glands (hepatopancreas) of zoea-I stage larvae of H. araneus both developing regularly,. *Supported by Deutsche Forschungsgemeinschaft (Sto 75, 4-9) and ...
HELGOL~NDER MEERESUNTERSUCHUNGEN Helgot~inder Meeresunters. 36, 67"75 (1983)
I n f l u e n c e of s t a r v a t i o n a n d f e e d i n g o n t h e h e p a t o p a n c r e a s of l a r v a l H y a s a r a n e u s (Decapoda, Majidae)* V. Storch 1 & K. Anger 2 1Zoologisches Institut; Im N e u e n h e i m e r Feld 230, D-6900 Heidelberg, Federal Republic of Germany 2Biologische Anstalt Helgoland (Meeresstation); D-2192 Helgoland, Federal Republic of Germany
ABSTRACT: Zoea-1 larvae of Hyas araneus were kept under different nutritional conditions. Their midgut glands were investigated with a transmission electron microscope. The glandular epithelium consists of the cell types known from adult decapods. It is mainly the R-cell type that undergoes ultrastmctural alterations which reflect nutritional conditions. R-cells of fed larvae are characterized by large lipid inclusions; after a certain perio d of food deprivation (point-of-noreturn) the original ultrastmcture cannot be reestablished. Refeeding results in large glycogen deposits in these cells.
INTRODUCTION Food is c o n s i d e r e d one of the k e y factors controlling survival i n m e r o p l a n k t o n i c larvae a n d a n extensive literature exists on the i n f l u e n c e of both quality a n d q u a n t i t y of nutrition. Effects of starvation periods, however, w h i c h must b e expected i n a patchy e n v i r o n m e n t such as the m a r i n e p l a n k t o n , are far less known. A n g e r & Dawirs (1981) e x p e r i m e n t a l l y i n v e s t i g a t e d impacts of food d e p r i v a t i o n o n the rate of larval developm e n t a n d survival of the spider crab, Hyas araneus. They r e v i e w e d our p r e s e n t knowl e d g e a b o u t starvation resistance i n d e c a p o d larvae. Later they s u g g e s t e d that some response patterns i n relation to a b s e n c e of suitable food m a y b e g e n e r a l features of b r a c h y u r a n larvae {Anger et al., 1981). A l t h o u g h there is a l r e a d y some information on c h a n g e s i n the e l e m e n t a l a n d b i o c h e m i c a l composition of larvae exposed to starvation {Anger & Dawirs, 1982), the physiological a n d ultrastructural basis of the effects observed has r e m a i n e d unclear. This lack of u n d e r s t a n d i n g is particularly obvious i n the p h e n o m e n a called " p o i n t - o f - n o - r e t u r n " a n d "point-of-reserve-saturation" (Anger & Dawirs, 1981), both characterized b y u n k n o w n irreversible processes. The p r e s e n t study was c o n d u c t e d to depict ultrastructural c h a n g e s i n the m i d g u t g l a n d s (hepatopancreas) of zoea-I stage larvae of H. araneus b o t h d e v e l o p i n g regularly, *Supported by Deutsche Forschungsgemeinschaft (Sto 75, 4-9) and Bundesministerium fiir Forschung und Technologie, Bonn (MFU-0502/7). © Biologische Anstalt Helgoland, Hamburg
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a n d exposed to differential periods of starvation. These c h a n g e s are c o m p a r e d with those after different schedules of food p r e s e n c e a n d absence.
