Digestion in a subtidal population of Mercenaria mercenaria (Bivalvia)

The digestive processes of Mercenaria mercenaria (Linn.) were studied in a natural subtidal population. Hourly sampling of 4 to 5 quahogs was conducted over one 25 h period in August, 1978, at Woods Hole, Ma., USA. Crystalline style length did not vary significantly with time, tide, particulate C, N or suspended material in the water. Changes in secretory activity of the style sac epithelium were not evident from histological sections. Based on cellular morphological differences, digestive tubules were individually classified into one of 4 categories, indicative of the state of intracellular digestion within the digestive cells. All 4 tubule types were present in individuals from each sample hour, signifying that intracellular digestion occurs continually within the digestive gland. A 3 h time lag is evident between times of peak C, N or suspended materials and apparent peak levels of absorptive (Type II) tubules.Contribution No. 76 Marine Science Institute, Northeastern University; Nahant, MA 01908, USA     

Structural changes in the hepatopancreas of the rock lobster,Jasus edwardsii (Crustacea: Palinuridae) during development from the puerulus to post-puerulus

There is evidence for non-feeding in pueruli of palinurid lobsters during their long-distance swim to the coast and the succeeding post-settlement phase until they molt to the juveniles. For a better understanding of the nutritional supply during the puerulus stage, structural changes in the hepatopancreas were studied in post-settlement pueruli and first-molt, postpueruli of the rock lobster, Jasus edwardsii, collected on the southeast coast of the North Island, New Zealand between 1989 and 1990. In newly settled (transparent) pueruli, the epithelium of the hepatopancreas is dominated by presumed young R-(resorptive) cells, which are devoid of lipid droplets, but show active uptake of material from the hemolymph through pinocytosis at the basal region. In the fully pigmented puerulus, the hepatopancreas is dominated by R-cells containing massive lipid droplets; also present are small numbers of F-(fibrillar) and B-(blister-like) cells. This increase of lipid inclusion in the R-cells corresponds to the reduction in size of the fat bodies as the lobster develops from the transparent to the pigmented puerulus. After the molt to the post-puerulus, the R-cells are compressed, and the lipid droplets almost disappear, while the B-cells increase both in size and number. A mechanism is proposed in the present paper to explain energy storage and utilization during the puerulus and post-puerulus stages, wherein the fat bodies and the lipid inclusions play a vital role.     

Observations on the biochemical changes in gonads and other organs of Uca annulipes,Portunus pelagicus and Metapenaeus affinis (Decapoda: Crustacea) during the reproductive cycle

The fluctuations in biochemical constituents such as water, nitrogen, non-protein nitrogen, protein, lipid and glycogen in gonad, muscle and hepatopancreas have been followed in 3 decapod crustaceans, Uca annulipes (Latreille), Portunus pelagicus (Linnaeus) and Metapenaeus affinis (Milne-Edwards). The water and ash content of the entire body show no systematic fluctuation in relation to the annual reproductive cycle. The water content of the ovary diminishes as it matures. In the ovary of these crustaceans, the lipid fluctuated greatly in relation to the reproductive cycle. The maturing ovary contains more lipid than an immature or spent ovary per unit tissue weight. The changes in the biochemical constituents in the testis are not so pronounced as in the ovary, since the testicular cycle is often drawn-out and almost continuous in these crustaceans. There is an inverse relationship between water content and lipid content of the hepatopancreas; the greater the fat content, the lesser the water content. The hepatopancreas in these crustaceans is apparently a storage organ and contains much lipid and glycogen. At the height of the breeding season, when gonad production is intense, there is an indication of the mobilisation of at least a part of the lipid from hepatopancreas to gonad.Dissertation for the award of a Doctorate Degree of the Kerala University.Formerly the Marine Biological Laboratory.     

