Crystalline Style Research Articles

Time-course of digestive-enzyme acclimation in the cockle Cerastoderma edule

We determined the temporal evolution of amylase, cellulase, laminarinase and protease in the digestive gland and crystalline style of cockles Cerastoderma edule held over 9 to 12 d in the presence and absence of food. Cockles were fed a constant diet of 1.5 mm3 l−1 of Tetraselmis suecica for 9 to 12 d and were then starved for 6 to 8 d in late summer (September 1992) and in winter (January 1993). Feeding increased the dry weight and total cellulase, laminarinase and protease activities of the digestive gland irrespective of season, whereas amylase activity remained unchanged. In winter (i.e. when cockles are metabolically weak) the response was faster and stronger, especially for protease. An additional experiment in September starved cockles for 20 d before resuming feeding. In agreement with the seasonal differences, the presence of food after prolonged starvation induced a rapid and marked increase in protease in the digestive gland of the cockles. In winter, the possible effects of the biochemical composition of food on their enzymatic response were tested by feeding two groups of cockles with the same ration of T. suecica but harvested at different growth phases. A compensatory induction of cellulases occurred in cockles fed on T. suecica with a lower carbohydrate content. In the crystalline style, the protein level and carbohydrase fell during the first day of feeding and increased during the first day of subsequent starvation. These results indicate that the release of enzymes from the style prevails over the incorporation of enzymes during the early stages of feeding, whereas the opposite occurs during starvation.

Seasonal variation of digestive enzyme activities in the digestive gland and the crystalline style of the common cockle Cerastoderma edule

Seasonal variation of amylase, cellulase, laminarinase and protease activities of the main digestive organs (crystalline style and digestive gland) of the cockle Cerastoderma edule have been investigated. The simultaneous increment of specific enzyme activities (mg end product/mg protein per h), mass (mg), and protein content of digestive organs yield a remarkable enhancement of total enzyme activities (mg end product per organ/h) during Spring–Summer as compared with Autumn–Winter. Although extracellular enzymes of the crystalline style have typically received more attention than intracellular enzymes from the digestive gland, the present study shows the latter to release as much as ten times higher total activities than the crystalline style, highlighting the relevance of intracellular digestion in cockles. The sole effect of temperature on enzyme activity does not explain recorded seasonal variability. Additionally, the present study has shown that enzyme activities in the digestive gland increased rapidly (3 days) in the presence of food at any time of the year, suggesting that seasonal variation of enzyme activities would reflect a process of continuous adjustment of digestive enzyme levels to environmental food availability. Further shaping of the seasonal cycles may be influenced by requirements of specific biochemical components and past feeding history.

Stability of an infectious pancreatic necrosis virus (IPNV) isolate stored under different laboratory conditions.

An infectious pancreatic necrosis virus (IPNV) belonging to the Sp serotype, isolated from scallops Pecten maximus, was propagated and suspended in sterile water and cell culture medium with different salinities and incubated at temperatures ranging from -80 to 40 degrees C. Virus stability was examined by measuring virus titers under different storage conditions. Virus titers were also measured after repeated freezing and thawing, and in incubated sterile filtered scallop hepatopancreas, haemolymph and crystalline style samples, and salmon Salmo salar kidney homogenate. The virus was stable under most storage conditions. Temperatures ranging from -80 to +20 degrees C, as well as salinities from 0 to 40/1000, did not seem to influence the stability of the virus. A reduction was observed above 20 degrees C. Each freezing and thawing procedure resulted in a reduction of the virus titer. This reduction was larger at -80 than at -20 degrees C. The IPNV persisted for a long period in sterile filtered scallop haemolymph, dissolved crystalline style and hepatopancreas. In kidney homogenate from IPNV-infected salmon the virus titers were reduced at least 10-fold during the first day of incubation at all temperatures tested. When virus was propagated in cell culture and subsequently mixed with non-infected salmon kidney homogenate, the virus proved more persistent. Our results illustrate the importance of rapid and standardised laboratory processing of potentially virus-containing tissue samples, and are relevant when considering laboratory storage of samples containing IPNV.

