ISSN 0300.9629/97/$17.00. PI1 SO300-962Y(97)00024.8. ELSEVIER. Digestive Enzyme Activities of Penaeus notiuh During. Reproduction and Moulting Cycle.
Camp. Biochem. Phystol. Vol. 118A, No. 4, pp. 1267-1271, Copyright 0 1997 Elsevier Science Inc. All rights reserved.
1997
ISSN 0300.9629/97/$17.00 PI1 SO300-962Y(97)00024.8
ELSEVIER
Digestive
Enzyme Activities of Penaeus notiuh Reproduction and Moulting Cycle
Isis Fernhndezf
Mario Oha:
Olympia Curdo,
During
7 and Ahin Van Wormhoudt#
*CENTRO DE INVESTIGACIONESMARINAS, UNIVERSIDAD DE LA HABANA, CALLE TREINTA 2808
ENTRE PRIMERA Y
HABANA, CUBA; TFACULTAD DE BIOLOGI& UNIVERSIDAD DE LA HABANA, CALLE VEINTICINCO ENTRE
TERCERA, CIWAD
1 Y J, CIIJDAD HABANA, CUBA; AND $LABORATOIRE DE BIOLOGIE MARINE IX COLLEGE DE FRANCE, BP 225, 29900 CONCARNEAU, FRANCE
ABSTRACT. The degree
of ovarian
amylase, trypsin, chymotrypsin,
maturation
and the specihc
carboxypeptidase
in hepatopancreas
activities
and stomach
ot
A and B and leucine aminopeptidase
were determined at differOvarian maturation appeared from
ent stages of the moulting cycle of wild female pink shrimp Penaeus not&. intermoult stage C,-C: to early premoult stage D,. The highest values for amylase in stomach and hepatopancreas ocurred at stages D, and B2 to Cj-C+ in stages B: to Cl-C+,
highest value was for trypsin TAME exopeptidases
respectively.
and in the stomach
in stages C,-C:
1997. 0
1997 Elsevier
Science
KEY WORDS. Panaeusnotialis,amylase, protease, stomach,
reproduction,
digestive
velopment
during
enzymes
with the stages of de-
the life cycle (10,24,29),
tion (23) and the moulting mon origins
change
cycle (1,21,25).
of the alterations
of enzymatic
ovarian
matura-
The most comvalues
are food
composition (28), season of the year (25) and circadian rhythm (26). Enzyme activities are also influenced by factors released stalk. These
by neurosecretory factors
can stimulate
in turn may result in increases Because
of the importance
zation of nutrients,
it is essential change
this
knowledge
artificial
culture
MATERIALS Animals
present
protein
in enzymatic
of digestive
digestive
enzymes
glands
synthesis, activities
which (27).
in the utili-
to know how the principal
can be used as a reference
Subsequently, pattern
Inc. trypsin,
chymorrypsin,
carhoxypeptidase,
in circulating
water
and classified
in accordance
scribed
aminopeptidase.
for up to 2 hr. Females
for this species
the post-orbital segment,
curve
with
(16).
Animals
to the spine
and the weight
were selected
the moulting of the
of the intact
stages
were measured animal
defrom
last abdominal was recorded.
Tissues Once
the substage
gut gland), The
stomach
organs
stomach
were
was identified, and ovaries weighed,
the hepatopancreas were dissected
and
the
to select
3 animals
(mid-
at 10:00 A.M.
hepatopancreas
were stored at - 60°C until analysis;
were processed
the
maintained
showed
and
150 specimens
for each moulting
stage.
The gonadosomatic index (GSI) was determined to estahlish the degree of gonadal maturity in females according to
METHODS
were then
aminopeptidase
and DJ. I‘OMP RIOCHEMPHYSIOI.
Ovarian Maturity Determination
Penaeus notidis were ohtained from Cienfuegos Bay, situated in the southern and central part of Cuba. Animals were captured by a commercial ship. Nets were towed for 30 min, and the shrimp
Leucine
the The
in the
of this species.
AND
and Ci-C,.
at stage C,-C:.
in the eye-
enzymes
in wild animals.
was highest
moulting
INTRODUCTION Crustacean
in the hepatopancreas
values and peaks in stomach were observed at stages CI-Cq
118A;4:1267-1271,
hepatopancreas,
activity
value was observed in D,. For the endopeptidases,
in stomach during stage D, and in the hepatopancreas
showed peaks in hepatopancreas
the lowest proteolytic
Total proteolytic
the highest
on board
the vessel
Address reprint requests to: A. Van Wormhoudt, Laboratoire de Biologic Marine du Colkge de France, BP 225, 29900 Concameau, France. Received 27 April 1994; revised 30 August 1995; accepted 6 February 1997.
following
weight
-
formula:
ovarian
weight
GSI
=
(ovarian
weight/animal
X 100.