MATERIAL A N D
METHODS
In J a n u a r y 1982, ovigerous females were d r e d g e d n e a r the North Sea i s l a n d of H e l g o l a n d . Thereafter they w e r e m a i n t a i n e d i n a laboratory r e c i r c u l a t i n g system with n a t u r a l seawater (ca 32 %°) kept at constant 2 °C, u n t i l the first larvae hatched. These were collected at 6 °C as described b y A n g e r & Dawirs (1981) a n d t h e n transferred i n d i v i d u a l l y into vials with 25 ml of filtered seawater (Millipore m e m b r a n e filter, 0.4 ~tm pore size). W h e n fed, the larvae received fresh food (Artemia spec. n a u p l i i a n d Brachionus plicatilis) a n d fresh s e a w a t e r every second day. Further details on the m a i n t e n a n c e a n d t r e a t m e n t of both fed a n d starved larvae are g i v e n b y A n g e r & Dawirs (1981). All e x p e r i m e n t s were carried out at 12 °C. The e x p e r i m e n t a l d e s i g n is s h o w n i n Figure 1. For electron microscopy, larvae were s a m p l e d a n d treated i n the following way: to facilitate p e n e t r a t i o n of the fixative, the a b d o m e n , dorsal spine, a n d rostrum of each larva were cut off u n d e r a stereo microscope. The rest of the larval body was i m m e d i a t e l y transferred into cold 3.5 % g l u t a r a l d e h y d e i n 0.1 M SSrensen's buffer (pH 7.5) for 2 h. After r i n s i n g the m a t e r i a l five times for 5 rain, respectively, i n cold 0.1 M SSrensen's buffer, it was postfixed i n 1 % o s m i u m tetroxide solution for 2 h. At all stages of fixation, the solutions were m a i n t a i n e d at 2 °C. After
Days after hatching 0 |
a
1
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2 i
3 !
4
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6 i
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8 =
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'-
Obt
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~,
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fed control
IIIIIIIIIIII ,], ...........
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Sstarved ,,,c,ontr o l
]
no
C c ~ .... -~ d i"
~- e I,, ~ - f II IIIIII LU
g
IIIIIIIIIIIIIIIII II
I = samp[e = feeding
l period starvation I
Fig. 1. Experimental design: Schedule of feeding and starving zoea-1 stage larvae of Hyas araneus. Arrows: times of sampling larvae for electron microscopy
H e p a t o p a n c r e a s of l a r v a l H y a s araneus
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d e h y d r a t i o n t h r o u g h a g r a d e d series of e t h a n o l at room t e m p e r a t u r e , the m a t e r i a l was e m b e d d e d in araldite. Sections w e r e cut on to w a t e r w i t h a n u l t r a m i c r o t o m e (Ultracut Reichert) a n d p i c k e d u p on c o a t e d c o p p e r grids. After s t a i n i n g w i t h u r a n y l a c e t a t e (saturated solution in 70 % m e t h a n o l ) a n d l e a d citrate, t h e y w e r e e x a m i n e d w i t h a Zeiss EM 9 S-2 e l e c t r o n microscope. 1 ~m sections u s e d for l i g h t m i c r o s c o p y w e r e s t a i n e d w i t h a n a q u e o u s solution of 1 % t o l u i d i n e blue. RESULTS As in o t h e r d e c a p o d s so far i n v e s t i g a t e d w i t h t h e t r a n s m i s s i o n e l e c t r o n m i c r o s c o p e the e p i t h e l i u m of t h e m i d g u t - g l a n d s of H y a s araneus l a r v a e consists of c l e a r l y disting u i s h a b l e cell t y p e s (R-, F-, B-cells) w h i c h arise from E-cells l o c a t e d in the blind, distal tip of the h e p a t o p a n c r e a s tubules. T h e question of cell l i n e a g e w i t h i n t h e organ, however, has not y e t b e e n a n s w e r e d d e f i n i t i v e l y (for r e c e n t discussion see: G i b s o n & Barker, 1979; H o p k i n & Nott, 1980). A l l of t h e s e cells b e a r an a p i c a l microvillous border. Basally t h e y are c h a r a c t e r i z e d b y a t u b u l a r labyrinth, also t y p i c a l of other d e c a p o d species, w h i c h at least p a r t l y is c o n n e c t e d to the l a t e r a l a n d b a s a l p l a s m a m e m b r a n e (Fig. 2a). The g l a n d u l a r tubuli are s u r r o u n d e d b y a b a s a l