Diel changes in gut-cell morphology and digestive activity of the marine copepod Acartia tonsa

Morphometric measurements of the gut cells of the marine calanoid copepod Acartia tonsa were made over a 24 h diel cycle to test the hypothesis that feeding rhythms in this copepod are limited by the cycles of blishter-like cells (B-cells) in the gut. Copepods were collected from Long Island Sound, New York, USA, in June 1991. Experimental treatments included low (1 g chlorophyll al^-1) and high (10 g chlorophyll al^-1) food concentrations. Copepods were fixed and embedded in Epon at six time intervals over the 24 h period, and semi-thin sections through the length of Midgut Zone II were analyzed for gut-cell area and vacuole area, with area measurements integrated to yield estimates of gut-cell volume and vacuole volume. Concurrent measurements of gut fluorescence, a measure of gut fullness, and digestive enzyme activities also were made. A diel cycle in gut fluorescence was observed, most notably in the low-food treatment, which exhibited a 3-fold increase in gut pigments. There was little consistent change in digestive enzyme activities over this time span. Gaps in the gut wall indicative of spent B-cells were observed in 17 of 39 copepods, with no trend over time or food treatment. Generally, only a single gap was seen in any one copepod. B-cells were vacuolated throughout the 24 h period. Morphometric analyses revealed a correlation between gut fluorescence and both maximum vacuole area and vacuole volume, as well as percent vacuolated cell volume, in the low-food treatment. The high-food treatment, which had a relatively smaller increase in gut fluorescence (1.5-fold) during the night cycle, showed no significant increase in vacuole size. B-cells appear to have a life-cycle greater than the diel feeding period, and while B-cell vacuoles respond to the ingested food during the diel cycle, production of B-cells does not appear to be a limiting factor in A. tonsa's feeding cycle.     

Glycogen storage in relation to the moult cycle in the two crustaceansEmerita asiatica andLigia exotica

InEmerita asiatica, the quantitative fluctuations in the glycogen content of the hepatopancreas were markedly related to the moult cycle. The glycogen content of the hepatopancreas was maximum during premoult stages. It has been suggested that reserve glycogen, in addition to meeting general metabolic needs, may be especially involved in the genesis of the sugar precursors of chitin. The marked fall in the hepatopancreas glycogen values following moulting is probably due to its utilization in chitin synthesis during and immediately following moulting. InE. asiatica, the storage of glycogen was found to be meagre in muscles, and muscle glycogen had no bearing on chitin synthesis during moulting. InLigia exotica, glycogen is stored to a lesser extent than inE. asiatica, and was found in connective tissue and muscles. Significant fluctuations were noted in glycogen values correlated with the phases in the moult cycle when chitin synthesis took place. InL. exotica, the hepatopancreas does not serve as a storage depot for glycogen related to chitin synthesis.     

Variable transfer of detoxified metals from snails to hermit crabs in marine food chains

In any polluted marine environment, different invertebrate species contain markedly different concentrations of heavy metals. Primary producers take metals from seawater, but animals take additional metals from diets of animals, plants and detritus. Metals in a dietary organism of a food chain have varying reactivities and they follow different biochemical pathways. Excess metals are bound by ligands to form insoluble compounds within cytological compartments. These metabolic systems prevent the disruption of normal biochemical reactions by metals. The present work on Mediterranean invertebrates, initiated in Greece in 1993, used digestive glands from three species of marine snail,Monodonta mutabilis (Philippi),Cerithium vulgatum (Bruguière), andMurex trunculus (Linnaeus) as prey tissue, and hermit crabsClibanarius erythropus (Latreille) as predators; the digestive glands and faecal pellets from all animals were analysed by atomic absorption spectroscopy and x-ray microanalysis. Most metals detoxified by the snails are unavailable to the crabs and they pass straight through the gut and appear in the faecal pellets. This applies to significant proportions of the manganese, nickel, copper, zinc and silver which are bound electrostatically to phosphate or covalently to sulphur within membrane-bound intracellular compartments. Cadmium and chromium are transferred to the crabs. In digestive glands of snails, cadmium is bound to soluble highsulphur protein in the cytosol; the cytology of chromium in these animals is not known.     

