ments and Grace (1967) may perhaps have been due t o t h e p r e s e n c e of some s u c r a s e in t h e hemolymph i n c l u d e d in t h e i r c u l t u r e medium. It seems r e a s o n a b l e , t h e r e f o r e , t o i n c l u d e merely g l u c o s e as an energy s o u r c e , as Landureau and G r e l l e t (1972) h a v e d o n e , w h i l e s u c r o s e may be added if a r e l a t i v e l y inert i n g r e d i e n t is required f o r a d j u s t m e n t of o s m o t i c p r e s s u r e . With regard to the p r o p o r t i o n s of inorganic c a t i o n s , our a s s u m p t i o n t h a t t h e s e s h o u l d resemble t h o s e f o u n d in the hemolymph of the s p e c i e s b e i n g c u l t u r e d seems in general t o be born o u t . Whereas l e p i d o p t e r a n c e l l s f l o u r i s h in h i g h - p o t a s s i u m m e d i a , c o c k r o a c h t i s s u e s prefer media w i t h a s o d i u m : p o t a s s i u m r a t i o greater t h a n 1 ( T i n g and B r o o k s , 1965; Marks and R e i n e c k e , 1 9 6 5 ) . V e r y l i t t l e e f f o r t , h o w e v e r , has been put into s y s t e m a t i c t e s t i n g of the inorganic c o m p o n e n t s , and we are s t i l l i g n o r a n t of t h e optima or l i m i t s for g r o w t h of v a r i o u s t y p e s of i n s e c t c e l l s . Now t h a t i n s e c t t i s s u e c u l t u r e has become e s t a b l i s h e d , t h r o u g h d e v e l o p m e n t s d u r i n g the p a s t 20 y e a r s , as a s u c c e s s f u l a n d v a l u a b l e t e c h n i q u e , i t is e n c o u r a g i n g t o s e e some w o r k e r s u n d e r t a k i n g the c r i t i c a l r e - e x a m i n a t i o n of t h e c o m p o s i t i o n of c u l t u r e media ( e . g . Landureau and G r e l l e t , 1 9 7 2 ; M i t s u h a s h i , 1976; H i n k s , 1976). Such e f f o r t s may be e x p e c t e d to y i e l d media w h i c h are more r a t i o n a l l y c o m p o s e d , s i m p l e r , l e s s e x p e n s i v e and a t l e a s t as s u c c e s s f u l as t h o s e c u r r e n t l y in u s e . III.
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S a r l e t , H . , D u c h a t e a u , G . , a n d F l o r k i n , M . ( 1 9 5 2 ) . Arch. S c h n e i d e r , J . ( 1 9 6 4 ) . J. Exp. Zool. S c h n e i d e r , J . ( 1 9 6 6 ) . J. Embryo/.
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( 1 9 0 9 ) . SanjiHokoku35.
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H.
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Gardiner,
60,
126.
15, Exp.
271. Morphol.
23.
53.
501.
T i n g , K.Y., a n d B r o o k s , M . A . ( 1 9 6 5 ) . Ann. Tsuji, C
Physiol.
91.
S h i e l d s , G . , a n d S a n g , J . H . ( 1 9 7 0 ) . J. Embryo/. T r a g e r , W. ( 1 9 3 5 ) . J. Exp. Med.
Internat.
MEDIUM
Entomol.
Soc.
Amer. 58.
197.
1. G.R.
(1976),
In: Invertebrate
T i s s u e C u l t u r e : A p p l i c a t i o n in
B i o l o g y a n d A g r i c u l t u r e (E. K u r s t a k a n d K. M a r a m o r o s c h , e d s ) . A c a d e m i c
Press,
New York. V a g o , C. (ed.) (1971). Invertebrate T i s s u e C u l t u r e . V o l . 1 . A c a d e m i c Press, New York. W y a t t , G.R. (1961 ) . Ann.
Rev. Entomol.
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W y a t t , G.R., a n d K a l f , G . F . ( 1 9 5 8 ) . J. Gen.
75. Physiol.
40,
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W y a t t , G.R., L o u g h h e e d , T . C , a n d W y a t t , S.S. ( 1 9 5 6 ) . J. Gen. W y a t t , S.S. ( 1 9 5 6 ) . J. Gen.