l a m i n a u n d e r l a i n b y a m e s h w o r k of striated m u s c l e ceils a n d flat e x t e n s i o n s of cells c o n t a i n i n g e l e c t r o n - d e n s e g r a n u l e s p r o b a b l y r e p r e s e n t i n g p i g m e n t cells (Fig. 2b). A d d i t i o n a l l y , cells c o n s i d e r e d excretory or w h i t e p i g m e n t ceils ( G r e e n & Neff, 1972) w e r e f o u n d (Fig. 2b). R - c e 11 (Figs 2-4): R-cells or r e s o r p t i v e cells are t h e most n u m e r o u s of t h e a b o v e m e n t i o n e d cell types. T h e y a r e m a i n l y c o n c e r n e d w i t h t h e a s s i m i l a t i o n a n d storage of n u t r i e n t s a n d thus c o n t a i n m u c h l i p i d a n d / o r g l y c o g e n w h e n the a n i m a l s w e r e w e l l fed. Lipid i n c l u s i o n s differ in size a c c o r d i n g to e x p e r i m e n t a l conditions. Also g l y c o g e n can o c c u p y l a r g e a r e a s of this cell type. The e n d o p l a s m i c r e t i c u l u m is not a p r o m i n e n t f e a t u r e of R-cells b u t varies in e x t e n t a n d ultrastructure a c c o r d i n g to f e e d i n g / s t a r v a t i o n conditions. It is this cell t y p e that m a y contain calcium, phosphorus, a n d m a g n e s i u m w h i c h a c c u m u l a t e in c o n c e n t r i c a l l y structured g r a n u l e s ( H o p k i n & Nott, 1979; Storch et al., 1982). M i t o c h o n d r i a a g a i n v a r y ultrastructurally; the s a m e a p p l i e s to the n u m b e r of l y s o s o m e - l i k e inclusions. In n e w l y h a t c h e d l a r v a e this cell t y p e is c h a r a c t e r i z e d b y m a n y l i p i d inclusions the l a r g e s t of w h i c h m a y o c c u p y most of the cellular v o l u m e (Fig. 3a). The n u c l e u s occurs s i n g l y a n d t e n d s to b e s o m e w h a t flattened. The r o u g h e n d o p l a s m i c r e t i c u l u m occurs as i n d i v i d u a l c i s t e r n a e t h r o u g h o u t the c y t o p l a s m a n d m a y form b u l k y stacks. M i t o c h o n d r i a t e n d to b e in a p i c a l location a n d do not show peculiarities. T h e s m a l l or i n t e r m e d i a t e G o l g i a p p a r a t u s is n o r m a l l y l o c a t e d in a s u p r a n u c l e a r position; g l y c o g e n is lacking. R-cells u n d e r g o m a r k e d u l t r a s t r u c t u r a l a l t e r a t i o n s i n r e s p o n s e to food d e p r i v a t i o n . P r e s u m a b l y this cell t y p e e n a b l e s l i p i d storing c r u s t a c e a to w i t h s t a n d starvation p e r i o d s for m o r e t h a n o n e y e a r (Storch et al., 1982). T h e a l t e r a t i o n s i n c r e a s e g r a d u a l l y d u r i n g t h e course of starvation a n d are m o r e or less i d e n t i c a l in different s p e c i m e n s . After 16 d a y s (Fig. 3b) t h e i r p r o m i n e n t f e a t u r e s are: a r e d u c e d cell size, l a c k of stored lipid, r e d u c t i o n of m i t o c h o n d r i a , i n c r e a s e of t h e t h i c k n e s s of the b a s a l l a m i n a w h i c h exhibits m a n y i r r e g u l a r infoldings. T h e b a s a l t u b u l a r system is v o l u m i n o u s (Figs 3b, 4a).
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Fig. 2. (a) Basal portion of R-cells (12 000:1). {b) Cells immediately below the glandular cells of the midgut gland (M): pigment cells (P) and excretory or white pigment cells (E) (14 300:1) A starvation p e r i o d of 12 d a y s (which causes p r i n c i p a l l y i d e n t i c a l c h a n g e s as one of 16 days) followed b y a f e e d i n g p e r i o d of 4 days, results in a c o m p l e t e l y different ultrastructure (Figs 3c, 4b): G l y c o g e n fields a n d small l i p i d d r o p l e t s a r e t h e p r o m i n e n t features. The m i t o c h o n d r i a are h e a v i l y swollen, ER profiles a r e rare. E l e c t r o n - d e n s e g r a n u l e s a n d different i n c l u s i o n b o d i e s m a y occur s i n g l y or in groups. F e e d i n g i m m e d i a t e l y after h a t c h i n g followed b y a starvation p e r i o d of 4 or 8 d a y s is different again. Large crescent a n d r o u n d i s h l i p i d d r o p l e t s are existent, g l y c o g e n is lacking.
