The digestibility of the bivalve crystalline style

Comp. Biochem. Physiol., Vol. 65B, pp. 451 to 453

0305-0491/80/0201-0451502.00/0

© Pergamon Press Ltd 1980. Printed in Great Britain

THE DIGESTIBILITY OF THE BIVALVE CRYSTALLINE STYLE R. G. B. REID and BETSY SWEENEY Department of Biology, University of Victoria, Victoria, British Columbia, Canada, V8W 2Y2 (Received 25 May 1979) Abstraet--l. The effect of proteolytic enzymes on the crystalline styles of the bivalves Tresus capax and Crassostrea 9igas is examined. 2. 1000 unit initial concentrations of trypsin cause rapid physical disintegration of Crassostrea style and slower disintegration of Tresus style at pH 6.0 and 7.5. 3. The effect of the trypsin is to hydrolyse peptide bonds in the crystalline style protein. The effect is therefore digestive. 4. Bivalves, such as Crassostrea, which have soft crystalline styles which spontaneously break down during exposure of the organism by an ebb tide, would be unable to reconstitute the crystalline styles if natural levels of gastric trypsin or similar enzymes were to rise above 100 units.

INTRODUCTION

MATERIALS A N D M E T H O D S

The bivalve crystalline style is a concentrically laminated hyaline rod, composed largely of mucoprotein (Bailey & Worboys, 1960; Kristensen, 1972). It resides in a caecum, the style sac, which is open to the stomach. The end of the crystalline style which is exposed to the stomach softens and sols, releasing a variety of digestive enzymes into the stomach (Kristensen, 1972). The integrity of the crystalline style was originally believed to be incompatible with gastric protein digestion, the crystalline style being a proteinaceous structure (Yonge, 1926, 1930). This proposition was debated, since some zoologists believed that bivalves were capable of rapid gastric protein digestion (Yonge, 1946; Mansour, 1946). Recent studies of gastric protein digestion in bivalves confirm that for the most part gastric proteolytic levels are low (Reid & Rauchert, 1976; Reid, 1978). An exception is to be found in the Septibranchia. In the carnivore Cardiomya planetica extracellular proteolysis is of a higher order of magnitude than that of the herbivorous bivalves (Reid, 1977). However, Cardiomya retains the crystalline style, despite the high levels of proteolytic enzymes. This observation revives the question of the compatability of the crystalline style with gastric protein digestion. In order to resolve such questions we undertook studies of the crystalline styles of the horse clam Tresus capax and the oyster Crassostrea yigas. The minute crystalline styles of Cardiomya were not available in sufficient quantities for experiment. Tresus was chosen since it is the genus about whose proteinases we knew most (Reid & Rauchert, 1976). Crassostrea was chosen since there was published data on its natural rates of crystalline style breakdown and reformation (Bernard, 1973; Morton, 1977) and because of the contrast in the consistency of the oyster crystalline style, with that of Tresus.

Specimens of Tresus capax (Gould) were obtained by digging intertidally at Cherry Point, Satellite Channel, British Columbia. Their crystalline styles were washed in distilled water and then exposed to the experimental concentrations of trypsin. Some Tresus styles were pulverized and freeze-dried for use in "solid state" enzyme columns as described below. Observations on the crystalline styles of Crassostrea yigas (Thunberg) were carried out in the field at Chemainus, British Columbia. Specimens with completely formed crystalline styles were collected by diving at high water. The crystalline styles were dissected out and washed in distilled water. They were then subjected to treatment with trypsin. Tresus and Crassostrea styles were exposed to 100 unit and 1000 unit concentrations of commercial trypsin dissolved in buffered seawater. One unit of trypsin activity causes an absorbance increase of 0.001 per min at 37°C. pH levels were set at 6.0 and 7.5. pH 6.0 is within the range of pH values observed for the gastric juices of Tresus, Crassostrea and Cardiomya (Reid, 1978). pH 7.5 is the optimum for trypsin, an enzyme which has been discovered in the stomachs of the three genera (Reid, 1978). At pH 6.0 trypsin which had an initial activity of 1000 units dropped to 700 units. This lower activity was not adjusted back to the initial activity. Controls consisted of washed crystalline styles exposed to buffered sea water at pH 6.0 and 7.5 at the experimental temperature, 20°C. The course of disintegration of the experimental styles was observed and the times of disintegration noted. These experiments were repeated twice. Since crystalline styles are known to break down spontaneously under a variety of physico-chemical conditions it was decided to determine if actual breakage of peptide bonds was being effected. To demonstrate protein hydrolysis it was intended to subject aliquots of powdered, freezedried crystalline style to trypsin, and measure the release of amino acids and peptides spectrophotometrically, noting absorbance increase at 280nm. Two ditfculties were encountered: the first was the problem of removing undigested mucoprotein from the digest prior to measuring absorbance changes. Efforts were made to remove the