Anatomy and systematics of Anodontites Elongatus (Swainson) from Amazon and Parana Basins, Brazil (Mollusca, Bivalvia, Unionoida, Mycetopodidae)

The anatomy of Anodontiies elongatus (Swainson, 1823), a rare species restricted to the Amazon and Parana Basins, is described by first time, showing a group of conchological and anatomical characters exclusive of this species that may be analyzed to identify it. Diagnosis of A. elongatus: shell long antero-posteriorly, umbones prominent, periostracum opaque and smooth, two posterior radial striae; middle fold of mantle edge veiy tall; gill long antero-posteriorly and short dorso-ventrally, extending about a half of it total length beyond visceral mass; palps proportionally small, several furrows in its outer surface; stomach without esophageal transversal ridjp, dorsal hood and gastric shield poorly developed, major typhlosole entering in ddd , posterior pouch of sa³ very-long; style sac reduced, without crystalline style; distal region of intestine and rectum with a well developed typhlosole, "T" in section, other intestinal regions without folds; gonad gonochoristic.

Phylogenetic position of the spirochetal genus Cristispira.

Comparative sequence analysis of 16S rRNA genes was used to determine the phylogenetic relationship of the genus Cristispira to other spirochetes. Since Cristispira organisms cannot presently be grown in vitro, 16S rRNA genes were amplified directly from bacterial DNA isolated from Cristispira cell-laden crystalline styles of the oyster Crassostrea virginica. The amplified products were then cloned into Escherichia coli plasmids. Sequence comparisons of the gene coding for 16S rRNA (rDNA) insert of one clone, designated CP1, indicated that it was spirochetal. The sequence of the 16S rDNA insert of another clone was mycoplasmal. The CP1 sequence possessed most of the individual base signatures that are unique to 16S rRNA (or rDNA) sequences of known spirochetes. CP1 branched deeply among other spirochetal genera within the family Spirochaetaceae, and accordingly, it represents a separate genus within this family. A fluorescently labeled DNA probe designed from the CP1 sequence was used for in situ hybridization experiments to verify that the sequence obtained was derived from the observed Cristispira cells.

Feeding and digestive adaptations of bivalve molluscs to sulphide-rich habitats

Abstract From the standpoint of sulphide availability, marine bivalves are divided into three categories: Type 1, external environment and pallial cavities low in sulphides while submerged; Type 2, external environment containing sulphides but pallial cavity normoxic and low in sulphides due to irrigation via siphons; and Type 3, external habitat variably rich in sulphides and pallial cavity with some oxygen and also suphides, due to lack of siphons. This review presents the known anatomical and physiological adaptations associated with feeding and digestion in Type 3 bivalves, compared with Types 1 and 2. The principal anatomical adaptations of Type 3 bivalves to habitats of increasing sulphide availability are, in order of occurrence and degree: reduction of labial palps; reduction in length and volume of intestine; reduction in stomach volume; disappearance of crystalline style and style sac; reduction in digestive tubule volume; disappearance of digestive gland secretory cells; and total disappearance of the digestive system. In parallel with the above adaptations, an increasing physiological reliance on endosymbiotic bacteria is observed, as evidenced from δ 13C values. We conclude that, under similar conditions of environmental sulphide availability, similar adaptations are found in the feeding/digestive system, irrespective of taxon, depth or geographic location. The convergence between littoral and deep-sea vent and seep inhabitants is particularly striking.