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Y a m a f u j i , K. ( 1 9 3 7 ) . T a b u l a e B i o l o g i c a e (W. J u n k , J u n k , The H a g u e , V o l . 1 4 , p p . 3 6 - 5 0 .
255
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O p p e n h e i m e r a n d W e i s b a c h , e d s ) . W.
Chapter 22
INSECT CELL LINE: AMINO ACID UTILIZATION AND REQUIREMENTS
J . Mitsuhashi
I.
Introduction
257
II.
M a t e r i a l s and methods
257
III.
Results
258
1.
U t i l i z a t i o n o f a m i n o a c i d s by t h e c e l l l i n e
258
2.
R e q u i r e m e n t s o f a m i n o a c i d s by t h e c e l l l i n e
258
IV.
Discussion
260
V.
References
262
I.
Introduction
Recently s y n t h e t i c media h a v e been i n t r o d u c e d f o r c u l t u r i n g i n s e c t c e l l s . T h e n u t r i t i o n a l r e q u i r e m e n t s of i n s e c t c e l l s in vitro, h o w e v e r , have been s t u d i e d o n l y t o a l i m i t e d e x t e n t . The u t i l i z a t i o n of amino a c i d s by i n s e c t c e l l l i n e s has been eucalypti (Ae) (Grace and B r z o s t o w s k i , e x a m i n e d in c u l t u r e s o f t h e Antheraea 1 9 6 6 ) , Periplaneta americana (EPa) (Landureau and J o l i e s , 1 9 6 9 ) , and Carpocapsa pomonella c e l l l i n e s (CP 1268 and CP 169) ( H i n k et al, 1 9 7 3 ) . The sugar u t i l i z a t i o n has been s t u d i e d i n A e (Grace and B r z o s t o w s k i , 1966; C l e m e n t s and G r a c e , 1 9 6 7 ) , a n d CP ( H i n k et al, 1973). The v i t a m i n r e q u i r e m e n t s have been s t u d i e d o n l y in EPa ( L a n d u r e a u , 1969). To know t h e n u t r i t i o n a l r e q u i r e m e n t s of a c e r t a i n c e l l l i n e is n o t o n l y p r o f i t a b l e for t h e improvement o f t h e c u l t u r e medium but a l s o important f o r t h e c h a r a c t e r i z a t i o n o f the c e l l l i n e . In t h e p r e s e n t s t u d y , t h e u t i l i z a t i o n a n d requirements of amino a c i d s by t h e Papilio xuthus c e l l l i n e (Px 58) w e r e i n v e s t i g a t ed. II.
M a t e r i a l s and methods
A mixed c e l l p o p u l a t i o n of Px 58 c e l l l i n e , w h i c h has been e s t a b l i s h e d f r o m p u p a l o v a r i e s of the s w a l l o w t a i l , Papilio xuthus ( M i t s u h a s h i , 1 9 7 3 ) , w a s u s e d . The s t o c k c o l o n y has been m a i n t a i n e d in MGM-431 medium ( M i t s u h a s h i , 1972) at 2 5 ° C by subculturing twice a week. For t h e q u a n t i t a t i v e d e t e r m i n a t i o n of amino a c i d c h a n g e s in t h e m e d i u m , r e p l i c a t e t i s s u e c u l t u r e s were s e t up in r o l l e r t u b e s . F i v e ml of t h e c e l l s u s p e n s i o n , c o n t a i n 5 i n g about 1.0 χ 1 0 c e l l s per m l , w a s d i s t r i b u t e d to each t u b e . The c u l t u r e s w e r e m a i n t a i n e d at 2 5 ° C . S a m p l i n g w a s made on t h e 0 , 4 t h and 8 t h d a y s . A t one t i m e , 3 tubes w e r e h a r v e s t e d , and t h e amino a c i d a n a l y s i s w a s made f o r each t u b e . In order to prepare the samples for t h e a n a l y s i s . H i n k et a/'s procedure ( H i n k et al, 1973) w a s a d o p t e d . F i r s t the c e l l s w e r e spun d o w n a t 2,000 r . p . m . f o r 10 m i n u t e s . Four ml of t h e s u p e r n a t a n t w e r e m i x e d w i t h a n equal v o l u m e of 70% e t h y l a l c o h o l . T h i s mixture w a s kept at 5 ° C f o r 2 h o u r s . Then t h e p r e c i p i t a t e w a s removed by 257