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R.G.B. REID and BETSYSWEENEY

mucoprotein by precipitation, by heat-coagulation and by the use of the affinity compound concanavalin A. But we were not satisfied that all the undigested material had been removed. However, we found that if the digest was run through a Sephadex G25 column there was adequate separation of digestion products and undigested mucoprotein. This was recorded on an LKB flow-through, recording spectrophotometer. The problem of distinguishing between the products of style protein hydrolysis and trypsin autolysis was circumvented by the use of a "solid-state" enzyme column, in which the trypsin was attached to CNBr-activated Sepharose 4B (Pharmacia Co. Ltd), with the result that trypsin molecules were isolated from each other and autolysis prevented (Gabel & v. Hofsten, 1970; Axen & Emback, 1971). Free trypsin and any hydrolysis products were eluted prior to the use of the column. 145mg of reconstituted style mucoprotein were exposed to "solidstate" trypsin in a Pharmacia K16/20 column for different lengths of time at 4 and 20°C. RESULTS

Effects of trypsin on physical integrity of crystalline style Results are shown in Table 1. At the 70 and 100 unit levels the Tresus styles disintegrated within 13 hr at pH 6.0 and 9 h r at pH 7.5. Control styles were unaffected within 24 hr. The oyster styles softened and formed viscous sols within 2 h r in both the experimental and control tests. At the 700 and 1000 unit levels, at pH 6.0 and 7.5, the Tresus styles showed deep, irregular lesions within 1.5 hr and had disintegrated completely within 4 hr at pH 7.5 and 8 hr at pH 6.0. Control styles were unaffected within 24 hr. At 700 and 1000 unit levels the oyster styles had disintegrated completely with 30 min. Control styles took 2 hr to soften and disintegrate.

Hydrolysis of peptide bonds of crystalline style protein by trypsin While we conclude that the results of the previous experiment indicate that trypsin digests crystalline style protein, reservations have been expressed in the earlier literature as to the chemical effects concerned. Crystalline styles are known to break down spontaneously under a variety of physico-chemical conditions. It was therefore decided to determine if actual

breakage of peptide bonds was being effected. As noted above in Materials and Methods aliquots of Tresus style were digested in a "'solid state" trypsin column and the products of digestion were separated from the undigested style mucoprotein by passing them through a Sephadex G25 column. The results are shown in Fig. 1. In all cases, there was significant decrease in the proportions of mucoprotein and increase in the proportions of amino acids and peptidases. DISCUSSION We conclude that the physical integrity of the bivalve crystalline style is affected by proteolytic enzymes, and that this is indeed due to breakage of peptide bonds, and not simply to physical changes. The digestibility of the crystalline style seems to vary with its consistency. The harder, rigid crystalline style of Tresus is much more resistant to breakdown than that of Crassostrea. However, the natural concentrations of proteinases in the stomachs of Tresus and Crassostrea (less than 100 units) are not sufficient to affect their crystalline styles, especially since attrition can be compensated for by the secretions of the style sac. In the case of Crassostrea, because of the natural disintegration of the styles when the animals are exposed, levels of gastric proteinases over 100 units would probably interfere with the formation of a new crystalline style. However, the crystalline style of Tresus could tolerate much higher levels of proteolysis than are normally found in the gastric juices of

