• No results found

Digestibility of chitin in cod,Gadus morhua, in vivo

N/A
N/A
Protected

Academic year: 2020

Share "Digestibility of chitin in cod,Gadus morhua, in vivo"

Copied!
12
0
0

Loading.... (view fulltext now)

Full text

(1)

HELGOI.~NDER MEERESUNTERSUCHUNGEN Helgol~inder Meeresunters. 41,425-436 (1987)

D i g e s t i b i l i t y of chitin in cod,

Gadus morhua,

in v i v o

Eva Danulat

Instl'tut fiir Hydrobiologie und Fischereiwissenschaft; Z e i s e w e g 9, D-2000 Hamburg 50, Federal Republic of Germany"

ABSTRACT: Sixteen cod, Gadus morhua (L.), were individually fed a single ration of shrimps, Crangon allmanni. Four fish were killed and examined 6, 12, 24 and 48 h after the fish had been fed. Chitinase activities were measured in the extracts of stomach contents, stomach tissue, pyloric caecae, intestinal contents and intestinal tissue. The level of enzyme activity in different parts of the digestive tract was shown to be dependent on the phase of the digestive process. High concen- trations of the chitin degradation product N-acetyl-D-glucosamine were determined in the stomach and in the intestinal contents. Based on the chitin concentration in the food organisms and the individual food uptake, the amount of chitin consumed by each fish could be calculated. Only up to 9 % of the ingested chitin was recovered from the intestinal contents of the fish at any given time after feeding (6, 12, 24 and 48 h). In addition, only 2.4 % of the chitin consumed with the food could be recovered in the collected faeces of the fish. The 4 cod killed 48 h after feeding had completely emptied their stomach. Chitin digestion in these fish was calculated to have been 90 %.

INTRODUCTION

Early life stages of the majority of fishes d e p e n d on p l a n k t o n i c c r u s t a c e a n s as initial food. Insects a n d c r u s t a c e a n s m a y also constitute a n i m p o r t a n t part of the diet of a d u l t fish, e. g. m e m b e r s of the Cetorhinidae, C l u p e i d a e , S a l m o n i d a e a n d G a d i d a e a n d other famihes. T h e major c o m p o n e n t i n the exoskeletons of arthropods is the c a r b o h y d r a t e chitin (Muzzarelli, 1977) which m a y well b e the d o m i n a n t c a r b o h y d r a t e in the n a t u r a l diet of fish (Richards, 1951). The biological d e g r a d a t i o n of chitin is carried out b y a system of exocellular e n z y m e s (Jeuniaux, 1963), k n o w n as chitinases (EC 3.2.1.14) a n d chitobiases (EC 3.2.1.29, N-acetyl-glucosaminidases). Chitinases hydrolyze the p o l y m e r chitin to the level of di- a n d trisaccharides w h i c h i n t u r n are d e c o m p o s e d b y chitobiases to the s u g a r m o n o m e r b e t a - N - a c e t y l - D - g l u c o s a m i n e {NAG).

Over the past 25 years, the p r e s e n c e of chitinases in the guts of fishes h a s b e e n d e m o n s t r a t e d i n m a n y studies (Jeuniaux, 1961, 1963; Okutani, 1966; Cohn, 1972; M i c h a et al., 1973; P6r&s et a1.,'1973; Goodrich & Morita, 1977 a,b; King et al., 1977; F~inge et al., 1979; Lindsay, 1984; R e h b e i n et al., 1986). In fact, all b u t two of a p p r o x i m a t e l y 150 fish species tested for gut chitinases, gave positive results, the exceptions b e i n g the cyprinid Abramis brama a n d the African lungfish Protopterus aethiopicus (Micha et al., 1973).

T h e origin of chitinolytic e n z y m e s i n fish has b e e n discussed quite controversially: Some authors h a v e c l a i m e d that chitinase activities m e a s u r e d i n the g u t c o n t e n t s of the * Present address: University of British Columbia, Department of Zoology; 6270 University

(2)

426 Eva Danulat

fish species under study were of bacterial origin only (Goodrich & Morita, 1977b), which was generally accepted until Jeuniaux (1961) described chitinases as hydrolases that can also be synthesized in vertebrate tissues. Dandrifosse et al. (1965) were able to show that in vitro chitinases could indeed be synthesized and secreted by the gastric mucosa of

Anguilla ~nflgaris (Cuv.) and Salmo irideus (L.). The mechanism of chitinase secretion in vertebrates could be clarified in another study (Dandrifosse & Schoffeniels, 1967). Most searchers working on "fish gut chitinases" found indications of both chitinase originating from fish tissues and bacterial chitinase. The enzyme produced by chitinolytic bacteria is characteristically different from "fish own" chitinase, for example with regard to the optimum pH-range for its activity (Jeuniaux, 1963; Okutani, 1966; Lindsay et al., 1984; Danulat, 1986a). Considering the extreme abundance and the major contribution of chitin to the diet of many fishes, it is surprising that only a few publications exist on its digestibility in vivo (Geyer, 1940; Mann, 1948; Buddington, 1980; Lindsay et al., 1984). In contrast to the well documented fact that the guts of many fish constitute a rich source of chitinases, chitin is most often referred to as very poorly digestible or indigestible by fish (RSsch, 1985). Especially in the case of Salmo gairdneri, reports of the very poor digestibility of chitin (Buddington, 1980; Lindsay et al., 1984) seem puzzling since this species has been shown to have strong chitinolytic potential (Jeuniaux, 1963; Micha et al., 1973; Lindsay et al., 1984).

This paper describes experiments to determine the digestibihty of natural chitin in cod, Gadus morhua (L.). Cod are omnivorous marine fish, crustaceans being part of the diet during their whole life cycle. The extent to which cod feed on crustaceans is variable, depending on factors such as the age of fish and the local and seasonal food availability.