J. MITSUHASHI c e n t r i f u g a t i o n at 3,500 r.p.m. f o r 15 m i n u t e s . The supernatant w a s s l i g h t l y t u r b i d , a n d w a s p a s s e d s u c c e s s i v e l y through membrane f i l t e r s o f 1.2 jum, 0.6 jum, and 0.45 jum. The r e s u l t i n g f i l t r a t e w a s d i l u t e d w i t h l i t h i u m c i t r a t e buffer, p H 2 . 7 3 . The f i n a l d i l u t i o n rate of t h e s a m p l e w a s 50 t i m e s . For t h e amino a c i d s w h i c h w e r e in s m a l l amounts in the m e d i u m , o n l y 5 t i m e s d i l u t e d s a m p l e s were p r e p a r e d . The a n a l y s i s w a s made by Na-Li t w o c o l u m n method i n a Japan E l e c t r o n O p t i c s Laboratory 6 A H a u t o m a t i c amino a c i d a n a l y s e r . For t h e d e t e r m i n a t i o n of t h e e s s e n t i a l amino a c i d s f o r t h e g r o w t h o f Px 58 c e l l s , e x p e r i m e n t s w e r e made in w h i c h amino a c i d s were d e l e t e d f r o m MGM-431 medium o n e at a t i m e . By t h e r e p l i c a t e t i s s u e c u l t u r e method t h e c e l l number w a s recorded a t 0 day and the 8th d a y . The r e p l i c a t e t i s s u e c u l t u r e s w e r e p r e p a r e d by d i s t r i b u t i n g 2 ml of c e l l s u s p e n s i o n ( 1 . 5 χ 1 0 ^ c e l l s per ml) into each r o l l e r t u b e . The c u l t u r e w a s kept at 2 5 ° C for 8 days w i t h o u t c h a n g i n g the c u l t u r e m e d i u m . A t t h e b e g i n n i n g a n d at the end of c u l t i v a t i o n , 3 t u b e s w e r e s a m p l e d , and the c e l l enumeration w a s d o n e by t h e use of Thoma's he moc y to meter. The growth rate of t h e c e l l s in each t e s t medium w a s e x p r e s s e d a s t h e number o f c e l l s a f t e r 8 d a y s o f c u l t i v a t i o n . III. 1.
Results Utilization
of amino acids
by the cell
line.
D u r i n g 8 d a y s of c u l t i v a t i o n , t h e c e l l number i n c r e a s e d as s h o w n in F i g . 1 , and m o s t of t h e amino a c i d s in t h e medium d e c r e a s e d ( F i g . 2 ) . The rate of t h e d e c r e a s e , h o w e v e r , v a r i e d in d i f f e r e n t amino a c i d s . L y s i n e , h i s t i d i n e , t r y p t o p h a n , a r g i n i n e , t h r e o n i n e , s e r i n e , g l u t a m i c a c i d , and / 3 - a l a n i n e , d i d not d e c r e a s e m a r k e d l y . The amino a c i d which showed t h e greatest decrease was glutamine. Asparagine, c y s t i n e , a s p a r t i c a c i d , m e t h i o n i n e , t y r o s i n e , p r o l i n e , l e u c i n e , i s o l e u c i n e , and v a l i n e d e c r e a s e d 10 - 40 per c e n t d u r i n g 8 d a y s c u l t i v a t i o n , o o a l a n i n e w a s t h e o n l y amino a c i d w h i c h i n c r e a s e d d u r i n g c u l t i v a t i o n . In a d d i t i o n to t h e a m i n o a c i d s , ammonia w a s noticed to increase during c u l t i v a t i o n . 2.
Requirements
of amino acids by the cell
line.