Tresus. While it cannot be generally stated that the bivalve crystalline style is incompatible with gastric protein digestion it can be concluded that some kinds of crystalline style would be unable to tolerate more than 100 units of proteinase, e.g. the crystalline style of Crassostrea, which is soft, is exposed along its length to the intestinal groove, and which breaks down at regular intervals. Other kinds of crystalline style, e.g. the crystalline style of Tresus, resist digestion. The basis of this resistence is not known, but may, as was once proposed, be a function of the concentration of mucoproteins (Berkeley, 1935). At high natural levels of gastric proteinases, e.g. Cardiomya, the persistence

Table 1

Organism

pH 6.0

.Crassostrea gigas 7.5 6.0

Tresus capax 7.5

Strength of trypsin (units) 70 700 100 1000 70

Crystalline style disintegration time (hr) Experimental Control 1.5 0.5 1.5 0.5 13

1.5 1.5 1.5 1.5 ---

700

8

--

100

9

1000

4

Crystalline styles of Crassostrea gigas and Tresus capax were exposed to various trypsin solutions in buffered sea water. The initial proteolytic activities of the trypsin solutions were 70 and 700 units at pH 6.0 and 100 and 1000 units at pH 7.5. (One unit causes an absorbance change of 0.001 per ml per rain at 37°C.) Results are given as the number of hours taken for complete disintegration of the crystalline styles at 20°C. The dashes in the control column indicate that the control styles were intact after 24 hr.

Digestibility of bivalve crystalline style I0

453

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I

20

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20 40 sO Time in minutes

(a)

(b)

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Fig. 1. Redissolved freeze-dried crystalline style protein and mucoprotein was exposed to a "solid state" trypsin column, consisting of trypsin molecules in CNBr-activated Sepharose 4B, for different lengths of time at 4 and 20°C. The soluble contents of the trypsin column were then passed through G25 Sephadex to separate the undigested protein and mucoprotein from the products of hydrolysis. The figure indicates the proportions of undigested style protein and mucoprotein and hydrolysis products following G25 Sephadex separation, as recorded spectrophotometrically at 280nm. (a) Absorption pattern of undigested style protein and mucoprotein; (b) absorption pattern of style protein and mucoprotein and hydrolysates, following digestion by trypsin at 4°C for 4 hr; (c) absorption pattern of style protein and mucoprotein and hydrolysates following digestion by trypsin at 20°C for 4hr. h = hydrolysates; s = style protein and mucoprotein.

of the crystalline style may be due to a c o m b i n a t i o n of factors including a fast rate of secretion and protection of the style by the style sac. Acknowledgements--We are grateful to Lisa Jang and Joan Friesen for their assistance with preliminary experiments and field work. This study was supported by an operating grant of the Canadian National Research Council. REFERENCES

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bacterial proteinase chemically fixed to agarose. Eur. J. Biochem. 15, 410-414. KRISTENSEN J. H. (1972) Structure and function of crystalline style of bivalves. Ophelia 10, 91 108. MANSOUR K. (1946) Food and digestive processes of the lamellibranchs. Nature 157, 482. MORTON B. S. (1977) The tidal rhythm of feeding and digestion in the pacific oyster, Crassostrea .qigas (Thunberg). J. exp. mar. Biol. Ecol. 26, 135 151. REIn R. G. B. (1977) Gastric protein digestion in the carnivorous sept|branch Cardiomya planet|ca Dall; with comparative notes on deposit and suspension-feeding bivalves. Comp. Biochem. Physiol. 56A, 573 575. RE1D R. G. B. (1978) The systematic, adaptive and physiological significance of proteolytic enzyme distribution in bivalves. Veliger 20, 260-265. REID R. G. B. & RAUCHERTK. (1976) Catheptic endopeptidases and protein digestion in the horse clam Tresus eapax (Gould). Comp. Biochem. Physiol. 54B, 467472. YONGE C. M. (1926) The disappearance of the crystalline style. Nature 117, 691. YONGE C. M. (1930) The crystalline style of the Mollusca and a carnivorous habit cannot normally co-exist. Nature 125, 444~-45. YONC;E C. M. (1946) Digestion of animals by lamellibranchs. Nature 157, 729.