G. morhua does not grind the prey mechanically but swallows it whole. This is not only true for smaller organisms like shrimps (Crangon sp.) but also for large specimen of the brachyura (Liocarnicus, syn. Macropipus, and Cancer). Nevertheless, no macroscopic parts of the exoskeletons of the prey can be found in the hind gut of cod. In previous studies, strong chitinolytic activities have been measured, originating predominantly in the stomach of G. morhua (Danulat & Kausch, 1984; Lindsay & Gooday, 1985; Danulat, 1986a). Often, chitinase activities in the pyloric caecae and the intestine of cod seemed to be substantial as well (Danulat, 1986b). The study of the optimum pH-range of chitinase showed that the enzyme is active under physiological conditions in the cod: its optimum was determined at pH 5.1. The enzyme in the stomach extracts was very active also at pH values lower than 5.1 while the one in extracts of the intestine showed high activity at pH 7.5, but rapid decrease of activity at pH values below the optimum (Danulat & Kausch, 1984).

Now, additional information is presented on the time course of chitin digestion and the digestibihty rate, following one meal of a natural chitinous food organism of cod, i.e. shrimps Crangon allmanni.

MATERIAL AND METHODS

Fish m a t e r i a l

(3)

Digestibility of chitin in cod 427 Anstalt H e l g o l a n d . T h e y w e r e k e p t in a e r a t e d 330-1 t a n k s fed c o n t i n u o u s l y w i t h w a t e r p u m p e d from the sea. D u r i n g the p e r i o d of i n v e s t i g a t i o n {August 1985), t e m p e r a t u r e a n d salinity in the t a n k w a t e r r a n g e d from 18 to 18.5~ a n d from 31.3 to 32.3 %o S, respectively.

For two w e e k s the fish w e r e fed daily ad libitum w i t h frozen c r u s t a c e a n s a n d sprats, Sprattus sprattus. T h r e e days p r e c e d i n g the e x p e r i m e n t , f e e d i n g w a s s t o p p e d {i.e. the fish w e r e s t a r v e d in o r d e r to e m p t y their d i g e s t i v e tract}. For practical r e a s o n s two cod w e r e p u t in e a c h t a n k a n d s e p a r a t e d from o n e a n o t h e r b y a s h e e t of Plexiglas. Before the e x p e r i m e n t a l f e e d i n g , t h e w a t e r flow into t h e t a n k s w a s s t o p p e d to a v o i d loss of food or f a e c e s by circulation. T h e tanks w e r e s u p p h e d w i t h a d d i t i o n a l air stones, a n d a l t h o u g h the w a t e r w a s cut off, the o x y g e n c o n c e n t r a t i o n in the t a n k s r e m a i n e d close to saturation for at l e a s t 48 h. E a c h fish was offered a single m e a l (15 g} of shrimps, Crangon a11rnanni. Before w e i g h i n g t h e food portions, the shrimps h a d b e e n t h a w e d , i m m e r s e d in s e a w a t e r a n d b l o t t e d on filter paper. T h e fish i m m e d i a t e l y s w a l l o w e d m o s t of t h e shrimps. A f t e r 5 min, food leftovers w e r e h o s e d from the t a n k b o t t o m a n d w e i g h e d . Thus, for e v e r y fish the e x a c t a m o u n t of food a n d the time of u p t a k e w e r e k n o w n . F a e c e s w e r e c o l l e c t e d b y hose e v e r y 6 to 8 h. A f t e r centrifugation, the s e d i m e n t s w e r e frozen ( - 2 5 ~

In total, 16 cod w e r e t r e a t e d as d e s c r i b e d above. Four fish w e r e killed at e a c h of the s a m p l i n g t i m e s (6, 12, 24, 48 h; fish groups I to IV) after r e c e i v i n g the single m e a l of shrimps {Table 1).

Table 1. Length/weight characteristics of the four groups of cod under study. Also, the amount of food (g wet weight) and chitin (g) consumed by each fish in a single meal of Crangon allmanni are

presented. The fish were examined 6, 12, 24 or 48 h after food uptake (fish groups I to IV)

Group No. Fish No. Length Weight Food Chitin"

(exp. dur.) (cm) (g) (g) {g)

1 40 569 15.50 1.30

I 2 26 162 4.46 0.37

(6 h) 3 31 345 10.60 0.89

4 29 287 10.10 0.85

5 29 273 13.22 1.11

II 6 28 231 8.15 0.68

{12 h) 7 32 369 13.90 1.17

8 37 483 15.00 1.26

9 30 322 7.40 0.62

III 10 29 265 6.53 0.55

(24 h) 11 27 171 11.27 0.93

12 35 423 14.80 1.24

13 27 183 6.98 0.59

IV 14 28 242 8.50 0.71

(48 h) 15 36 475 15.00 1.26

16 34 416 15.00 1.26

(4)

428 Eva D a n u l a t S a m p l e p r e p a r a t i o n

T h e p r e p a r a t i o n of e n z y m e extracts has b e e n d e s c r i b e d p r e v i o u s l y i n detail (Danulat & Kausch, 1984; Danulat, 1986b). Extracts of stomach contents, s t o m a c h tissue, pyloric caecae, i n t e s t i n a l contents a n d i n t e s t i n a l tissue w e r e frozen at - 70 ~ for a m a x i m u m p e r i o d of 4 w e e k s after the addition of 1 % toluene.

C h i t i n a s e a s s a y

Chitinase activity was a s s a y e d according to the e n d p r o d u c t m e a s u r e m e n t of J e u n i a u x (1966), u s i n g n a t i v e chitin (Serva, FRG) as e n z y m e substrate. It w a s modified so that 4000 p / o f test mixture c o n t a i n e d 100 ~1 of t o l u e n e as a n t i b a c t e r i a l a g e n t ; a beta~ glucosidase (Sigma, FRG) c o n c e n t r a t i o n of 1.5 units p e r m l distilled w a t e r w a s used. Prior to use, the substrate, 5 m g chitin x m1-1 dist. w a t e r incl. 2 % toluene, w a s stirred o n a m a g n e t i c stirrer for 48 h, r e s u l t i n g in a h o m o g e n o u s s u s p e n s i o n of c h i t i n p a r t i c l e s r a n g i n g from 30 to 60 ~m.