The filtration and feeding physiology of the infaunal estuarine bivalve Solen cylindraceus Hanley 1843

Feeding (filtration) activity and filtration rate were determined in Solen cylindraceus subjected to a variety of natural seston concentrations (5–500 mg 1 −1). At concentrations 5–100 mg l −1 animals spent approximately 90% of the submerged periods filtering (filtration rate ca. 20 ml min −1). Above these seston concentrations filtration activity declined, until at 500 mg 1 −1 animals were active for only 30% of the time (filtration rate ca. 10 ml min −1). At seston concentrations below 50 mg 1 −1 pseudofaecal production was minimal (<10 mg h −1). Between 100 and 500 mg 1 −1 the amount of pseudofaeces produced increased significantly to about 45 mg h −1. The formation and dissolution of the crystalline style and activity of the digestive gland was investigated in animals under natural conditions. The volume of the crystalline style was constant at all phases of the tidal cycle, and it was estimated that the style was completely replaced every 48 h. However, the rate of style production was greater during flood tides. Histological sections of the digestive glands did not reveal any change in the appearance of the digestive cells.

Quantitative analysis of crystalline style carbohydrases in five suspension- and deposit-feeding bivalves

Amylase, laminarinase, and cellulase activities in the crystalline style were assayed in vitro 18°C at pH 4–8 for the suspension-feeders: Crassostrea virginica (Gmelin), Rangia cuneata (Sowerby), andv Geukensia demissa (Dillwyn), and the deposit feeders, Macoma balthica (L.), and M. mitchelli Dall. Interspecific differences were considerable, but detectable amylase, carboxymethyl-cellulase, and laminarinase activities were found for all five species. At pH 6, α-amylase activity accounted for between 30 and 70% of the total activity of the three carbohydrases, cellulase for 15–27%, and laminarinase for 14–47%. The specific activities for the three carbohydrase classes at pH 6 were similar in the two deposit- and the three suspension-feeders. Species-specific catalytic activity for one enzyme class was correlated with activity levels for the other two enzyme classes. Enzyme optima were obtained at pH values which were more similar for one enzyme class obtained from different species than for the three enzyme classes obtained from the crystalline style of one particular species. For any particular species, maximum activities for crystalline style carbohydrase and the corresponding digestive gland carbohydrase were obtained at pH values which differed no more than 0.5 pH units, while the total pH range was 6.5–7.5 for α-amylases and 5–6 for laminarinases. In 13 cases out of 15, the specific carbohydrase activity of the crystalline style exceeded that of the corresponding digestive gland. We suggest that crystalline style carbohydrases play an important role not only by catalyzing extracellular digestion in the stomach, but also intracellular digestion in the digestive diverticula.

Lysozyme-like enzyme from the scallop Chlamys islandica

A lysozyme-like enzyme isolated from the crystalline style of the scallop Chlamys islandica, was purified by cation exchange chromatography on carboxymethyl cellulose and Mono S columns. The enzyme was optimally active at pH 4.8, similar to that observed for the lysozyme of Mytilis edulis. The present enzyme has an unusual 67%, remainder activity at 4ºC when cell walls of Micrococcus lysodeikticus were used as substrate. The enzyme had a specific activity of 486,090 U/mg, corresponding to ten times higher than 1ha1 of hen egg while lysozyme.

Seasonal and tidal changes in the length of the crystalline style intertidally living Macoma balthica (Mollusca, Bivalvia)

Samples of Macoma balthica were collected during 1978 and 1979 from the westernmost part of the Dutch Wadden Sea. The length of the crystalline style in the stomach of this intertidal bivalve was studied through tidal and annual cycles. The length of the style was ca. half the shell length and changed slightly with the season. Maximum lengths were recorded in spring, the season of high food intake, and minimal values in autumn and winter. Changes in the length of the style also exhibited a tidal cycle, but with two maxima and two minima. Tidal fluctuations in style length amounted to ca. 5% only. The influence of enzymes and waste products from the digestive diverticula, the food intake as well as the influence of the pH on the length of the style are discussed.