H e p a t o p a n c r e a s of larval H y a s a r a n e u s
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Fig. 3. Diagrams of R-cells under different conditions. (a) fed with Artemia and Brachionus. (b) starvation period of 16 days. (c) 12 days starvation and subsequent feeding (4 days) F - c e 11 (Fig. 5a): F-cells or fibrillar cells are highly basophilic, w h i c h is characteristic of e n z y m e - p r o d u c i n g cells. The F-cells of H y a s a r a n e u s zoeae are rather uniform a n d more or less i n d e p e n d e n t on food deprivation a n d s u b s e q u e n t f e e d i n g procedures. They exhibit extensive r i b o s o m e - s t u d d e d e n d o p l a s m i c r e t i c u l u m the profiles of which are more or less p a r a l l e l to the l o n g axis of the cell. Mitochondria a n d n u m e r o u s Golgi bodies are present. As i n other cells, starvation results i n dilatation of ER cisternae. B - c e 11 (Fig. 5b): In shape the B-cells correspond more or less to goblet cells of the vertebrate intestine. They b e a r a single, large vacuole (blisterlike cells) w h i c h squeezes the cytoplasm to the periphery, the cell o r g a n e l l e s thus b e i n g c o n c e n t r a t e d i n a very t h i n m a r g i n a l layer of the cytoplasm. Basically a n d apically there is some more space for organelles left. A single cell n u c l e u s is located at the base, w h e r e the cell is r e d u c e d to a narrow stem w h i c h is t e n u o u s l y c o n n e c t e d to the b a s a l lamina.. The apical portion is characterized by n u m e r o u s m e m b r a n e i n v a g i n a t i o n s which form c h a n n e l s e x t e n d i n g deep into the cell (Fig. 5b). Alterations provoked b y f e e d i n g or starvation were not found.
DISCUSSION The p r e s e n t study demonstrates that the e p i t h e l i u m of the h e p a t o p a n c r e a s of larval H y a s a r a n e u s w h i c h is composed of the cell-types well k n o w n from adult decapods
(Gibson & Barker, 1979; H o p k i n & Nott, 1979, 1980; Loizzi, 1971; Storch & Welsch, 1977) u n d e r g o e s m a r k e d ulh'astructural c h a n g e s d e p e n d e n t on the n u t r i t i o n a l status. It is
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Fig. 4. (a) Basal portion of R-cell after 16 days without food with tubular system (T) and folded basal lamina (B). (b) Apical portion of R-cell (12 days starvation, 4 days refeeding) with irregular microvillous border, swollen mitochondria (M), and large amounts of glycogen (14 300:1). Inset: inclusion body (3800:1)
H e p a t o p a n c r e a s of larval H y a s araneus
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Fig. 5. (a) Central part of F-cell (fed}. (b) Apical part of B-cell (fed) (14 300:i) m a i n l y the R-cell that is affected, R-cells of freshly h a t c h e d zoeae are stuffed with lipid i n c l u s i o n s r e p r e s e n t i n g yolk reserves. They r e s e m b l e the R-cells of adult l a n d - d w e l l i n g hermit crabs (Storch et al., 1982; Lawrence, 1970) which are well a d a p t e d to tolerate severe starvation periods e v e n for more t h a n one year. As has b e e n shown b y A n g e r
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& Dawirs (1981), this reserve i n zoea-1 larvae of H . a r a n e u s is not sufficient to reach the next instar w h e n the larva suffers from c o n t i n u a l lack of food. In contrast to all other decapods so far i n v e s t i g a t e d electron microscopically, g l y c o g e n was n e v e r f o u n d i n conspicuous a m o u n t s i n R-cells of regularly fed i n d i v i d u a l s . This is r e m i n i s c e n t of the liver of teleost fish species the m e t a b o l i s m of w h i c h c a n b e p r e d o m i n a n t l y more lipid- or more carbohydrate-oriented. During starvation the a m o u n t of lipid decreases steadily. After more t h a n 4 days Rcells appear small due to d e p l e t i o n of lipid. As i n other crustacea a n d i n other a n i m a l p h y l a the decrease of lipid is a c c o m p a n i e d by ultrastructural alterations that b e g i n to a p p e a r soon after the cessation of feeding. As has b e e n found by A