After i n c u b a t i o n for 180 rain at 37 ~ the c o n c e n t r a t i o n of e n d p r o d u c t (NAG) i n the test mixtures w a s d e t e r m i n e d b y the m e t h o d of Reissig et al. (1955), modified as i n D a n u l a t (1986b). T h e s a m p l e s w e r e r e a d at 585 n m i n a s p e c t r o p h o t o m e t e r (Uvikon 810; Kontron, Switzerland). A s t a n d a r d curve was u s e d to convert OD r e a d i n g s to ~g N-acetyl- D - g l u c o s a m i n e . T h e correlation coefficient is 0.9996, m = 0.0129 a n d b = 0.0009. R e a d i n g s of two b l a n k s were s u b t r a c t e d from the s a m p l e r e a d i n g s (Jeuniaux, 1966): r e p l a c i n g the chitin s u s p e n s i o n b y distilled water, the c o n c e n t r a t i o n of e n d p r o d u c t (NAG) in the fish extract could b e d e t e r m i n e d (Blank 1). F u r t h e r m o r e , b r e a k d o w n of chitin d u e to some r e a s o n other t h a n the action of chitinase d u r i n g the i n c u b a t i o n period could b e r u l e d out b y a n a l y s i n g a test mixture in w h i c h the e n z y m e extract h a d b e e n r e p l a c e d by distilled w a t e r (Blank 2). Chitinase activities are reported as ~g N A G x h -1 x 9 -I w e t w e i g h t of sample.

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 c h i t i n

(5)

Digestibility of chitin in cod 429

C o m b u s t i o n l o s s

T h e p e r c e n t a g e of o r g a n i c m a t e r i a l w a s d e t e r m i n e d b y c o m b u s t i o n of d r i e d

Crangon

shells at 470 ~ for 2 h. Since s o m e d a r k o r g a n i c rests could b e f o u n d after this p r o c e d u r e , the c o m b u s t i o n w a s r e p e a t e d once after a d d i n g 25 ~d 10 % a m m o n i u m n i t r a t e solution (DEV, 1981) to the s a m p l e s in o r d e r to o b t a i n i n o r g a n i c m a t e r i a l s only.

RESULTS

T a b l e I shows t h e l e n g t h - w e i g h t characteristics of t h e 4 g r o u p s of cod, e x a m i n e d 6 (1), 12 (II), 24 (Ill) a n d 48 h (IV) after t h e e x p e r i m e n t a l f e e d i n g . T h e m e a n l e n g t h a n d w e i g h t of t h e fish in e a c h of the 4 groups, as w e l l as the a v e r a g e c o n s u m p t i o n of

Crangon

al/mann/, w e r e v e r y similar. T h e chitin c o n t e n t of t h e food w a s c a l c u l a t e d on the b a s i s of the food analysis. T a b l e 2 gives a v e r a g e dry w e i g h t (%), chitin c o n t e n t (% w.wt; % d.wt) a n d c o m b u s t i o n loss (%) of 5 s a m p l e s of C. aHmann/. At a chitin c o n t e n t of 8.37 % (w.wt shrimps) a n d a n a v e r a g e c o n s u m p t i o n of 11.0 + / - 3.7 g shrimps, e a c h cod h a d c o n s u m e d 923 + / - 309 m g chitin (n=16) in a single meal.

T a b l e 3 p r e s e n t s t h e results of the analysis of the s t o m a c h a n d i n t e s t i n a l c o n t e n t s of

Gadus morhua

6, 12, 24 a n d 48 h after food u p t a k e . T h e d a t a s h o w t h a t t h e d i g e s t i o n

process a n d e v a c u a t i o n of the s t o m a c h h a d p r o c e e d e d v e r y rapidly. As e a r l y as 6 h after the f e e d i n g , r e l a t i v e l y h i g h c o n c e n t r a t i o n s of the chitin d e g r a d a t i o n e n d p r o d u c t N- A c e t y l - D - g l u c o s a m i n e (NAG) could b e m e a s u r e d in extracts of the s t o m a c h a n d intesti- nal contents. At t h e t i m e of e x a m i n a t i o n , the a m o u n t of chitin in the i n t e s t i n a l c o n t e n t s v a r i e d b e t w e e n a n a v e r a g e of 18.08 m g (group I) a n d 42.66 m g (group IV). C h i t i n a c c o u n t e d for 9.28-% (group I) to 30.28 % (group IV) of the dry w e i g h t of t h e i n t e s t i n a l contents. T h e s e results s h o w that s o m e chitinous m a t e r i a l h a d b e e n e v a c u a t e d from t h e s t o m a c h at a v e r y e a r l y s t a g e (i.e. 6 h after the feeding). T h e a v e r a g e p e r c e n t a g e of chitin in the d r i e d i n t e s t i n a l contents (groups I to IV) is h i g h e s t after the s t o m a c h s h a d b e e n c o m p l e t e l y e m p t i e d , d e m o n s t r a t i n g t h a t chitin is d e g r a d e d at a s l o w e r r a t e t h a n o t h e r food c o m p o n e n t s . T h e a m o u n t s of chitin d e t e r m i n e d in the i n t e s t i n a l c o n t e n t s r e p r e s e n t only v e r y small p o r t i o n s of the total chitin i n g e s t e d with the food: O n l y 0.58 % (fish no. 1) to 9.10 % (no. 11) of t h e

Crangon-chitin

w e r e r e c o v e r e d in the cod i n t e s t i n e s ( c o m p a r e T a b l e s 1 a n d 3).