Feeding and extracellular digestive rhythms in some intertidal bivalve molluscs

The tidal rhythms for changes in morphology, length, dry weight and protein content of the crystalline style are studied in two venerid clams,Meretrix meretrix andKatelysia opima and in a donacid clam,Donax cuneatus. In the two venerid clams the style is housed in a sac conjoined with midgut and there is a tidal rhythm of feeding and extracellular digestion involving a partial dissolution of the style in the ebb tide and reformation in flood tide. But in the donacid clam where the style sac is completely separated from midgut, no detectable changes in the style parameters are recorded during a tidal cycle. However, the rhythmicity in feeding activity is indicated, by the presence of ‘mudcap’ and food particles in the style during flood tide and their subsequent absence during ebb tide. Hence the tidal rhythm of feeding and digestion inDonax cuneatus may be without corresponding changes in the crystalline style. Also this study throws light on the mode of style dissolution.

Novel bacteriolytic activity associated with the style microflora of the mussel Mytilus edulis (L.)

Previous studies have shown that bacterioplankton are retained by the mussel Mytilus edulis (L.), and that lysozyme-like enzymes are associated with the crystalline style of the mussels. This study has demonstrated the presence of a lytic agent which is produced by bacteria associated with the crystalline style. The production of the agent is inhibited by chloramphenicol, and anaerobic growth conditions. The agent is heat-sensitive and nonfilterable. Attempts at the isolation of the agent in a cell-free extract have been unsuccessful. The presence of bacteria-like cells in the outer laminae of the crystalline style, coupled with the nonfilterable nature of the lytic agent, make it seem possible that these bacteria are responsible for lysis of gram-negative bacteria which are taken in with the food supply.

Role ofCristispira sp. and other bacteria in the chitinase and chitobiase activities of the crystalline style ofCrassostrea virginica (Gmelin)

Activity was found for chitinase and chitobiase in the crystalline styles of American oysters (Crassostrea virginica Gmelin) collected from the Chesapeake Bay (Maryland, USA). The oysters were maintained in tanks on natural food from a constant flow of unfiltered estuarine water. Chitinase and chitobiase specific activities were compared with total, viable, and chitinoclastic bacterial counts andCristispira counts. Regression analyses revealed that one correlation, chitobiase vsCristispira, was significant (P < 0.05). Several oysters were fed chitin in the presence or absence of chloramphenicol. Although no chitinoclasts were present in the antibiotic-treated oysters, the treatment means did not differ significantly (P > 0.05) for either chitinase or chitobiase activity. In several cases with both chitin-fed and naturally fed oysters, enzyme activity was found when noCristispira were present. The results of the investigations suggest that the oyster produces chitinase and chitobiase endogenously.

The stomach in the bivalvia

The stomach in the bivalvia

Comparison of the enzymatic and structural characteristics of β-1,3-glucanases from marine mollusks

A comparison has been made of the enzymatic and structural characteristics of two endo-β-1,3-glutamases from the crystalline styles of bivalve mollusks. The enzymes possess similar enzymatic properties but differ in thermal stability. The molecules of the enzymes have differences in the N-terminal sequences and in the numbers of disulfide bonds, tryptophan residues, and neutral sugar residues. An homology of the primary structures is to be expected in the functionally important sections of the enzyme molecules.

Crystalline style proteins from bivalve molluscs

1. 1. Proteins from crystalline styles of twelve species of bivalve mollusc were examined under different gel electrophoresis conditions and stained to reveal both protein and carbohydrate. 2. 2. Native extracts of styles produced relatively few protein bands, however denaturation with SDS resulted in much more complex zymograms. 3. 3. All species possessed several prominent high mol wt glycoproteins. 4. 4. Eulamellibranchia all had a major non-glycosylated protein at approx. 62,000 mol. wt. 5. 5. Most Filibranchia had a major non-glycosylated protein at 37,000–50,000 mol. wt. 6. 6. Eulamellibranchia were a much more homogeneous group than the Filibranchia.