Tissue Extracts and Protein
Detewninution
Extraction buffer solutions were sodium phosphate (10 mM, pH 7.0) for hepatopancreas and ammonium acetate (10 mM, pH 8.5) for stomach. Tissue disruption was attained by homogenization (Ultraturrax) for 30 set, followed by
1268
I. Fern6ndez
et al.
TABLE 1. Enzymes and assay procedures Enzyme
(reference)
Amylase Protease Trypsin
Substrate Glycogen (1.5%) Yellow caseine 1% BAI’NA lo-’ M TAME 10-l M BTEE 10-j M
(2) (3) (5)
( 11)
Chymotrypsin Carboxypeptidase
A (7)
HPLA
10ml M
Carboxypeptidase
B (6)
BzGA
lo--’ M
LPNA
10-l
Leucine
aminopepticlase
(20)
Temperature
Wavelength
(0°C)
(nm)
37 37 20 25
540 420 410 247 256
25
254
25
410
Buffer and pH Sodium Sodium
phosphate, phosphate,
10 mM, pH 7.0 10 mM, pH 7.0
Tris-HCI 50 mM CaC12 20 mM, pH 8.0 Tris-HCI 50 mM CaC& 20 mM, pH 8.0 Tris-HCl 25 mM NaCl 0.5 M, pH 8.0 Tris-HCI 25 mM NaCl 0.1 M, pH 8.0 Tris-HCI 0.1 M MgCl: 5 mM, pH 8.0
M
All substrates are from Sigma Chemical Co. Synthetic ones are dissolved m dimethylsulfoxide before being diluted. A total volume of 1 ml was taken for each measurement. BAPNA, benzoyl-L-arginine-p-nitroanillde; TAME, tosyl-L-arginine methyl ester; BTEE, b enzoyl-L-tyrosine ethyl ester; HPLA, hippurylLx_-phenyllactic acid; BzGA, hippuryl-L-arginine; LPNA, leucine p-nitroanilide.
sonication
(60
set).
Homogenates
were
centrifuged
20,000 g for 10 min at 4 “C and supematants Protein
concentration
was determined
at
were used.
by Lowry’s method
(14).
Digestive Enzyme Activities in the Stomach and Hepatofiuncreas
minimum Enzyme
The protocols shown
used to analyze
in Table were
amylases
1. The
expressed
were
results as
expressed
tidases
(trypsin,
expressed
as units/min/mg
to an increase length.
(SA).
The
= mg liberated
proteases were expressed as casein/min/mg protein. For chymotrypsin,
carboxypep-
aminopeptidase), protein.
of 0.001
SA
are
the SA were
One unit corresponded
absorbance
SA peaked
in activity,
In hepatopancreas,
afterward,
D:, with a light
a decrease increase
amylase
in D+ Variability
a
until ecdysis
increased
in the activity
at Do-D,
until reaching
in B! and
was noted
until
was low.
determina-
activities
as amylase
A and B and leucine
activities
of the enzyme
specific
maltose/min/mg protein. The proteases SA = mg hydrolyzed the rest of the enzymes
the enzymatic
amylase
by a decrease
at D,,; this low level was maintained
took place.
Determinations
C,-C,;
tions
In the stomach,
AMYLASES.
(Fig. 2a), followed
at the specified
GENERAL
PROTEASES.
The
maximum
values
of specific
proteolytic activity in stomach were in stages D,, D4 and B, (Fig. 2b) whereas in hepatopancreas, the maximal values were C,-C?
and C\-C,,
and the minimal
are values
were
at Do and B,. Tryptic activity in stomach attained at D, with both benzoyl-L-arginine-p--nitroani-
ENDOPEPTIDASES.
its maximum
wave8
T Statistical Analysis GSI data were arcsin were one-tail
ANOVA
of P < 0.05. Only cussed.
transformed.
The statistical
and Duncan’s
results
presented
tests used
test for a probability at these
levels are dis-
RESULTS Ovarian Maturation and the Moulting Cycle During the moulting the immature stages
cycle, the GSI changed from 0.2 for to 7 for specimens with totally devel-
oped ovaries (Fig. 1). During the post-moult cycle and the late pre-moult cycle, the gonadosomatic levels well lower and of similar magnitude; the values were significantly higher in stages C,-CL and C,-C, of intermoult, followed by Do and D,.
Bl
BZ
Cl-C2
O-C4
DO
Di
Dt
D3
D4
Moultingstages FIG. 1. Gonadosomatic index variation in relation to the moulting stages in Penaeus notiaks. Results are given as mean of three measurements 2 SE. Three animals were used at each stage.
Digestive
of P. notialis
Enzymes
q
1269
Figure 4c shows the relatively
q
Stomach
a
Heprfopmcreas
tine
aminopeptidase;
and
D,--D2
maximum
for hepatopancreas
low specific
activity
of leu-
values were attained and
in B?
in D2 and C\-C,
for
stomach. Minimal values were found in B2 and D4 for stomach and in B,, C,-C2 and D1 for hepatvpancreas
.e d d
n
700 a
Q
Stomach
Hepatopancreas
.e 2
e a M E
0.3 Bl
2 6 2 u 3
DI
D2
D3
D4
E 3500, .?c 0)
T
f
0.2
h
0 # bOL m p
B2 Cl-C2 C3-C4 DO
k? ‘c ._ e 2
0.1
3000 2500 2000
% t! 0
Bt
BI
Cl-C2 C3-C4
DO
Dl
D2
D3
D4
cb WI Y z .. .E,
1500 1000
Moultingstages FIG. 2. Variation of the specific amylase and protease activities in the stomach and hepatopancreas from Penaeusnothdii during the intermoult cycle. Results are given as the mean of three measurements -C SE. Three animals were used at each stage. (a) Amylase activity; (b) total protease activity.
Bl
Bt
Cl-C2
81
82
Cl-C2 C3-C4 DO
C3-C4
DO
Dl
D2
D3
D4
Dl
D2
D3
D4
lide (BAPNA) and tosyl-r_-arginine methyl ester (TAME) as substrates. Minimal values were found at C,-C2 and D, for BAPNA
and at CI-CI
and D4 for TAME
h). In the hepatopancreas,
the maximal
DO and 1),-D,
and C,-Cr
for BAPNA
Chymotryptic in stomach found
activity (Fig.
in C-C2
in stomach,
3~).
was higher Maxima
and in D-D\
the maxima
were in C,
for TAME.
in hepatopancreas
for hepatopancreas with minima
were noted
EXOPEPTIDASES. Activities B are presented
(Fig. 3a and
values
than were
at B2 and D+
at B, and D+
of carboxypeptidases
in Fig. 4a and b, respectively.
A and
In hepatopan-
cress, the carboxypeptidase A peak occurred at CI-C2 and minima at D, and B,; carhoxypeptidase B peaked at C&Z4 and minima at B:, DO and 1):. In the stomach, carboxypeptidase A activity
was highest
peptidase H activity reached and D,; mmima were found
in D, and lowest in D,; carboxymaximum values in Bz, C,-CI in B, and Do.
Moaitiag stages FIG. 3. Variation of endoprotease activities in the stomach and hepatopancreas from Penaeus notiah during the intermoult cycle. Results are given as the mean of three mea. surements f SE. Three animals were used at each stage. (a) Trypsin activity measured with BAPNA as the substrate; (b) trypsin activity measured with TAME as the substrate; (c) chymotrypsin activity measured with benzoyl+tyrosine ethyl ester (BTEE) as the substrate.
1270
1. Fernandez et al.
DISCUSSION EJ
Aepatopancreas
A well-known
relationship
cle and ovarian
900
ing moulting
so0 700 600
exists between
maturation. stages
Ovarian
in which
the highly
demanding
concluded
preceding
ecdysis.
processes
takes place at stages D, (4,17), it occurs during stages C,-C, females
in advanced
phases
is hard and
immediately
In Palaemon
maturation
cy-
occurs dur-
the exoskeleton
metabolic
nus maenas, we found
the moulting
maturation
serratus,
has
ovarian
whereas
in Carci-
(19). In this study, of ovarian
maturity
in intermoult (C,-C,) and early premoult (DC-D,) stages, whereas immature ovaries appeared in late post-moult (B,BJ and late pre-moult Bl
B2
Cl-C2
C3-C4
DO
DI
D2
D3
maturation
D4
occurs
(DI-D,).
Thus, in P. no&&s, ovarian
in agreement
with the abovementioned
relationship. Amylase b
enzymes.
is one of the best studied An amylase
maenus
( 1). Two maxima
D: in P. serrattts, of the activity pre-moult with
100
0
sr
the
intermoult
that
cia
cic4
00
dl
d2
d3
D‘l
to the feeding
chronically ties build
I
moulting
process
in P. notialis
cells
associated
secreted
change
that protease
synactivi-
after ecdysis
with
the
activity
(9). of the
of the
by the Y organ (21). Our studies
are coincident is considered
previ-
are closely
by the stimulation
with the abovementioned
ies in the case of hepatopancreas Trypsin
activities they
the
in which
it was found
and that
at D: may be caused
hormone
that
coincide
and early pre-moult
Coincidentally,
up in D, and Di and decrease
hepatopancreas
B and in the
we found
and stomach
(22). It has been observed
The increase
I c
Here
and proteolytic
and
(25). A maximum
in the post-moult
stages
amyolytic
in stages A?, C,
kerathurus
in hepatopancreas
feed actively.
related
liz
was found
maxima
animals ously
in Penaeus
digestive in C+ in C.
in stages C,, D, and
three peaks,
D, in P. elegans (30).
amylase
crustacean
peak was found
were found
whereas
Dr, were observed 200
activity
at intermoult
to be the most
stud-
stages.
important
crusta-
cean digestive enzyme, accounting for 50-60% of the digestive proteolysis (9). In P. juponicus and P. kerathurtcs, trypsin contributes from 40’)/0 to 50%1 (8), whereas chymotryptic activity makes a smaller contribution in some penaeids (9,12,15). On the contrary, some studies did not shown any tryptic activity and only maxima of chymotrypsin activity Bl
B2
Cl-C2
C3-C4
DO
Dl
D2
D3
D4
Moulting stages
in the
hepatopancreas
(18,19).
It has heen
pattern FIG. 4. Variation of exoprotease activities activities in the stomach and hepatopancreas from Penaeus not&u% during the intermoult cycle. Results are given as the mean of three measurements -C SE. Three animals were used at each stage. (a) Carboxypeptidase A activity measured with hippuryl-mphenyllactic acid (HPLA) as the substrate; (b) carboxypeptidase B activity measured with hippuryl-L~arginine (BzGA) as the substrate; (c) leucine amino peptidase (LAP) activity measured with 1eucineMpnitroanilide (LPNA) as the substrate.
at the found
as total proteolytic
substages
C,,
trypsin
follows
that activity
Do and
in P. serrams
D
the same (25).
In this study, the highest tryptic activity appeared at D, in the stomach, whereas in the hepatopancreas, maximum values were observed in intermoult and late pre-moult stages. This leads us to the possibility that the inclusion of proteins from the environment during the feeding period causes a rise of the specific enzymatic activity in these stages for both stomach and hepatopancreas. Moreover,
subbe-
cause the digestive enzymes are produced only in the hepatopancreas, the peaks in the stomach are explained by a diffusion of the enzymes. With regard to penaeid exopeptidases, it has heen found that these activities have a maximum
at Dz and
L),. Leucine
aminopeptidase
had highest
Digestive
Enzymes
1271
of P. not&s
activity, followed by carboxypeptidase dase B (8). Opposite
A and carboxypepti-
to these reports, in this study, in P.
not&, the peaks of exopeptidase activities were found in intermoult stages and early pre-moult. The lowest activity corresponded
to leucine aminopeptidase.
Carboxypeptidase
A activity was higher than that of carboxypeptidase
B.
There are several causes pointed out here that could have changed the different activities of digestive enzymes in relation to the moulting
cycle and the ovarian
maturation.
Among these causes we may mention the absence of feeding in the extreme moulting stages (A, B,, Di and D4), the coincidence
of the beginning
of the feeding process (from Bz
until D,) with the oocyte development stimulating
and inhibiting
and the influence of
substances derived from eyestalk
neurosecretions. Special thanks to Dr. Yves Le Gal from the Luburatoire de Biologic Marine du Coll+e de France, Concarneau, for his support and advice. Thanks also tcj Drs. Albert0 Huberman and Manuel Aguilar from the Department of Biochemistry, I.N.N.S.Z., Mexico City, for their cornments on the manuscript.
References 1. Bauchau, A.G.; Mengeot,
2. 3.
4.
5.
6.
7. 8.
9.
10.
11.
12.
13.
J.C. Proteases et amylases de l’hepatopancreas des Crabes au tours du cycle de mue et d’intermue. Ann Sec. R. Zool. Belgique 95:29-37;1965. Bernfeld, I’. Amylases aand /I. Methods Enzymol. 1:149-154; 1955. Charney, 1.; Tomarelli, R.M. A calorimetric method for determination of the proteolytic activity of duodenal juice. J. Biol. Chem. 171:501-505;1947. Drach, P. Systeme endocrinien du pedoncule oculaire, duree l’intermue et vitellogenese chez Lean&r serratus. C.R. Sot. Biol. 149:2079-2083;1955. Erlanger, S.; Kokowsky, N.; Cohen, W. The preparation and properties of two chromogenic substrates of trypsin. Arch. Bi