F i g u r e s l a - d p r e s e n t the a v e r a g e chitinase activities as ~g N A G x h - t x g-1 w e t w e i g h t in the guts of e a c h 4 fish that w e r e k i l l e d 6, 12, 24 a n d 48 h after the f e e d i n g . No m a t t e r h o w m u c h t i m e h a d e l a p s e d since food u p t a k e , activities w e r e a l w a y s h i g h e s t in

Table 2. Mean dry weight (% w. wt.), chitin content (% w. wt.; % d. wt.) and loss of dry weight during combustion (%) of

Crangon

a//rnann/. Means (~) and standard deviation ( _ s.d.) based on 5

determinations. (Chitin assay according to Bishop et al. 1982)

Mean __ s.d. Dry weight Chitin Chitin Combustion

(% w. wt.) (% w. wt.) (% d. wt.) loss (%)

24.44 8.37 34.25 70.82

(6)

4 3 0 E v a D a n u l a t

Table 3. Stomach a n d intestinal content of 4 groups of Gadus morhua, e x a m i n e d 6, 12, 24 a n d 48 h after food uptake. Wet weights (g w. wt.} a n d content of N-Acetyl-glucosamine (mg NAG) are presented, in the case of the intestinal contens also dry w e i g h t (g d. wt.) a n d chitin content (mg)

Fish Fish Stomach contents Intestinal c o n t e n t s

group No. g w. wt. m g NAG* g w. wt. g d . wt. m g chitin* * m g N A G "

1 12.88 6.74 2.60 0.135 7.50 5.95

I 2 3.86 2.53 1.63 0.171 15.13 6.77

(6 h) 3 8.47 6.81 2.04 0.191 29.22 6.73

4 8.74 6.70 2.57 0.277 20.47 7.50

5 7.60 13.34 2.55 0.209 41.49 7.90

II 6 2.50 3.51 1.23 0.076 22.13 0.56

(12 h) 7 8.10 23.90 2.75 0.292 56.31 9.25

8 9.73 24.53 2.99 0.284 79.86 2.50

9 0.95 0.62 1.15 0.188 23.65 1.61

Ill I0 - - 1.33 0.119 40.I0 1.76

(24 h) I I 3.15 3.24 1.99 0.323 84.73 6.01

12 2.86 2.51 2.36 0.202 44.08 2.13

13 - - 0.77 0.080 29.67 1.59

IV 14 - - 1.05 0.100 28.29 2.78

{48 h) 15 . . . .

16 - - 2.72 0.275 70.02 3.23

- no stomach or intestinal contents p r e s e n t w h e n e x a m i n e d " N A G - c o n t e n t d e t e r m i n e d according to Reissig et al. (1955) " ' Chitin-con.tent d e t e r m i n e d b y the m e t h o d of Bishop et al. (1982)

e x t r a c t s of t h e s t o m a c h t i s s u e . A c o m p a r i s o n of t h e r e s u l t s for t h e 4 f i s h g r o u p s r e v e a l s t h a t c h i t i n a s e a c t i v i t i e s v a r i e d a c c o r d i n g to t h e t i m e e l a p s e d s i n c e t h e f o o d u p t a k e (i.e. t h e s t a g e of d i g e s t i o n } , a s w e l l a s w i t h i n t h e d i f f e r e n t p a r t s of t h e g u t s . I n t h e s t o m a c h c o n t e n t s , m a x i m u m a c t i v i t i e s w e r e o b t a i n e d 12 h a f t e r t h e f e e d i n g , w h i l e i n t h e s t o m a c h t i s s u e t h e h i g h e s t a c t i v i t i e s w e r e f o u n d a f t e r 24 h. In e x t r a c t s of t h e i n t e s t i n a l c o n t e n t s , c h i t i n a s e r e s u l t s 4 8 h a f t e r f e e d i n g w e r e a l m o s t t w i c e a s h i g h a s 6 h a f t e r f e e d i n g , r a n g e s of s t a n d a r d d e v i a t i o n n o t o v e r l a p p i n g ( d u e to t h e s m a l l s i z e , s t a t i s t i c a l s i g n i f i c a n c e of t h e s e r e s u l t s w a s n o t t e s t e d ) . O v e r a l l t h e r e a p p e a r s to b e a d e f i n i t e i n t e r d e p e n d e n c e b e t w e e n c h i t i n a s e s e c r e t i o n a n d t h e s t a g e of d i g e s t i o n .

T a b l e 4 s h o w s t h e r e s u l t s of t h e c h i t i n a n a l y s i s i n t h e p o o l e d f a e c e s s a m p l e s . U n f o r t u n a t e l y , c h i t i n i n t h e s t o m a c h c o n t e n t s w a s n o t d e t e r m i n e d q u a n t i t a t i v e l y , b u t it Table 4. Dry w e i g h t (mg) a n d chitin content (mg) of pooled faeces of 3 groups of Gadus morhua (n =

4, each), after a single m e a l of C. allmanni.

Fish group Faeces (mg) Chitin c o n t e n t (rag)

II (12 h) 65 20

Ill (24 h) 202 77

(7)

D i g e s t i b i l i t y of chitin in cod 431

6 0 0

4 0 0 -

2 0 0

6 0 0

4 0 0 84

2 0 0

6 0 0

4o0.

2 0 0

1

I

4 8 h

2 4 h

1 2 h

6 0 0 84

4 0 O

2 0 0

SC ST PC IC

|

IT

6 h

Fig. I. A v e r a g e chitinase activity (Bg N A G x h -I x g-* w e t w t of sample) _+ S.D. of each 4 Gadus

morhua that w e r e e x a m i n e d 6, 12, 24 a n d 48 h after a single m e a l of Crangon allmanni (chitinase assay according to Jeuniaux, 1966). S C = stomach contents; S T = stomach tissue; P C = pyloric

(8)

432 Eva D a n u l a t

can b e a s s u m e d t h a t s o m e chitin w a s left in the fish that w e r e k i l l e d w i t h i n 24 h after t h e feeding. H o w e v e r , cod e x a m i n e d 48 h after food u p t a k e h a d c o m p l e t e l y e v a c u a t e d their stomachs. Here, t h e s u m of chitin in t h e i n t e s t i n e a n d in the f a e c e s reflects t h e a m o u n t of n o n h y d r o l y z e d c a r b o h y d r a t e :

G r o u p IV: 128 m g (faeces) + 252 m g (guts) = 380 m g n o n - d e g r a d e d chitin.