MGM-431 media l a c k i n g one amino a c i d were p r e p a r e d . S i n c e MGM-431 medium c o n t a i n e d 10 per c e n t f e t a l b o v i n e s e r u m , and t h e serum c o n t a i n e d f r e e amino a c i d s , i t w a s i m p o s s i b l e to d e l e t e any one of t h e amino a c i d s c o m p l e t e l y . A n a n a l y s i s , h o w e v e r , showed t h a t t h e f r e e amino a c i d s d e r i v e d from f e t a l b o v i n e serum w e r e a l m o s t n e g l i g i b l e in MGM-431 medium ( T a b l e 1). The growth rate of t h e c e l l s in t h e media l a c k i n g a s p a r a g i n e , o o a l a n i n e , g l u t a m i c a c i d , g l y c i n e , and p h e n y l a l a n i n e w e r e not s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t in t h e c o m p l e t e MGM-431 medium ( F i g . 3 ) . In t h e medium l a c k i n g / 3 - a l a n i n e , t h e c e l l s g r e w much f a s t e r compared w i t h t h e c o n t r o l . D e l e t i o n of t h e o t h e r amino a c i d s res u l t e d in t h e r e d u c t i o n of t h e g r o w t h rate of t h e c e l l s c o m p a r e d w i t h t h e c o n t r o l . A m o n g t h e m , t h e d e l e t i o n of a r g i n i n e , a s p a r t i c a c i d , c y s t i n e , and t r y p t o p h a n s t i l l p e r m i t t e d l i m i t e d g r o w t h of t h e c e l l s , w h i l e t h e d e l e t i o n o f g l u t a m i n e , h i s t i d i n e , isoleucine, leucine, l y s i n e , methionine, proline, serine, threonine, tyrosine, and v a l i n e stopped t h e c e l l growth c o m p l e t e l y or even k i l l e d t h e c e l l s . The long-term c u l t i v a t i o n of Px 58 c e l l s w a s performed w i t h media l a c k i n g one of amino a c i d s . In t h e media l a c k i n g a s p a r a g i n e , o o a l a n i n e , / 3 - a l a n i n e , g l u t a m i c a c i d , g l y c i n e or p h e n y l a l a n i n e , t h e c e l l s c o u l d be s u b c u l t u r e d f o r a n i n f i n i t e p e r i o d . T h e s e amino a c i d s c o u l d be removed a l t o g e t h e r w i t h o u t d e l e t e r i o u s e f f e c t on t h e
258
INSECT CELL LINE: AMINO ACID UTILIZATION AND REQUIREMENTS
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Days Fig. 1. The growth curve of the Px 58 c e l l s during 8 days of experimental culture. Medium, MGM-431. Temperature, 25°С.
TABLE 1 Composition of amino acids, added to MGM-431 medium, and free amino acids derived fetal bovine serum added to MGM-431 medium (mg per /iter MGM-431 medium). Amino acids
A.M.*
F.B.S.**
Amino acids
A.M.*
from
F.B.S.**
(x-alaninę
263
13.7
Leucine
63
5.7
/3-alanine
167
trace
Lysine
521
3.3
nil
Methionine
Arginine
583
Aspartic acid
293
Asparagine
293
nil
Proline
292
2.9
21
nil
Serine
917
4.7 3.7
Cystine
0.9
Phenylalanine
42
0.2
125
3.1
Glutamic acid
500
14.7
Threonine
146
Glutaminę
500
5.5
Tryptophan
83
nil
Glycine
542
8.4
Tyrosine
42
2.0
2083
0.8
Valine
83
5.5
42
1.6
Histidine Isoleucine * **
amino acids in MGM-431 medium. free amino acids derived from fetal bovine serum (Microbiological Associates Inc., Bethesda, U.S.A.). (After Mitsuhashi, 1976).