Hepatopancreatic endosymbionts in coastal isopods (Crustacea: Isopoda), and their contribution to digestion

Three isopod species (Crustacea: Isopoda), commonly found in the intertidal and supratidal zones of the North American Pacific coast, were studied with respect to symbiotic microbiota in their midgut glands (hepatopancreas). Ligia pallasii (Oniscidea: Ligiidae) contained high numbers of microbial symbionts in its hepatopancreatic caeca. Numbers of endosymbionts were strongly reduced by ingestion of antibiotics. By contrast, the hepatopancreas of Idotea wosnesenskii (Valvifera: Idoteidae) and Gnorimosphaeroma oregonense (Sphaeromatidea: Sphaeromatidae) did not contain any microbiota. Results of feeding experiments suggest that microbial endosymbionts contribute to digestive processes in L. pallasii, the most terrestrial of the three isopods that we studied. The acquisition of digestion-enhancing endosymbionts may have been an important evolutionary step allowing isopods to colonize terrestrial habitats where relatively indigestible leaf litter is the primary food source. By contrast, the ability to digest phenolic compounds was most developed in one of the more marine species, suggesting that this trait may have evolved independently in isopod species that consume a phenolic-rich diet, whether in marine habitats or on land. Received: 28 August 2000 / Accepted: 8 December 2000     

Endocrine regulation of carbohydrate metabolism during the moult cycle in crustaceans I. Effect of eyestalk removal in Ocypoda platytarsis

The endocrine regulation of carbohydrate metabolism in the decapod Ocypoda platytarsis was investigated by eyestalk ablation at all stages of the moult cycle. Eyestalk removal (and hence removal of a substance probably produced by it) profoundly influences the carbohydrate level of the major storage organ, the hepatopancreas. The changes taking place storage organ, the hepatopancreas. The changes taking place in hepatopancreatic glycogen are reflected in the blood sugar (glycemic) level. The blood-sugar level is also related to dynamic events taking place in the cuticle during the moult cycle. At the intermoult and postmoult stages, the eyestalk factor directs the sugar supply for energy metabolism, and prevents sugar deposition as glycogen in the hepatopancreas. At the premoult stage, the activity of the eyestalk is suppressed by the moulting gland (the Y organ), as eyestalk ablation does not alter the blood-sugar value or the hepatopancreatic glycogen-level. After the increase at the freshmoult stage due to eyestalk ablation, the blood-sugar level tends to re-attain its previous low level at the postmoult stage.     

Subcellular distribution of zinc and cadmium in the hepatopancreas and gills of the decapod crustacean Penaeus indicus

The decapod crustacean Penaeus indicus accumulated Cd and Zn in different subcellular compartments of hepatopancreas and gill cells. Most of the Cd and part of the Zn accumulates within the soluble fraction of the cells, while the remainder of the Zn is found in insoluble inclusions, associated with P, Ca, Mg and Si in B-, F- and R-cells in the hepatopancreas, and haemocytes, nephrocytes and epithelial cells in the gills. No presence of Cd was observed in metal-rich inclusions in any cell analysed. Metallothionein-like proteins (MTLP), analysed by differential pulse polarography, were present in the hepatopancreas (12–18 mg g⁻¹) and gills (7–8 mg g⁻¹) of metal-exposed prawns. Binding to MTLP is the detoxification mechanism for cadmium, while the detoxification of zinc involves both binding to MTLP and incorporation into insoluble metal-rich inclusions.     

Chitobiase activity in the epidermis and hepatopancreas of the fiddler crab Uca pugilator during the molting cycle

The activity of chitobiase, also known as N-acetyl-β-glucosaminidase, in the epidermis and hepatopancreas of the fiddler crab Uca pugilator (Bosc, 1802), during the molting cycle, was investigated. A pH optimum of 5 to 6 was found for the enzymatic activity in both the epidermis and hepatopancreas. The temperature optimum for epidermal and hepatopancreatic chitobiase activities was 50 to 60 °C. The K _m values for epidermal and hepatopancreatic chitobiase activities at 19 °C were 0.190 ± 0.027 and 0.203 ± 0.016 mM 4-methylumbelliferyl-N-acetyl-β-glucosaminide, respectively. Hepatopancreatic chitobiase activity was significantly higher than epidermal enzymatic activity in all the molt cycle stages tested except Postmolt Stage A-B. Chitobiase activity varied significantly during the molting cycle, with the epidermal enzymatic activity in Premolt Stage D_3–4 significantly higher than in Stage C (intermolt) and Premolt Stage D₀, whereas hepatopancreatic chitobiase activity in Premolt Stage D_3–4 was significantly higher than in all other molt stages tested. The patterns of chitobiase activity in the epidermis and hepatopancreas correlate well with the hemolymph titer of ecdysteroids in U. pugilator during the molting cycle; this suggests that chitobiase activity in both tissues is regulated at least in part by the steroid molting hormones. Received: 6 May 1998 / Accepted: 12 September 1998     

Tissue distribution and excretion of 99mTc-disofenin in three marine species: Pleuronectes americanus (winter flounder), Homarus americanus (lobster), and Mya arenaria (soft-shell clam)

To determine the pharmacokinetics of a small lipophilic molecule in vivo, the distribution and accumulation of ^99mTc-radiolabelled disofenin (diisopropylacetanilide iminodiacetic acid) were traced during 1991–1992 by scintigraphy and gamma well counting in winter flounder (Pleuronectes americanus collected from Boston Harbor and Long Island Sound in 1992), lobsters (Homarus americanus collected from Massachusetts Bay in 1991), and soft-shell clams (Mya arenaria purchased in 1991). The agent was distributed throughout the bodies of lobsters within 12 s, throughout flounder within 40 s, and throughout clams within 2 min. It was concentrated most strongly by the liver of flounder, which contained 61.2±7.8% of the injected dose within 1 h of injection, and by the lobster hepatopancreas. Accumulation also occurred in the flounder kidney, lobster antennal glands, and the kidney and pericardial glands of clams. The compound was rapidly excreted from the flounder liver into the gall bladder, and from the lobster hepatopancreas into the stomach. The data suggest its excretion from the lobster antennal glands and clam kidneys. The rate of clearance of disofenin from the body varied among species: 99±2.1% of the initial dose remained in flounder sampled 16 to 24 h after injection, compared to 80.5±7% remaining in the lobster after 15 h, and 87.4±5.9% remaining in clams after 27 h. The clearance rates in flounder and lobster are considered to be minimum values because of the lack of gut activity in unfed individuals. Overall, these in vivo tracer studies establish the utility of scintigraphy for assessing the uptake and excretion of a lipid soluble compound in different taxa, and may be applicable for evaluating disease and/or environmental effects on organ function in marine animals.     

Cnidosac morphology in dendronotacean and aeolidacean nudibranch molluscs: from expulsion of nematocysts to use in defense?

Nudibranchs (Mollusca, Gastropoda) feeding on tentacles and polyps of Cnidaria thereby ingest both latent and discharged nematocysts (NCs) along with the food mass. In eolid nudibranchs (Opisthobranchia, Aeolidacea), many of the undischarged NCs are transported to terminal cnidosacs in their body appendages (cerata) and incorporated as kleptocnidae for defense. In the present report, the occurrence and fate of NCs in the digestive tracts of eolids is compared with hydrozoan-feeding dendronotacean nudibranchs (Opisthobranchia, Dendronotacea), which may show more basic stages in the evolution of cnidosacs. Tomographic reconstructions of the distal tips of cerata were composed from series of semithin light microscopic sections, utilizing 3D-surface rendering software. Doto acuta (Dendronotacea, Dotidae) does not have cnidosacs; transmission electron micrographs show that the NCs are digested in lysosomes of digestive gland cells. In contrast, species of the genus Hancockia (Dendronotacea, Hancockiidae) have several small cnidosacs in each ceras; they accumulate NCs in the digestive cells, as well as in the cnidosacs. Many of the cnidosacs were found open to the exterior with NCs in the process of expulsion. These and other structural details suggest assigning a function of expelling the NCs to the Hancockia spp. cnidosacs. It is proposed that cnidosacs similar to those of Hancockia spp. provide a clue to understanding how the defensive function of eolid cnidosacs may have evolved. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Short-term changes in feeding and digestion by the copepodCalanus pacificus

The planktonic marine copepodCalanus pacificus exhibits an enhanced feeding rate, or hunger response, when exposed to food following short periods of starvation. In a scries of laboratory experiments with copepods collected from the main basin of Puget Sound, Washington, during 1982 and 1984, we measured maximum ingestion rate, assimilation efficiency, and digestive enzyme activity to determine the time scales over which the feeding behavior ofC. pacificus responds to increases in food. These laboratory results were then compared to field studies of diel fluctuations in digestive enzymes and gut fluorescence ofC. pacificus in Dabod Bay, a fjord of Puget Sound, during September, 1980, and the closely relatedC. marshallae off the Washington coast, in August, 1981. Laboratory experiments demonstrated that the hunger response ofC. pacificus lasts approximately 6 h before ingestion rate returns to a steady state level of about one-half maximum. On the order of 12h of starvation were required to induce the maximum ingestion rate of the hunger response. Digestive enzyme activities did not change over these time scales. Assimilation efficiency peaked within a few hours of the onset of feeding, with low initial rates possibly related to the period of starvation prior to feeding. These results were consistent with diel patterns observed in the field. The hunger response ofC. pacificus appears to be controlled by processes within the gut, and our results are discussed in relation to recent studies of the digestive processes of calanoid copepods.Contribution No. 1772 from the School of Oceanography, University of Washington, Seattle, Washington 98195, USA     

Biochemical correlates of dissolved mercury uptake by the oyster Ostrea edulis

From previous work, the equilibrium concentration factor for dissolved mercury in the digestive gland of Ostrea edulis Linnaeus was found to be three to four times higher than that in the gills. In the present study, an analysis of soluble protein revealed values of 49.3±14.2 mg g wet tissue^-1 for the digestive gland and 0.7 ±0.1 mg g wet tissue^-1 for the gills. Starvation significantly reduces the soluble protein level of the digestive gland to 31.1±6.4 mg g^-1 and that of the gills to below the limit of detection. These results suggest that the difference in concentration factors between the gills and digestive gland may be based on a quantitative difference in macromolecular binding sites. However, the uptake of dissolved mercury over a period of 48 h was considerably greater in the gills, so that although the soluble protein content of the tissue may influence the final concentration factor, it does not appear to affect the rate at which this equilibrium is achieved. A more detailed investigation of the mechanism of dissolved mercury uptake by oyster gills has been carried out using isolated tissues. The process is inhibited by 5mM 2–4 dinitrophenol, by the absence of a readily metabolizable substrate (dextrose) in the uptake medium, and by 30mM K⁺. The effect of K⁺ necessitated further investigation with a specific inhibitor of K⁺ transport. Strophanthin G (ouabain), at a concentration of 0.01 mM, caused a significant increase in mercury uptake.     

Influence of physiological condition on cadmium transport from haemolymph to hepatopancreas inCarcinus maenas

Accumulation and binding of cadmium in the tissues of the shore crabCarcinus maenas (L.) were investigated in a series of laboratory experiments. The relation between the physiological condition of individual crabs and their ability to transport cadmium from haemolymph to hepatopancreas was specifically addressed. Cadmium was removed from the haemolymph with a half-life of approximately 10 h, and half of the cadmium removed from the haemolymph was taken up in the hepatopancreas. The efficiency with which individual crabs transported cadmium from the haemolymph to the hepatopancreas was strongly related to physiological parameters, such as concentrations of calcium and magnesium in the hepatopancreas and haemolymph volume and haemolymph protein concentration. The transport of cadmium from the haemolymph to the hepatopancreas was saturated in crabs exposed to more than 2 to 4 mg Cd l^–1 in the sea water. In the hepatopancreas of unexposed crabs, cadmium was bound mainly in the insoluble tissue fraction (40%) and in the protein fraction with a molecular weight of approximately 6000 D (50%). Exposure to 0.25 to 1.5 mg Cd l^–1 for 2 w led to dose-dependent increases of the amounts of cadmium bound in the high-molecular weight protein fraction and in the insoluble tissue fraction. Modes of internal cadmium transport and accumulation in tissues and variability in physiological parameters are discussed.     

Ultrastructural changes in the lysosomal-vacuolar system in digestive cells of Mytilus edulis as a response to increased salinity

The effects of increasing salinity on the ultrastructural morphology of the lysosomal-vacuolar system in digestive cells of the common mussel Mytilus edulis were investigated in order to relate structural changes to previous biochemical and cytochemical observations. After 3 h of increased salinity, from 21 to 35%., the digestive cells showed an increase in numbers of heterolysosomes. There was some evidence of digestive cell breakdown, the contents forming membrane-bound bodies and being released into the tubule lumen. After 12 h of increased salinity, heterolysosomes were prevalent in the digestive cells. There was increased evidence for digestive-cell breakdown, many of the tubule lumina being packed with membrane-bound bodies. It is concluded that increasing salinity from 21 to 35%. stimulates the lysosomal-vacuolar system, as a result of autophagocytosis or apoptosis; this is consistent with the hypothesis that intracellular, lysosomally-mediated, catabolism of proteins is a source for free amino acids during the adaptation of mussels to increased salinity.     

Fatty acid composition of organs and tissues of the tiger prawn Penaeus esculentus during the moulting cycle and during starvation

The fatty acids (FA) in neutral lipid (NL) and polar lipid (PL) of digestive gland, muscle and integument of Penaeus esculentus Haswell were analysed and compared during the moulting cycle and during starvation. The prawns were collected from Moreton Bay, Queensland, Australia, by trawling during 1985–1987, and were fed with a standard semi-purified diet. Compared with a natural diet, the artificial diet had much higher levels of 18:1n-9 and 18:2n-6, but only trace amounts of 20:4n-6, but there was no evidence of dietary imbalance. The fatty acid composition (percentage of total lipid) of the digestive gland changed markedly during the moulting cycle and during starvation, but the small changes observed in both muscle and integument suggested that these tissues maintained their composition under both conditions. When the fatty acids were calculated as absolute amounts, muscle composition, as well as that of the digestive gland, changed significantly. In the digestive gland, saturated FA (SFA), monounsaturated FA (MUFA), diunsaturated FA (DUFA) and polyunsaturated FA (PUFA) all increased up to the middle of the moulting cycle and then declined; with starvation all groups decreased. In muscle, SFA, MUFA and DUFA all increased during the moulting cycle; starvation caused SFA, MUFA and PUFA to decrease, whereas DUFA did not vary. Starvation caused both 18:2n-6 (linoleic) and 18:3n-3 (linolenic) in the digestive gland to reach or almost reach zero. The other essential PUFA, 20:4n-6 (arachidonic), 20:5n-3 (eicosapentaenoic) and 22:6n-3 (docosahexaenoic), decreased during the moulting cycle, but during starvation 20:4n-6 did not decrease as much. In muscle, the levels of 18:2n-6 and 18:3n-3 increased, while 20:4n-6, 20:5n-3 and 22:6n-3 remained approximately constant during the moulting cycle. Starvation reduced 20:5n-3 and 22:6n-3 to about 60%. The data suggest that levels of 18:3n-3, 20:4n-6, 20:5n-3 and 22:6n-3 are regulated, and that 20:4n-6 can be synthesised from 18:2n-6. There is no clear evidence that 20:5n-3 and 22:6n-3 are essential in P. esculentus, but tissue catabolism of cell membranes during starvation may have provided sufficient amounts for maintenance.     

Polonium-210 and lead-210 in marine food chains

²¹⁰Po and ²¹⁰Pb have been measured systematically in whole animals, muscle and hepatopancreas of crustaceans and of molluscan cephalopods representative of a pelagic and benthic food chain. The same nuclides were also measured in liver, pyloric caecum, stomach contents and muscle of tuna. The concentration factors from sea water to whole animals were approximately constant along both food chains, being of the order of 10⁴ for ²¹⁰Po and 10² for ²¹⁰Pb. The highest concentration factors were found in shrimp of the genus Sergestes. In muscle, the concentration factors were an order of magnitude less; in the hepatopancreas, they were an order of magnitude higher, reaching 10⁶ in shrimp of the genus Sergestes. Such concentrations imply alpha-radiation doses of the order of 10 rem per year and more in this organ, which contains about 50 to 90% of the ²¹⁰Po in the whole animal in the 11 species analyzed. A detailed study of the intracellular behaviour of ²¹⁰Po in the hepatopancreas is clearly indicated. ²¹⁰Po can be used as a sensitive natural tracer in biological systems. Thus, feeding Meganyctiphanes norvegica in the laboratory on food low in ²¹⁰Po led to an approximate value of about 61/2 days for the biological half-life of ²¹⁰Po in the hepatopancreas of this euphausiid. Furthermore, the data on ²¹⁰Po and ²¹⁰Pb in the cephalopod hepatopancreas allowed the time of conservation of frozen squid which had been bought at the market to be estimated.     

Intracellular digestion in two sublittoral populations of Ostrea edulis (Lamellibranchia)

The rhythmicity of intracellular digestion was examined in two sublittoral populations of Ostrea edulis L. On the West coast of Ireland. During 12 h cycles at each station, 20 oysters were collected each hour, grouped as sub-samples of 5, facing each of the 4 cardinal points of the compass. A segment of digestive diverticula from each oyster was examined histologically and classified according to the digestive phases of the tubules. Oysters at both stations exhibited fluctuations in digestive activity which were not correlated with tidal ebb and flow nor with orientation to tidal currents. A relationship between variations in suspended particulate matter concentration in the water body and digestion is proposed. It is suggested that increases in the levels of particulate matter, by stimulating feeding, cause a significant increase in the proportion of absorptive-phase tubules 4 to 6 h later.     

Cell cycle and toxin production in the benthic dinoflagellate Prorocentrum lima

Profiles of diarrhetic shellfish poisoning (DSP) toxins produced throughout the growth cycle and the cell cycle of the toxigenic marine dinoflagellate Prorocentrum lima were studied in triplicate unialgal batch cultures. Cells were pre-conditioned at 18 ± 1 °C, under a photon flux density (PFD) of 90 ± 5 μmol m⁻² s⁻¹ on a 14 h light:10 h dark photoperiod. In exponential growth phase, cultures were synchronized in darkness for 17 d. After dark synchronization, cultures were transferred back to the original photoperiod regime. Cells were harvested for DSP toxin analysis by LC-MS (liquid chromatography with mass spectrometry), and double-stranded (nuclear) DNA was quantified by flow cytometry. The cell populations became asynchronous within approximately 3 d after transition from darkness to the 14 h light:10 h dark photoperiod. This may be due to the prolonged division cycle (5 to 7 d) that is not tightly phased by the photoperiod. Unlike other planktonic Prorocentrum spp., cytokinesis in P. lima occurred early in the dark and ceased by “midnight”. Cellular levels of the four principal DSP toxins, okadaic acid (OA), OA C8-diol-ester (OA-D8), dinophysistoxin-1 (DTX1) and dinophysistoxin-4 (DTX4), ranged from 0.37 to 6.6, 0.02 to 1.5, 0.04 to 2.6, and 1.8 to 7.8 fmol cell⁻¹, respectively. No toxin production was evident during the extended period of dark synchronization nor during the initial period when NH₄ was consumed as the major nitrogen source. Soon after the cells were returned to the 14 h light:10 h dark cycle and they began to take up NO₃, cellular levels of all four toxins gradually increased. This increase in DSP toxins usually occurred in the light, marked by a rise in DTX4 levels that preceded an increase in the cellular concentration of OA and DTX1 (delayed by 3 to 6 h). Thus, DTX4 synthesis is initiated in the G1 phase of the cell cycle and persists into S phase (“morning” of the photoperiod), whereas OA and DTX1 production occurs later during S and G2 phases (“afternoon”). No toxin production was measured during cytokinesis, which happened early in the dark. The evidence indicates that toxin synthesis is restricted to the light period and is coupled to cell cycle events. Received: 3 September 1998 / Accepted: 30 March 1999