In total, 3806 m g chitin h a d b e e n c o n s u m e d b y this fish g r o u p (Table 1). S u b t r a c t i n g 380 m g n o n - h y d r o l y z e d chitin from this value, a m e a n d i g e s t i b i l i t y of 90 % can b e c a l c u l a t e d for chitin. C o n s i d e r i n g the s a m p h n g t e c h n i q u e of t h e f a e c e s m a t e r i a l , it s e e m s h k e l y t h a t s o m e of t h e f a e c e s could b e m i s s e d w h e n h o s i n g t h e t a n k b o t t o m s , r e s u l t i n g in a n i n c o r r e c t l y h i g h d i g e s t i b i l i t y v a l u e for chitin. N e v e r t h e l e s s , t h e s m a l l p e r c e n t a g e s of chitin t h a t w e r e r e c o v e r e d from the i n t e s t i n a l contents (Table 3 ) c o n f i r m t h a t the d i g e s t i b i l i t y of chitin in G. morhua m u s t h a v e b e e n v e r y h i g h i n d e e d .

D I S C U S S I O N A N D C O N C L U S I O N S

Long b e f o r e s a m p l e s of v e r t e b r a t e s w e r e t e s t e d for chitinolytic e n z y m e activity, G e y e r (1940) a n d M a n n (1948) i n v e s t i g a t e d the digestibility of the " a n i m a l f i b e r chitin" in fish. H a v i n g fed c h i r o n o m i d s , g a m m a r i d s a n d Daphnia to Umbra lacustris (Salmonifor- mes), G e y e r (1940) d e t e r m i n e d d i g e s t i b i h t y rates b e t w e e n 12 a n d 16 %, w h i l e M a n n (1948) f o u n d 15 % d i g e s t i b i l i t y in a similar s t u d y with Cyprinus carpio. M a n n (1948) r e m a i n e d c o n v i n c e d t h a t chitin w a s c o m p l e t e l y i n d i g e s t i b l e a n d a c c o u n t e d his results to difficulties w i t h the chitin d e t e r m i n a t i o n . In case of C. carpio, c h i t i n a s e tests w e r e p e r f o r m e d b y M i c h a et al. (1973): C o m p a r e d to most o t h e r fish s p e c i e s e x a m i n e d in t h e s a m e study, C. carpio h a d v e r y w e a k c h i t i n a s e activities, a n d the s a m e w a s t r u e for o t h e r m e m b e r s of the C y p r i n i d a e . Thus the low chitin d i g e s t i b i l i t y f o u n d for C. carpio (Mann,

1948) m i g h t b e a c c o u n t e d to w e a k e n z y m e activities in this species.

Recently, B u d d i n g t o n (1980) p e r f o r m e d studies on the d i g e s t i o n of chitin a n d cellu- l o s e in Salmo gairdneri a n d t h r e e s p e c i e s of Tflapia (T. aurea, T. mossambica a n d T.

nilotica). T h e a u t h o r f e d t h e fish w i t h p e l l e t s c o n t a i n i n g 2 to 10 % " p u r e chitin". T h e a m o u n t s of c a r b o h y d r a t e s r e c o v e r e d from t h e intestine a n d the f a e c e s of t h e fish w e r e not significantly r e d u c e d c o m p a r e d to t h e p e l l e t mix. From t h e s e results, B u d d i n g t o n (1980) c o n c l u d e d t h a t n e i t h e r of t h e two p o l y s a c c h a r i d e s can b e d i g e s t e d b y a n y of t h e 4 species, a n d c a l l e d chitin a n d c e l l u l o s e " h y d r o l y s i s r e s i s t a n t m a t t e r " . T h e a p p a r e n t i n d i g e s t i b i h t y of chitin a p p e a r s c o n t r a d i c t o r y to the v e r y h i g h l e v e l of c h i t i n a s e in the s t o m a c h tissue of

S. gairdneri, r e p o r t e d p r e v i o u s l y b y M i c h a et al. (1973): in vitro, t h e s t o m a c h m u c o s a of S.

gairdneri r e l e a s e d 16290 ~tg N A G x h -1 • g-1 (chitin b e i n g u s e d as e n z y m e substrate). L i n d s a y et al. (1984), c o n d u c t i n g v e r y e x t e n s i v e f e e d i n g s t u d i e s with S. gairdneri,

(9)

Digestibility of chitin in cod 433 Results o b t a i n e d in the p r e s e n t s t u d y s h o w t h a t c h i t i n a s e s in the g u t s of cod h y d r o l y z e chitin not only in vitro b u t also in vivo: (a) Strong c h i t i n a s e activities w e r e m e a s u r e d in g u t contents a n d tissues of' the cod, s h o w i n g g r e a t v a r i a t i o n s d u r i n g the course of t h e d i g e s t i v e process. (b) In extracts of the s t o m a c h as w e l l as the i n t e s t i n a l c o n t e n t s of t h e cod, h i g h c o n c e n t r a t i o n s of t h e chitin d e g r a d a t i o n p r o d u c t N A G w e r e m e a s u r e d . (c} F e e d i n g a single m e a l of shrimps, only u p to 9 % of t h e chitin from t h e food could b e r e c o v e r e d from the i n t e s t i n a l contents a n d the f a e c e s of t h e fish, chitin from

Crangon allmanni w a s d e t e r m i n e d to b e 90 % d i g e s t i b l e in Gadus morhua. E v e n t h o u g h further, m o r e e x t e n s i v e s t u d i e s w i t h a m o r e e l a b o r a t e s y s t e m for t h e collection of t h e fish f a e c e s m a y s h o w t h a t t h e p r e s e n t results slightly o v e r e s t i m a t e t h e true d i g e s t i b i l i t y of chitin, it has b e c o m e clear t h a t in cod, chitin is d i g e s t i b l e to a v e r y l a r g e extent.