n g e r & Dawirs (1981) 16 days is close to the m a x i m a l survival time of zoea-1 larvae at 12 °C. At this stage of starvation the b a s a l l a m i n a shows a n increase i n thickness as has b e e n reported b y earlier authors i n bivalves, crustaceans, insects, a n d teleosts s u b j e c t e d to starvation (for references see Storch et al., 1982). As i n a variety of cells of different p h y l a affected by starvation, the r e s i d u a l bodies i n d i c a t i n g autolysis increase i n n u m b e r . These f i n d i n g s correspond to results o b t a i n e d e. g. b y Schmekel (1972) for molluscs, J a n s s e n & M611er (1981) for cnidarians, a n d Storch et ai. (1982) for crustaceans. The e n d o p l a s m i c reticulum, w h i c h is regularly d e v e l o p e d i n w e l l - f e d larvae, c a n be found b r o k e n u p into r o u n d e d vesicles or short a n d w i d e n e d cisternae. Again, similar observations have b e e n m a d e i n different p h y l a (for references see David, 1967; Schmekel, 1972; Storch et al., 1982). These findings are i n accordance with the observations of H a l l b e r g & Hirche (1980) that i n m a r i n e c a l a n o i d copepods d i s c o n t i n u a t i o n of f e e d i n g d u r i n g o v e r w i n t e r i n g is associated with low e n z y m e activities. Mitochondria b e c o m e s w o l l e n e v e n after a relatively short period of starvation. This is p a r a l l e l e d by findings, for example, i n teleost hepatocytes (Storch & Juario, 1983) w h e r e m i t o c h o n d r i a can surpass the cell n u c l e u s i n size after cessation of feeding. Refeeding after 8 a n d 12 days, respectively, does not result i n a complete recovery of the affected R-cells. As is shown i n Fig. 4b the m i t o c h o n d r i a are still s w o l l e n a n d occur m a i n l y in a n apical position. T h e y are u n d e r l a i n b y c o n s i d e r a b l e q u a n t i t i e s of glycogenparticles and lipid droplets which are smaller i n size t h a n i n well fed larvae. G l y c o g e n is a w i d e - s p r e a d carbohydrate reserve material. W h e n required, for e x a m p l e to supply the e n e r g y for m u s c u l a r contraction, the glycogen is b r o k e n down a n d transported to the muscle as glucose. O n l y the latter is f o u n d i n the blood, a n d the glucose of the blood (or haemolymph) of several species is m a i n t a i n e d at a steady level d u r i n g adverse conditions, such as starvation (Veldhuijzen, 1975). Thus, the g l y c o g e n deposit m a y help to e x p l a i n why larvae w h i c h have starved for some time can survive a n d move for rather long periods. A n g e r & Dawirs (1981) found that after a 12 days starvation period still b e l o w the lethal threshold at 12 °C no larva recovered a n d r e a c h e d the second stage, b u t some of them lived for u p to 29 days before they died. Their a s s u m p t i o n that these zoeae h a d not completely lost their ability to take up food is thus corroborated by our electron microscopic findings. Additionally, the data p r e s e n t e d here indicate that a r e e s t a b l i s h m e n t of the R-ceil uttrastructure s e e m s to b e impossible after a certain period of initial starvation i m m e diately after hatching. W h e n larvae were fed i m m e d i a t e l y after h a t c h i n g (and prior to starvation, e x p e r i m e n t s f a n d g i n Fig. 1), the R-cells were f o u n d to b e ultrastructurally
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d i f f e r e n t f r o m t h o s e t r e a t e d i n t h e o p p o s i t e w a y ( e x p e r i m e n t s d, e, h i n Fig. 1). F o u r d a y s of i n i t i a l f e e d i n g a r e e v e n s u f f i c i e n t to b u i l d u p a n e w c u t i c l e . T h i s , a g a i n , c o r r o b o r a t e s t h e f i n d i n g s of A n g e r & D a w i r s (1981). A c k n o w l e d g e m e n t s . We are grateful to Miss C. Pfischel and Miss R. Mummert for technical
assistance. Miss B. L~immel und Mrs. I. Ranker made the line drawings. Our thanks are also due to Dr. J. Bolton for correcting the manuscript.
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