Quantitative analysis of carbohydrases in the crystalline style of some intertidal bivalve molluscs

Amylase activity in different pH ranging from 3·6 to 8·0 and activity of sucrose, maltose, trehalase, raffinase, lactase, glycogenase, pectinase, chitinase, carboxy methyl cellulase and cellulase at their respective optimum pH were quantitatively estimated in the crystalline style of intertidal bivalves,Crassostrea madrasensis, Meretrix meretrix, Meretrix casta, Katelysia opima andDonax cuneatus. Amylase was the most active among all enzymes tested. A maximum activity of 137·5 μg/mg/h was recorded in the style ofKatelysia opima while the activity was minimal inDonax cuneatus (39·3 μg/mg/h). Two pH optima were recorded for style amylase ofCrassostrea madrasensis (6·0 and 6·8) andKatelysia opima (5·6 and 6·8). But the activity was higher at higher optimum pH. The enzymes hydrolysing oligosaccharides were weak in most cases or absent. The enzymes acting on reserve polysaccharides such as amylase and glycogenase showed maximum activity in the species tested. However the activity of enzymes degrading structural polysaccharides such as cellulase, carboxy methyl cellulase, pectinase and chitinase was either at a low level or absent. The role of the style enzymes in the extracellular digestion of bivalves is discussed.

Implications for the assessment of crystalline style activity in bivalves when using the Bernfeld and Nelson-Somogyi assays for reducing sugars

The crystalline style enzymes of Choromytilus meridionalis Krauss and other bivalves include amylase, laminarinase and cellulase which are potentially capable of liberating reducing sugars from a wide variety of structural carbohydrates. However, although colorimetric analyses of reducing sugars by the Bernfeld and Nelson-Somogyi assays give qualitative agreement, during quantitative assays using glucose for calibration purposes, there is a two to seven-fold discrepancy between the two methods, depending on the enzyme and substrate reagents used. The Bernfeld assay yields the higher reducing sugar estimate, although the Nelson-Somogyi assay is some eight times more sensitive. It seems likely that the quantitative discrepancy between the two methods can be accounted for by variability in the colour response to different end products. However, when the appropriate calibration standard is used, the two methods are in close agreement. Since these two reducing sugar assays have been previously used with an inappropriate standard (glucose) to make quantitative estimates of reducing sugar release, in an effort to calculate the energetic gain from crystalline style activity, it seems likely that such energy balance studies may be in error and these have been reevaluated.

A study of the pattern of action of endo-(1→3)-β- d-glucanases from marine bivalves on a polymer substrate labelled at the reducing end

The pattern of action of L-IV and L-O endo-laminarinases (EC 3.2.1.6) from the crystalline style of the marine bivalves Spisula sachalinensis and Chlamys albidus, respectively, on Smith-degraded laminarin labelled with 3H at the reducing end has been studied. The average d.p. of the substrate was 25. On the basis of relative bond-cleavage frequencies. The L-IV enzyme was shown to attack the substrate molecule more than once per encounter. The degree of this repetitive attack and the subsite affinities of the aglycon part of the active centre of both (1→3)-β- d-glucanases have been evaluated.

Filtration, lysis and absorption of bacteria by musselsChoromytilus meridionaliscollected under upwelling and downwelling conditions

Choromytilus meridionalis Krauss is able to filter single bacterial cells from surrounding water, even at low bacterial concentrations (4,16 × 106·mℓ−1). Under upwelling conditions (cold), filtration of bacteria is only slightly more efficient than under downwelling conditions (warm). However, upwelling conditions correspond with a very much higher efficiency of incorporation of bacterial biomass into body tissues owing to the production of a bacteriolytic factor by the crystalline style of the mussel, which only happens during upwelling. During upwelling, the concentration of lytic factor rises to 3 100 units·mg−1 style protein but falls to < 1 unit·mg−1 during downwelling. The lytic factor cause lysis of a wide range of marine bacterial strains, those isolated from the water column being particularly sensitive. The lytic factor is stable at 80°C. It does not lyse Micrococcus luteus, Escherichia coli or Bacillus subtilis and is therefore not a true lysozyme.