It has to b e t a k e n into a c c o u n t t h a t t h e d i g e s t i b i l i t y ~ l i k e l y v a r y d e p e n d i n g on the p r e y o r g a n i s m s t h a t are t a k e n : t h e chitin of s h r i m p shells m a y b e d i g e s t i b l e to a g r e a t e r e x t e n t t h a n t h a t of a l a r g e s p e c i m e n of Cancer, u n l e s s the d i g e s t i o n p e r i o d is m u c h l o n g e r in t h e l a t t e r case, i.e. the s t o m a c h e v a c u a t i o n is s l o w e d d o w n c o n s i d e r a b l y . S t o m a c h s of fish t h a t h a d b e e n c a u g h t for e a r l i e r studies, often still c o n t a i n e d c o n s i d e r a b l e a m o u n t s of food {crustaceans; fish) after 3 d a y s of m a i n t e n a n c e , a n d s t o m a c h e v a c u a t i o n w a s n e v e r o b t a i n e d in less t h a n 48 h. O n e cod of t h e p r e s e n t s t u d y h a d e m p t i e d its s t o m a c h c o m p l e t e l y w i t h i n only 24 h. T h e difference m a y b e e x p l a i n e d b y t h e fact t h a t p r e v i o u s l y frozen s h r i m p s are m o r e r e a d i l y d i g e s t e d t h a n fresh shrimps. O t h e r results a g r e e w e l l w i t h e a r l i e r e x p e r i m e n t a l findings: For e x a m p l e , w h e n a g r o u p of c o d w a s f e d e x c l u s i v e l y on c r u s t a c e a n shells for 3 w e e k s , their condition factor w a s s i g n i f i c a n t l y h i g h e r t h a n the o n e of the control g r o u p {starved over the s a m e period), i n d i c a t i n g t h a t the shells w e r e d e g r a d e d a n d the d i g e s t i v e p r o d u c t s w e r e a b s o r b e d b y the i n t e s t i n e (Danulat, 1986b).

So, w h a t are p o s s i b l e e x p l a n a t i o n s for the conflicting results o b t a i n e d for cod a n d for r a i n b o w trout (Lindsay et al., 1984}, b o t h fish s p e c i e s b e i n g e q u i p p e d w i t h e n z y m e s that are h i g h l y specific for h y d r o l y z i n g chitin?

(10)

434 l~va D a n u l a t

i n d e e d a p p h c a b l e in t h e c a s e of S. gairdneri. C o n c e r n i n g G. morhua, c h i t i n a s e tests w e r e p e r f o r m e d s i m u l t a n e o u s l y at 6 different t e m p e r a t u r e s b e t w e e n 5 ~ a n d 55 ~ Surprisingly, t e s t i n g extracts of s t o m a c h a n d i n t e s t i n a l contents of a cod t h a t w a s c a u g h t at 4.4 ~ w a t e r t e m p e r a t u r e , c h i t i n a s e activities m e a s u r e d at 5 ~ r e a c h e d u p to 4 5 - 5 0 % of t h e m a x - i m u m activity d e t e r m i n e d at 37 ~ (Danulat, u n p u b l i s h e d data). Since c h i t i n a s e activities a r e e v e n c o n s i d e r a b l y h i g h e r in S. gairdneri (Micha et al., 1973) t h a n in G. morhua, o n e s h o u l d a s s u m e t h a t chitin c a n b e d i g e s t e d e v e n b e t t e r b y the s a l m o n i d s p e c i e s . Possibly, structural c h a n g e s in the chitin m o l e c u l e d u r i n g t h e purification p r o c e s s a r e t h e r e a s o n w h y c h i t i n a s e c o u l d not a t t a c k the p u r i f i e d chitin w h i c h w a s i n c l u d e d in t h e trout feed. It c a n n o t b e e x c l u d e d t h a t t h e r e a r e c e r t a i n factors or activators in chitinous p r e y o r g a n i s m s w h i c h s t r o n g l y e n h a n c e their d i g e s t i o n in the fish stomach. Also L i n d s a y et al. (1984) r e p o r t chitinolytic activities of t i s s u e extracts only, not of extracts of s t o m a c h a n d i n t e s t i n a l c o n t e n t s , so for s o m e r e a s o n the e n z y m e m a y not h a v e b e e n s e c r e t e d into t h e g u t l u m e n .