259
J. M I T S U H A S H I
0
4
8
0
4
8
Days Fig. 2. Q u a n t i t a t i v e c h a n g e s i n a m i n o a c i d s in M G M - 4 3 1 m e d i u m d u r i n g 8 d a y s c u l t i v a t i o n o f Px 58 c e l l s .
c e l l g r o w t h . In media l a c k i n g other amino a c i d s , t h e c e l l s d e t e r i o r a t e d sooner o r l a t e r . The c e l l s , h o w e v e r , c o u l d adapt t o t h e medium l a c k i n g s e r i n e . In t h i s medium t h e c e l l s s t a y e d a l i v e f o r a long t i m e , g r a d u a l l y m u l t i p l i e d , and f i n a l l y t h e g r o w t h rate of the c e l l s became comparable t o t h e c o n t r o l . In t h e medium l a c k i n g g l u t a m i n e , t h e c e l l s a t t a c h e d t h e m s e l v e s t o t h e g l a s s , and s t a y e d a l i v e for s e v e r a l m o n t h s , b u t a p p a r e n t l y d i d not m u l t i p l y .
IV.
Discussion
The u t i l i z a t i o n o f amino a c i d s by i n s e c t c e l l l i n e s has been reported in A e c e l l s ( G r a c e and B r z o s t o w s k i , 1 9 6 6 ) , EPa c e l l s (Landureau and J o l i e s , 1 9 6 9 ) , and CP c e l l s ( H i n k et al, 1973). In t h e p r e s e n t s t u d y , it became e v i d e n t t h a t t h e use of a m i n o a c i d s by Px 58 c e l l s f o l l o w e d d i f f e r e n t p a t t e r n s f r o m t h o s e reported b e f o r e . In t h e c u l t u r e of Px 58 c e l l s , most of t h e a m i n o a c i d s d e c r e a s e d as t h e c u l t u r e s i n c r e a s e d in a g e . The d e c r e a s i n g p a t t e r n of e a c h a m i n o a c i d , h o w e v e r , v a r r i e d . The t h r e e b a s i c amino a c i d s , l y s i n e , h i s t i d i n e , a n d a r g i n i n e in MGM-431 medium d i d not d e c r e a s e m a r k e d l y . T h i s does not mean t h a t t h e s e a m i n o a c i d s are not i m p o r t a n t . If any one of t h e s e amino a c i d s w a s d e l e t e d , t h e growth o f t h e c e l l s w a s s e v e r e l y i m p a i r e d . These t h r e e amino a c i d s , t h e r e f o r e , are e s s e n t i a l f o r t h e
260
INSECT CE L L L I N E : A M I N O A C I D U T I L I Z A T I O N A N D R E Q U I R E M E N T S
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Media Fig. 3. Number o f c e l l s a f t e r 8 d a y s c y l t i v a t i o n o f Px 58 c e l l s i n M G M - 4 3 1 m e d i a l a c k i n g o n e a m i n o a c i d s . ( A f t e r M i t s u h a s h i , 1976). g r o w t h of t h e c e l l s . In a d d i t i o n to t h e b a s i c amino a c i d s , t h r e o n i n e , s e r i n e , g l y c i n e , v a l i n e , p h e n y l a l a n i n e , / ^ - a l a n i n e , t r y p t o p h a n , and g l u t a m i c a c i d d e c r e a s e d o n l y s l i g h t l y . A m o n g t h e s e amino a c i d s , t h r e o n i n e , s e r i n e , v a l i n e and t r y p t o p h a n w e r e f o u n d t o be e s s e n t i a l w h i l e g l y c i n e , p h e n y l a l a n i n e , / 3 - a l a n i n e , and g l u t a m i c a c i d w e r e found to be n o n - e s s e n t i a l . G l u t a m i n e , a s p a r a g i n e , and c y s t i n e d e c r e a s e d c o n s i d e r a b l y i n t h e g r o w i n g c u l t u r e . G l u t a m i n e and c y s t i n e w e r e f o u n d t o be e s s e n t i a l , w h i l e a s p a r a g i n e w a s none s s e n t i a l . Following this group, aspartic a c i d , methionine, t y r o s i n e , isoleucine, l e u c i n e , and p r o l i n e w e r e c o n s u m e d . A l l o f t h e s e amino a c i d s w e r e f o u n d to be essential. The common p a t t e r n s of amino a c i d c h a n g e s i n g r o w i n g c e l l c u l t u r e s , w h i c h c a n b e summarized f r o m t h e data on t h e four c e l l l i n e s h i t h e r t o s t u d i e d , are t h e i n c r e a s e o f
