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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