W i t h o u t d o u b t c e r t a i n fish s p e c i e s do exist that are u n a b l e to d e g r a d e n a t u r a l chitin from n a t u r a l o r g a n i s m s : For e x a m p l e , c o m p l e t e e x o s k e l e t o n s of g a m m a r i d s c o u l d b e f o u n d in the f a e c e s of t h e f l o u n d e r Platichthys flesus (Karpevitch & Bokoff, 1937), a n d e v e n v e r y fine e x t r e m i t i e s of Daphnia pulicaria could b e r e c o v e r e d from t h e i n t e s t i n e of t h e s a l m o n i d Coregonus lavaretus (R6sch, 1985; Kausch, pers. comm.). T h i s m a y b e t h e c a s e w h e r e t h e r e is a l a c k of chitinolytic e n z y m e s , a n d / o r specific c o n d i t i o n s in t h e d i g e s t i v e tract of t h e fish. In g e n e r a l , " T h e r e is, h o w e v e r , no r e a d y e x p l a n a t i o n for t h e failure of a n i m a l s p o s s e s s i n g c h i t i n a s e to d i g e s t chitin" (citing from: L i n d s a y et al., 1984). F u t u r e e x p e r i m e n t s on the d i g e s t i b i h t y of chitin s h o u l d b e b a s e d o n t h e f e e d i n g of r a w chitinous m a t e r i a l i n s t e a d of p u r i f i e d chitin: for e x a m p l e , a d d i n g c e r t a i n p e r c e n t a g e s of g r o u n d c r a b shells to the p e l l e t m i x t u r e of 5. gairdneri m a y l e a d to v e r y d i f f e r e n t results c o n c e r n i n g t h e d i g e s t i b i l i t y of chitin t h a n t h e ones o b t a i n e d with the s a m e s p e c i e s a d d i n g h i g h l y p u r i f i e d chitin to t h e f e e d (Buddington, 1980; L i n d s a y et al., 1984). Results of e x p e r i m e n t s with G. morhua a r e p r o m i s i n g a n d s h o u l d e n c o u r a g e further, l a r g e scale s t u d i e s of the d i g e s t i b i l i t y of chitin in fish. P r o v i d e d that c e r t a i n fish s p e c i e s (or o t h e r organisms) t h a t a r e of i n t e r e s t for a q u a c u l t u r e h a v e t h e a b i l i t y to d e g r a d e chitin, f e e d i n g chitinous w a s t e s from t h e c r a b a n d s h r i m p i n d u s t r i e s m a y p r o v e v a l u a b l e in s e v e r a l w a y s : A l o n g w i t h chitin, p r o t e i n s in the w a s t e m a t e r i a l (Knorr, 1984) m i g h t f u n c t i o n as a n e n e r g y source. T h e c a r o t i n o i d a s t a x a n t h i n e could p l a y a role as a p r e c u r s o r of v i t a m i n A in fish (Love, 1970). In N o r w e g i a n a q u a c u l t u r e , a s t a x a n t h i n e h a s b e e n u t i l i z e d for a l o n g t i m e to g i v e colour to t h e flesh of salmonids. A d d i t i o n a l l y , t h e r e m a y b e s u r p r i s i n g i n d i r e c t effects w h e n s o m e chitin is i n c l u d e d in the food, a n e x a m p l e of w h i c h is r e p o r t e d b y A u s t i n et al. (1981): T h e y f o u n d in f e e d i n g e x p e r i m e n t s t h a t chitin at 2 % in t h e food e n h a n c e s t h e d e v e l o p m e n t of b i f i d o - b a c t e r i a in the i n t e s t i n e s of c h i c k e n s , w h i c h in t u m s y n t h e s i z e t h e e n z y m e lactase. Thus, t h e lactose in the food m i x t u r e b e c o m e s d i g e s t i b l e a n d the g r o w t h of t h e c h i c k e n s is e n c o u r a g e d .

(11)

D i g e s t i b i l i t y of c h i t i n i n c o d 435

Acknowledgements. The project was f u n d e d by the Deutsche Forschungsgemeinschaft (DFG) and, in the final phase, by a grant from the University of H a m b u r g to the author. I wish to t h a n k Prof. Dr. H. Kausch for his continuing support and greaf interest .in the project. Special thanks are also due to scientists and staff of the Biologische Anstalt Helgoland (Helgoland and Hamburg) w h o supported me in every possible way, and Dr. H. Rehbein at the Bundesforschungsanstalt ftir Fischerei (Hamburg) for m a n y helpful discussions. Last but not least, I would like to thank J. Philip A d a m s for his suggestions concerning the manuscript.

L I T E R A T U R E C I T E D

Austin, P.R., Brine, C.J., Castle, J. E. & Zikakis, J . P . , 1981. Chitin; n e w facets in research. - Science, N.Y. 212, 749-753.

Barnard, E. A., 1973. Comparative biochemistry of digestive enzymes. I. Digestion. In; Comparative animal physiology. Ed. by C. L. Prosser. Saunders, Philadelphia, 133-139.

Bishop, R. H., Duncan, C. L., Hvancho, G. M. & Young, H., 1982. Estimation of fungal contamination in tomato products by a chemical assay for chitin. - J. Fd Sci. 47, 437-444.

Brine, C. J. & Austin, P. R., 1981. Chitin variability with species and m e t h o d of preparation. - Comp. Biochem. Physiol. 69 (B), 283-286.

Buddington, R. K., 1980. Hydrolysis resistant organic matter as a reference for m e a s u r e m e n t of fish digestive efficiency. - Trans. Am. Soc. Fish. 109, 653-656.

Colin, D.A., 1972. Recherches sur la digestion de la chitine chez quelques T414ost4ens marins. Th~se du Troisi~me Cycle, Universit~ Claude Bernard, Lyon, 61 pp.

Dandrifosse, G. & Schoffeniels, E., 1967. M e c h a n i s m of chitinase secretion by gastric mucosa. - Biochim. biophys. Acta 148, 741-748.

Dandrifosse, G., Schoffeniels, E. & Jeuniaux, C., 1965. Secretion de la chitinase par la m u q u e u s e gastrique isol4e. - Biochim. biophys. Acta 94, 153-164.

Danulat, H., 1986a. Role of bacteria with regard to chitin degradation in the digestive tract of the cod,

Gadus morhua. - Mar. Biol. 90, 335-343.

Danulat, H., 1986b. The effects of various diets on chitinase and beta-glucosidase activities a n d the condition of cod, Gadus morhua. - J. Fish Biol. 28, 191-197.

Danulat, E. & Kausch, H., 1984. Chitinase activity in the digestive tract of the cod, Gadus morhua

(L.). - J. Fish Biol. 24, 125-134.

Deutsche Einheitsverfahren zur Wasser-, Abwasser- u n d Schlamm-Untersuchung, 1981. Hrsg. yon der Fachgruppe Wasserchemie in der Gesellschaft Deutscher Chemiker. Verl. Chemie, Weinheim, 2.

F~inge, R., Lundblad, G., Lind, J. & Slettengren, K., 1979. Chitinolytic enzymes in the digestive system of marine fishes. - Mar. Biol. 53, 317-321.

Geyer, F., 1940. Der ungarische Hundsfisch. - Z. Morph. Okol. Tiere 36.

Goodrich, T. D. & Morita, R. Y., 1977a. Incidence a n d estimation of chitinase activity associated with marine fish a n d other estuarine samples. - Mar. Biol. 4I, 349-353.

Goodrich, T. D. & Morita, R.Y., 1977b. Bacterial chitinase in the stomachs of marine fishes from Yaquina Bay, Oregon, USA. - Mar. Biol. 41,355-360.

Herth, W. & Barthlott, W., 1979. The site of beta-chitin fibril formation in centric diatoms. I. Pores and fibril formation. - J. Ultrastruct. Res. 68, 6-15.

Jeuniaux, C., 1961. Chitinases. An addition to the list of hydrolases in the digestive tract of v e r t e b r a t e s . - Nature, Lond. 192, 135-136.

Jeuniaux, C., 1963. Chitine et chitinolyse. Un ch~pitre de la biologie mol4culaire. Masson, Paris, 174 PP.

Jeuniaux, C., 1966. Chitinases. In: Methods in enzymology. Ed. by Sidney P. Colowick & N a t h a n O. Kaplan. Acad. Press, N e w York, 8, 644-650.

Karpevitch, A. & Bokoff, E., 1937. The rate of digestion in marine fishes. - Zool. Zh. 16, 28--44. King, P.A., Wissing, T.E., DeviUez, E.J., Chimney, M. J. & Randall, W. B. jr, 1977. Nutritional

(12)

4 3 6 E v a D a n u l a t

Knott, D., 1984. The use of chitinous polymers in food. - Fd Technol., C h a m p a i g n , 1984 (Jan.), 85-89; 92-97.

Lindsay, G. J. H., 1984. Distribution a n d ftinction of digestive tract chitinolytic e n z y m e s in fish. - J. Fish Biol. 24, 529-536.

Lindsay, G. J. H., Walton, M. J., Adron, J. W., Hetcher, T. C., Cho, C. Y. & Cowey, C. B., 1984. T h e growth of r a i n b o w trout (Salmo gairdnen') g i v e n diets containing chitin a n d its relationship to chitinolytic e n z y m e s a n d chitin digestibility. - Aquaculture 37, 315-324.

Lindsay, G. J. H. & Gooday, G.W., 1985. Chitinolytic e n z y m e s a n d the bacterial microflora in t h e digestive tract of cod, Gadus morhua. - J. Fish Biol. 26, 255-265.

Love, R. M., 1970. The chemical biology of fishes. Acad. Press, London, 549 pp.

M a n n , H., 1948. Ober die R o h f a s e r v e r d a u u n g des Karpfens. - Arch. FischWiss. I, 68-78.

Micha, J . C . , Dandrifosse, G. & Jeuniaux, C., 1973. Distribution et localisation tissulaire de la synth~se des chitinases chez les vert~br4s inf4rieurs. - Archs int. Physiol. Biochim. 81, 4 3 9 4 5 1 . Muzzarelli, R. A. A., 1977. Chitin. P e r g a m o n Press, Oxford, 309 pp.

Okutani, K., 1966. Studies of chitinolytic systems in the digestive tract of Lateolabrax japonicus. -

Bull. Misaki mar. Biol. Inst. 10, 1--47.

P4r~s, G., Bog4, G., Colin, D. & Rigal, A., 1973. Effets de la t e m p e r a t u r e sur les processus digestifs des poissons, activit4s e n z y m a t i q u e s et absorption intestinale. - Revue Tray. Inst. P&ches marit.

37, 223-232.

Rehbein, H., Danulat, E. & Leinemann, M., 1986. Activities of chitinase a n d p r o t e a s e a n d concen- tration of fluoride in the digestive tract of Antarctic fishes feeding o n lu-ill (Euphausia superba

Dana}. - Comp. Biochem. Physiol. 85 (A}, 545-551.

Reissig, J. L., Strominger, J. L. & Leloir, L. F., 1955. A modified, colorimetric m e t h o d for the estima- tion of N-acetyl-amino-sugars. - J. biol. Chem. 217, 9 5 9 9 6 6 .

Richards, A. G., 1951. T h e i n t e g u m e n t of arthropods. Univ. of M i n n e s o t a Press, Minneapolis; 411 pp. RSsch, R., 1985. N a h r u n g s a u f n a h m e u n d N a h r u n g s a u s n u t z u n g bei S a n d f e l c h e n (Coregonus fera

Jurine) bei Ffitterung mit L e b e n d p l a n k t o n (Daphnia pulicaria}. Diss., Univ. Freiburg, 122 pp. Senstad, C. & Almas, K. A., 1986. Use of chitosan in recovery of protein from s h r i m p p r o c e s s i n g

Figure

Table 1. Length/weight characteristics of the four groups of cod under study. Also, the amount of food (g wet weight) and chitin (g) consumed by each fish in a single meal of Crangon allmanni are presented
Table 2. Mean dry weight (% w. wt.), chitin content (% w. wt.; % d. wt.) and loss of dry weight during combustion (%) of Crangon a//rnann/
Table 3. Stomach and intestinal content of 4 groups of Gadus morhua, examined 6, 12, 24 and 48 h after food uptake
Fig. I. Average chitinase activity (Bg NAG x h -I x g-* wet wt of sample) _+ S.D. of each 4 Gadus morhua that were examined 6, 12, 24 and 48 h after a single meal of Crangon allmanni (chitinase assay according to Jeuniaux, 1966)

References

Related documents

Field experiments were conducted at Ebonyi State University Research Farm during 2009 and 2010 farming seasons to evaluate the effect of intercropping maize with

The article will investigate four expressions of philanthropy as ideology: consumer philanthropy, in which we are asked to consume with good conscience; corporate philanthropy,

Time changing nature of pediatric practice imas been further illustrated in a recent article on time types of cases seen in practice oven the past 25 years.3 This report indicates

In this thesis, an efficient algorithm for mining high utility itemset called EUP-growth + is proposed for mining high utility itemsets with a set of effective strategies for

The current prospective observational study of six months duration was designed to assess the rate of adverse drug reactions ADRs induced by anti tubercular drugs in patients

ADD: Attribute Driven Design; AI: Artificial Intelligence; BMI: Body Mass Index; BPEL: Business Process Execu- tion Language; CDSS: Computerized decision support systems; CfMS:

Methods: A follow-up study 5.5 years after the explosion, 330 persons aged 18 – 67 years, compared lung function, lung function decline and airway symptoms among exposed

T1D: type 1 diabetes; DD: diabetes related distress; MDD: major depression disorder; DSM-5: Diagnostic and Statistical Manual of Mental Disorders-5th edition; HAD-D: