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Ventricles isolated from clams (Mercenaria mercenaria) that had been acclimated to 1000 mOsm seawater (SW) release amino acids when incubated in 500 mOsm SW. Taurine, glycine, and alanine account for nearly all of the released amino acids, and total about 37 μmol/g dry tissue weight during a 2-h incubation. The release of amino acids is increased to 69 μmol/g by the addition of 10-6 M 5-hydroxytryptamine (5HT) to the hypoosmotic SW, and to 83 μmol/g by the addition of 10-6 M FMRFamide to the medium. The potentiation of the release by 5HT is blocked by methysergide. The amino acid release is increased by two phorbol esters--phorbol 12,13-diacetate and phorbol 12-acetate, 13-myristate--to 97 and 83 μmol/g, respectively. Forskolin and other cyclic 3',5' adenosine monophosphate agonists have no effect on the release of amino acids in hypoosmotic SW. Phorbol esters, 5HT, and FMRFamide have no effect on the release of amino acids from ventricles incubated in 1000 mOsm SW. Ventricles, first isolated from clams acclimated to 1000 mOsm SW, and then transferred to 500 mOsm SW, increase in wet weight by 20-25%. The increase is maintained for 30 min, and the tissues return their original weight in the ensuing 30 min. The addition of 5HT, FMRFamide, or phorbol esters to the hypoosmotic SW decreases the time necessary for the tissues to return to pre-transfer weights. These results implicate protein kinase C in the responses of bivalve tissues to hypoosmotic media, and suggest that these responses may be modified by neuronal or neurohumoral control.

The "brown tide" alga, Aureococcus anophagefferens, had no effect on the activity of the lateral cilia of isolated gills of the bivalve mollusks Argopecten irradians, Geukensia demissa, and Mya arenaria. A. anophagefferens caused a significant decrease in the activity of lateral cilia of five other bivalve mollusks, Crassostrea virginica, Ostrea edulis, Mercenaria mercenaria, Modiolus modiolus, and Mytilus edulis. Exposure of isolated gills of M. edulis to the water from which A. anophagefferens had been removed, or to polystyrene beads at the same concentration as A. anophagefferens, had no effect upon the activity of the lateral cilia. Thus, the inhibition of the lateral cilia is not caused by a compound excreted into the water, nor is it the result of the high density of cells. The response of lateral cilia to dopamine was identical to the response to A. anophagefferens; lateral cilia that were inhibited by dopamine were also inhibited by A. anophagefferens. Pretreatment of the gills of M. edulis with the dopamine antagonist ergometrine blocked the inhibition of the lateral cilia by both dopamine and A. anophagefferens. A water-soluble inhibitory compound was released from A. anophagefferens by exposing the cells to amylase, and then removing the cells by filtration. The effect of this inhibitory compound was also blocked by ergometrine. We propose that the isolated gills are digesting the extracellular coat of A. anophagefferens releasing a water soluble dopamine-mimetic compound that causes inhibition of lateral cilia.

The SCPs3 are a small peptide family, characterized in gastropods, and implicated in the control of the cardiovascular system and the muscles involved in feeding and gut motility. We aimed to determine the manifestation of this peptide family in the class Bivalvia. Acetone extracts of whole bivalves were fractionated by high pressure liquid chromatography (HPLC), and reactive peaks were identified by radioimmunoassay (RIA). After purification, sequencing, and analysis by mass spectroscopy, three peptides were identified in the clam Mercenaria mercenaria: IAMSFYFPRMamide, AMSFYFPRMamide, and YFAFPRQamide4. SCP-related peptides from two other species were also sequenced: APKYFYFPRMamide and SAFYFPRMamide from an oyster, Crassostrea virginica; and AMSFYFPRMamide (identical to one of the clam peptides) from a cockle, Dinocardium robustum. The tissue distribution and pharmacological actions of the clam SCPs were determined in M. mercenaria, as follows. The levels of peptide in extracts of 12 tissues were estimated by RIA. The largest concentrations of SCP occur in the palps and the visceral ganglia; the levels in the cerebral and pedal ganglia, the rectum, intestinal typhlosole, and gills were substantially lower; and the smallest amounts were found in the heart and the style sac typhlosoles. Immunohistochemistry revealed many cell bodies in the periphery of the ganglia and fibers in the neuropil. Immunoreactive, varicose fibers also occur in the typhlosoles of the intestine and style sac, and in the rectum, gill, and palps. The atrioventricular valves, but not the atria or ventricle proper, contain immunoreactive fibers. Synthetic clam SCPs were assayed on the rectum, the typhlosoles of the intestine and style sac, and the ventricle, all isolated in an organ bath. At low to moderate doses, the SCPs relaxed the muscles of the rectum; higher doses had biphasic actions. The muscles of the intestinal and style sac typhlosoles were relaxed, and spontaneous rhythmicity was slowed by the SCPs. Most ventricles were unresponsive. We conclude that the SCPs isolated in bivalves--though distinctive--are true homologs of those in gastropods. Moreover, the bivalve peptides also serve similar roles, controlling feeding and digestion, and perhaps even cardioactivity.

In marine sediments, many of the processes associated with high post-settlement mortality of infauna have similar effects on the sediment surface. In most cases the original sediment surface is either removed, buried, or mixed with subsurface sediment. The experiments reported here tested the ability of new juvenile infauna to discriminate between undisturbed and recently disturbed sediment surfaces (i.e., subsurface sediment exposed). Recently settled juveniles of two polychaete species (Nereis vexillosa and Arenicola cristata) and one bivalve species (Mercenaria mercenaria) were exposed to simulated erosional and mixing events as well as to fresh feces, burrow tailings, and feeding tracks. Where the disturbance buried or removed several millimeters of the sediment surface, the time to initiate burrowing or the percentage of individuals failing to burrow increased significantly over times and percentages for juveniles on undisturbed surfaces. In all cases the results were consistent with the hypothesis that new juveniles reject (or are significantly slower to burrow into) disturbed sediment surfaces, if the disturbance is less than several hours old. For example, 51% of nereid juveniles did not burrow when placed on subsurface sediments, whereas 100% burrowed into surface sediments; their average burrowing time on surface sediments was 29.3 s compared with 109.7 s on fecal mounds of arenicolid polychaetes or 106.1 s on burrow tailings of thalassinid crustaceans. Individuals that did not indicate acceptance of a sediment surface by burrowing were all rapidly eroded from the surface in the presence of flow. Erosion of nonburrowing individuals occurred within 90 s of initiation of flow. Burrowing individuals were not eroded. The decision as to the acceptability of a sediment was made within 30 s. These data imply that the new juveniles are utilizing cues associated with a process, the disturbance of surface sediments, in addition to the species-specific cues described elsewhere.

The importance of chemical signals as foraging and orientation cues has been demonstrated for many marine organisms. It is still unclear whether sea stars use chemical signals during orientation and whether chemoreception occurs in the absence of macroscale flow. To determine whether the sea star Asterias forbesi can perceive chemical signals in the absence of flow and what role such signals play in orientation and foraging behavior, we tested the orientation behavior of sea stars to prey and nonprey items under conditions of nondirectional flow. Prey items were whole and broken clams (Mercenaria mercenaria) and mussels (Mytilus edulis); the nonprey item was squid flesh. Asterias forbesi showed the ability to successfully locate odor sources irrespective of the type of odor. Only in trials with the broken clam did the animals reveal an initial directional choice towards the odor source. There were significant changes in the movement rates and heading angles during orientation for all three stimuli. In addition, orientation paths were different for each of the chemical stimuli tested. From these results, we conclude that sea stars can detect and respond to chemicals in the absence of macroscale flow. Orientation paths appear to be more of a taxis, in which heading is directly guided by the stimulus field.

The in vivo response of adult northern qua- hogs, Mercenaria mercenaria ,t oAureococcus anophagef- ferens (brown tide) at the level of the gill was determined using video-endoscopy. Feeding activity, particle-approach velocities, and ventral-groove-transport velocities were documented after the quahogs were exposed to Isochryis galbana (baseline observations) supplemented with either toxic or nontoxic A. anophagefferens at two bloom concen- trations (8 10 5 or 2 10 6 cells ml -1 ). Externally, there was no evidence of adverse effects of brown tide on feeding, as siphons remained extended and dilated. Toxic brown tide at both concentrations elicited gill muscular contractions, intermittent cessation of water flow, and decreased particle loading within the pallial cavity. The 8 10 5 cell ml -1 toxic treatment had no significant effect on approach velocities or ventral-groove-transport velocities after 2 h, although time- averaging showed significant reduction of the latter during the last 30 min of exposure. The higher concentration of toxic brown tide caused a significant decrease in these velocities after only 1 h. Nontoxic brown tide produced none of these effects. Thus, A. anophagefferens compro- mised quahog feeding by stimulating contractions of the branchial musculature and interfering with lateral and ven- tral groove ciliary beating. These effects were both time- and concentration-dependent and could be caused by either a dopaminergic or a serotonergic toxic factor.

The activities of both the lateral and frontal cilia of Mercenaria mercenaria were unaffected, either by the two endogenous SCP-related peptides AMSFYFPRMamide and YFAFPRQamide, or by FMRFamide (all at 10(-6) M). Dopamine (DA) inhibited the lateral cilia; the mean EC50 was 2 x 10(-6) M. The peptide YFAFPRQamide--but neither AMSFYFPRMamide nor FMRFamide--antagonized the inhibition induced by DA; this effect was dependent on both time and dose. At a DA concentration of 5 x 10(-7) M, the effect of YFAFPRQamide appeared within 20 min and became maximal within 40-60 min; the mean EC50 at these times was 4.7 x 10(-11) M. If the concentration of DA was increased to 10(-6) M, the maximal effect of the peptide was delayed to 50 min, and the mean EC50 increased to 1.1 x 10(-7) M. Particle transport by the frontal cilia was inhibited by 5-hydroxytryptamine (5HT); the mean EC50 was 5.7 x 10(-7) M. Again, only YFAFPRQamide had an antagonistic effect on the 5HT-induced inhibition. At a 5HT concentration of 10(-6) M, the effects of YFAFPRQamide did not appear until 45 min; the mean EC50 was 10(-6) M. When radioimmunoassayed with an SCP antiserum, the elution profile of a gill extract overlapped those of the SCP-related peptides that had previously been identified in extracts of whole animals. These data suggest that all three SCP analogs occur in the gill. Immunohistochemistry of the gill, carried out with a monoclonal antibody raised to SCPB, stained many varicose neuronal fibers. Most of these were associated with the gill musculature, but a sparse innervation of the filaments underlying the cilia was also observed. Some fluorescent nerve cell bodies were also seen in the gill tissue. Our results are consistent with the hypothesis that YFAFPRQamide modulates branchial activities--muscular as well as ciliary--that are associated with feeding.

Calcium and small organic molecules (e.g., tyrosine, MW 181 Da) introduced into the extrapallial fluid (EPF) of the quahog Mercenaria mercenaria exhibit rapid fluxes across the outer mantle epithelium and are distributed throughout the circulatory system within 3 h. Larger molecules (e.g., bovine serum albumin, MW 66,000 Da) are less readily exchanged between EPF and blood. The protein compositions of blood plasma and EPF are different, with at least seven protein bands expressed more prominently in the EPF. Equilibrium dialysis experiments reveal that Ca2+ constitutes only 2% of the total Ca in plasma; most of the Ca (85%) is bound to macromolecules, and the remaining 13% is present as dialyzable low molecular weight moieties. This distribution cannot be explained by speciation of inorganic Ca alone, since the MINTEQA2 equilibrium speciation model predicts that 79%-86% of the Ca should be present as Ca2+, with the remainder as CaSO4 (20%-13%). However, inclusion of a weakly Ca-binding organic molecule (log10 Ka approximately 2 M-1) into MINTEQA2 could fully reconcile modeling with experimental measurements. Results suggest that calcium transport in blood plasma and EPF is mediated by a suite of proteins and small organic ligands with a low affinity for Ca.

Previously we showed that when the gill muscles of the venerid clam Mercenaria mercenaria are stimulated to contract by 5-hydroxytryptamine (5HT), the contraction is about doubled when another identical dose of 5HT is applied after washout. Furthermore, this "endogenous potentiation" is mimicked by nitric oxide (NO), which is synthesized in the gill. We now report that the isolated gills also synthesize H2S; the basal rate of synthesis was 0.70 micromol.g(-1).h(-1) (se = 0.14; n = 24), but in the presence of 5HT (10(-2) M), the rate increased markedly to 35.82 micromol.g(-1).h(-1) (se = 4.93; n = 4). In addition, dithiothreitol (DTT; 2.2 mM) increased the rate of synthesis significantly to 4.9 micromol.g(-1).h(-1) (se = 0.8; n = 8). Stimulation of H2S synthesis by 5HT (5 x 10(-3) M) was seasonal; that is, the rates measured monthly from December through June are significantly lower than those measured from July through November. We also found that if isolated gills were pretreated with the H2S donor, sodium hydrosulfide (NaHS), their contractions in response to 5HT were potentiated. The threshold of the potentiation was 10(-8) M NaHS, and the largest effect was at 10(-6) M. During August, however, when endogenous and NO-induced potentiations are both absent, 10(-6) M NaHS was also ineffective. Like the effect of NO, that of NaHS (10(-6) M) was blocked by oxadiasoloquinoxalin (ODQ; 5 x 10(-5) M), an inhibitor of soluble guanylate cyclase (sGC). Moreover, Rp-8-CPT-cGMPS (10(-5) M), which inhibits protein kinase-G, also blocked the effect of NaHS (10(-6) M). When isolated gills were treated with 2.2 mM DTT, the endogenous potentiation of a second 5HT-induced contraction more than doubled in comparison to untreated controls. In conclusion, H2S is synthesized in the gill and, along with NO, is a seasonal, endogenous modulator of branchial muscle contraction; its action may be mediated through a sGC/cGMP signaling cascade.

In bivalves, water-pumping potential is determined both by ciliary activity and by the geometry of the system of passageways that acts as a conduit for water flow. Smooth muscles intrinsic to the gills of eulamellibranch bivalves possess the anatomical organization needed to regulate the dimensions of these water passageways. The tone of these muscles can be controlled experimentally using excitatory neurotransmitters to elicit muscle contraction and by removing Ca++ from the Ringer's solution to induce muscular relaxation. These experimental methods were used to investigate the effects of smooth muscle tone on the gill dimensions of two freshwater bivalves, Dreissena polymorpha and Corbicula fluminea, and one marine bivalve, Mercenaria mercenaria. In addition, endoscopic observations were made from the suprabranchial chamber of a freshwater unionid, Lampsilis anodontoides. Contraction of gill muscles led to a significant reduction in interfilament width, internal ostial area, and the cross-sectional area of the water tubes. Endoscopic observation from minimally disturbed L. anodontoides revealed rapid constriction of the water tubes upon contraction of the muscles of the gill and gill axis. Taken together, these data support the idea that alteration of smooth muscle tone in the gill provides a mechanism for controlling water-pumping activities.

The origin of hemocytes, the circulating “blood cells” of bivalve molluscs, including hard clams (Mercenaria mercenaria) has not been identified (1, 2). Proliferation of hemocytes, however, can be recognized through their increased numbers in diseased animals. Proliferating cell nuclear antigen (PCNA), or cyclin, is a protein produced during the late G1 and S phases of the cell cycle (3, 4). Using antibodies that recognize PCNA in mice, we attempted to identify the origin of hemocytes in the hard shell clams. Quahog parasite unknown (QPX) is a protist that causes severe inflammation and mortality in infected clams (5, 6). We attempted to induce hemocyte proliferation by exposing clams to QPX in a 10-l water column in which 12 ml of undiluted QPX culture (at a concentration of 7p10 cells/ml) were added every 10 days; by injecting QPX between the membranous mantles and the right valves, 3 cm ventral anterior to the siphon and into the pericardial cavities (0.25 ml of undiluted QPX culture); and by injecting an inert particle (India ink, 1:10 dilution in sterile seawater [7, 8]) into the pericardial cavities. The controls consisted of two groups of clams. One group was injected with sterile seawater in the pericardial cavities; the other was untreated. Groups were sampled at 24 h, and at 1, 4, and 8 weeks after the start of the experiment. At sampling, the animals were shucked, fixed in 10% neutral buffered formalin (NBF) for 24 h, and embedded in paraffin. Sections were cut (4–6 mm), mounted onto positively charged slides (Fisherbrand, Superfrost/Plus and ProbeOn Plus slides) and stained either with hematoxylin and eosin (H&E) (9) or with anti-PCNA with a hematoxylin counterstain (Zymed, PCNA Staining Kit). Clams injected with QPX in the pericardial cavities showed mild focal inflammation associated with viable and necrotic QPX organisms. At 2 months post-injection, viable QPX organisms were no longer identified. QPX organisms and associated inflammation were not observed in clams injected in the mantle cavity. After 2 months of water column exposure, only very rare infection by QPX organs with minimal inflammation was observed in mantle tissue. India ink injection caused a minimal inflammatory response. Pools of injected ink in the tissues and vascular spaces were either engulfed by individual hemocytes or surrounded and sequestered by hemocytes (encapsulation), forming thin-walled granulomas (6, 10). Numerous individual hemocytes containing India ink were eliminated from the clams by diapedesis over lumenal epithelial surfaces (Fig. 1A). Thick-walled granulomas (6, 10) were also identified in the gills, pericardial sacs, and other 1 Worcester Polytechnic Institute, Worcester, MA.

Examination of Mercenaria mercenaria hemocyte preparations on glass coverslips showed that adherent, macrophage-like cells tended to form multinucleated giant cells morphologically similar to classical Langhans cells and foreign body inflammatory giant cells, as seen in vertebrates. The frequency of giant cell formation varied from clam to clam, possibly reflecting each animal's physiological state, as influenced by pollutant exposure, other environmental stressors and disease. Treatment of the hemocytes with concanavalin A promoted hemocyte aggregation and increased the rate of giant cell formation. Attempts to increase polykaryon formation by in vivo or in vitro phagocytic stimulation of the hemocytes were generally unsuccessful.

The hemocytes of the hard clam M. mercenaria were of three types: an agranulocyte, a small, and a large granulocyte. The agranulocyte, with only a thin periphery of cytoplasm surrounding the nucleus, had no visible cytoplasmic granules in living preparations but did exhibit a few centers of nonspecific esterase activity. This cell type represented 2% of the hemocyte population. The small granulocyte possessed four distinct granule types and comprised 61% of the total cell population. Large granulocytes accounted fro 37% of all hemocytes. While they contained the same four granule types identified in the small granulocyte, only one-third the total number were present. The nucleus of all three hemocyte types appeared morphologically similar. The four types of granules observed were a blunt, dot-like, a refractile and a filamentous granule. Blunt granules were identified as mitochondria, based on their ability to reduce Janus Green B to diethyl safranin, the presence of NADH dehydrogenase activity and boundary staining with Sudan black B. Dot-like granules were identified as lysosomes on the basis of neutral red staining, localization of acid phosphatase and nonspecific esterase activity and staining with Sudan black B. Refractile granules were demonstrated to be membrane-bound, lipid-filled structures that reacted positively with Sudan black B and Oil red O, respectively; these granules act as lipid storage centers. Nuclear similarity of the three cell types suggest that these cells might represent different stages of maturity, rather than three distinct cell lines. This was also indicated by the similar yet graded cytochemical reactions and the varying degree of motility and phagocytic activity demonstrated by hemocyte types.

Thirteen monthly measurements of individually marked juvenile (16 mm long) and adult (60 mm long) specimens of Mercenaria mercenaria (L.) from field plots in North Carolina demonstrated similar seasonal patterns in size-adjusted monthly growth rates in shell volume: a large absolute maximum occurred in spring (April or May) with smaller relative maxima in mid summer and late autumn. The ratio of juvenile to adult size-adjusted growth rates in shell volume was nearly constant for ten months but then increased eight-fold in December and January. This growth anomaly between size classes could not be explained by examining dry weights of soma and gonads from additional marked juvenile and adult Mercenaria that were sacrificed monthly. Juveniles differed from adults by possessing negligible gonadal mass on all dates. However, knowledge of monthly changes in adult gonadal mass did not explain a significant amount of the residual variation in the regression of monthly juvenile volumetric growth on monthly adult volumetric growth. Seasonal changes in growth of adult gonadal mass and quarterly examinations of gonad histology both suggested a winter period of negligible gametogenesis followed by a spring burst of intense reproductive activity. The best explanations for the anomalously high volumetric growth of juveniles relative to adults in December and January are: (1) winter availability of a food source accessible only to juveniles or (2) biochemical storage of energy during winter by adults in preparation for the process of rapid gametogenesis in spring. If the latter explanation is correct, adult Mercenaria exhibit a large seasonal change in the allocation of resources between somatic growth and reproduction with maximal allocation to reproduction in winter months before gonad histology and growth of gonadal mass indicate reproductive effort.

The hematological parameters of Mercenaria mercenaria of similar size from two geographical areas as well as the morphology and behavior of their leucocytes were studied. On the basis of qualitative and quantitative characteristics, three types of leucocytes, designated as granulocytes, fibrocytes, and hyalinocytes, can be distinguished in both living and stained preparations.A correlation matrix computed between all parameters considered has revealed an insignificant correlation between the dimensions of whole animal and the differential count, packed cell volume, and total cell count as well as an insignificant correlation between the hematological parameters themselves. The only exception is that there is a positive correlation between the packed cell volume and the total cell count.

The extrapallial fluid (EPF), the fluid component of the shell-forming system of molluscs, has been examined for its effect on the rate of CaCO3 crystal formation in vitro. The medium was an artificial inorganic extrapallial fluid supersaturated with respect to CaCO3. EPF of the bivalves Crassostrea virginica and Mercenaria mercenaria strongly inhibited CaCO3 crystallization in high dilution. The inhibitory material had negatively charged groups as indicated by the removal of inhibition subsequent to passage through a DEAE-Sephadex column and the restoration of inhibition in material eluted from the column with 2.0 M NaCl. Polyaspartic and polyglutamic acids with COO- groups also inhibited crystal formation whereas polyamino acids lacking those groups and free amino acids were without effect. The inhibition by the acidic polyamino acids suggests that polypeptides known to be present in EPF are one possible cause of inhibition of crystal formation by EPF. Mg present in EPF was also strongly inhibitory. The...

TLC analysis of the neutral lipids in the hemolymph cells and serum of twelve specimens of Mercenaria mercenaria was performed. The serum fractions contained sterols (81%), free fatty acids (6%), sterol esters (11%), and a small amount of triglyceride (2%). Absolute amounts of chloesterol and triglyceride were 5.2-7.5 mg/100 ml and about 1 mg/100 ml, respectively. More total lipid and nearly all triglyceride was found to be in the cell fraction of the hemolymph. Cholesterol comprised 18% of the hemolymph cell sterols and 13% of the serum sterols.The five major fatty acids, in order of decreasing abundance, in the sera of specimens of Mercenaria mercenaria harvested in March and June from Buzzards Bay, Massachusetts, were C20:5, C16:0, C22:6, C18:1, and C20:1. A total of 22 fatty acids were resolved with tentative identification made for all of them. Standard deviations of the per cent composition of the major fatty acids were greater among specimens harvested in June than among those harvested in March.Th...

Laboratory studies were conducted to compare growth rates of the estuarine sand shrimp Crangon septemspinosa on various natural and artificial diets. Field studies examined the types of food consumed by the shrimp.In nature, 85% of the material in the stomach is organic debris; sand, crustacean parts, copepods, plant material and polychaetes make up the remainder. Some of the organic debris results from trituration of ingested tissues, but an undetermined portion is ingested as detritus.In the laboratory, shrimp grow rapidly on diets of Artemia salina, Mercenaria mercenaria and hard-boiled egg. Fish meal, copepods, beef liver, tropical fish food, agar enriched with glycogen, bacteria, and Spartina alterniflora detritus with associated microflora are also utilized, but growth is retarded.Crangon septemspinosa utilizes a variety of foods. The shrimp have a preference for animal tissues of marine origin and grow best on these foods, but they have the ability to utilize food of microbial and terrestrial origins.

1. The mean setting dates for larvae of Venus (Mercenaria) mercenaria grown at constant temperatures of 30.0°, 27.0°, 24.0°, 21.0° and 18.0° C. were found to lie on an almost perfectly straight line according to equation Y' = - 1.00 X + 37.91, where Y' is the predicted setting date and X is the temperature.2. There were no significant differences among the five temperature groups with respect to mean length of larvae at time of setting.3. There was no apparent relationship between maximum length of larvae at time of setting and temperature.4. The correlation between mean width and mean length of larvae at time of setting was very high (r = .95).5. No apparent relationship was found between shape of larvae (i.e., ratio of mean length to mean width) at time of setting and temperature.6. The average daily growth increment for all cultures at given temperatures under the conditions prevailing during the experiments was determined.7. The rate of growth of larvae of the southern clam, Venus (Mercenaria) campech...

In the summers of I909 and I9IO, while engaged in the study of the parasites of fishes at the laboratory of the Bureau of Fisheries, Woods Hole, Mass., I examined a number of invertebrates for larval stages of trematodes. The results of these examinations, although rather meagre with respect to the number of species found, are not without interest. The following species of mollusks were examined: Crepidula fornicata, C. plana, Ilyanassa obsoleta, Littorina littoria, L. rudis, Modiolus plicatulus, Mya arenaria, Mytilus edulis, Neverita duplicata, Pecten irradians, Purpura lapillus, Urosalpinx cineria, Venus mercenaria. Besides these several species of crustaceans were examined, also one annelid, Hydroides dianthus. Larval trematodes were found in only two species of mollusks, viz. Ilyanassa obsoleta and Pecten irradians. No trematode parasites were found in any of the crustaceans. A sporocyst found in the annelid, Hydroides dianthus, has already been reported.2

Rapid progress in studies of the morphology, physiology and ecological requirements of lamellibranch larvae, as well as other forms, can be achieved only if these organisms are easily available for study. This is seldom so in the case of lamellibranchs because the majority have a breeding period of comparatively brief duration lasting only a few months (Lebour, 1938; Thorson, 1946). During this period the larvae can be collected in plankton tows but, unfortunately, the early stages of many lamellibranchs are so alike in size and appearance that it is often impossible to distinguish with any degree of certainty the larvae of different species, or even genera. Thus, unless these larvae can be grown to metamorphosis their specific identity may remain in doubt. It is preferable, therefore, to work with larvae raised in the laboratory from fertilized eggs because in that case their identity cannot be questioned. In obtaining material for morphological and physiological studies of larvae the advantages of the direct method, i.e., the method of raising larvae in the laboratory, over the indirect method of collecting them in plankton and often only guessing their identity, are clear. However, because lamellibranch larvae are small, and because their free-swimming period is relatively long, they are difficult to culture. As a result, with the exception of a few successful attempts, mostly confined to commercially important species, such as oysters and mussels, few lamellibranchs have been grown in laboratories past the early veliger stage (Thorson, 1946). Therefore, the need for a simple but reliable method for culturing them is, of course, obvious. It is hoped that the simple but efficient method described in this article for conditioning the hard shell clam, V. mercenaria, to spawn out of season and for culturing its larvae will be applicable to many other species of lamellibranchs permitting their successful cultivation in the laboratory, where their morphological features and various aspects of behavior can be studied under controlled conditions. Realizing the importance of having a good supply of larvae for studies of the life history of Venus mercenaria, Belding (1912) tried to raise them in the laboratory. He was not successful because most of the larvae in his cultures died either before they reached the straight hinge veliger stage or soon afterwards. Belding concluded that there was no practical method for raising hard shell clams to the setting stage because of the small size and delicate nature of the eggs. Several years later, nevertheless, Wells (1927) succeeded in growing clam larvae to the setting stage. Wells, however, was mostly interested in oysters and did not continue the clam work.

1. The pH range for normal embryonic development of oysters was 6.75 to 8.75, and for clams, 7.00 to 8.75.2. More than 68% of the larvae of both clams and oysters survived at pH 6.25 to 8.75. The lower pH limit for survival of oyster larvae was 6.00 and for clam larvae, 6.25.3. The pH range for normal growth was 6.75 to 8.50 for clam larvae and 6.75 to 8.75 for oyster larvae. The rate of growth of both species dropped rapidly at pH levels below 6.75.4. The optimum pH for growth was 7.50 to 8.00 for clam larvae and 8.25 to 8.50 for oyster larvae.5. At pH 9.00 to 9.50 the percentage of eggs that developed normally, the percentage of larvae that survived, and the percentage increase in mean length of both species decreased rapidly.

1. Continuous kymograph recordings of the opening and closing of the valves of common quahogs, Venus mercenaria, secured from the Virginia coast and from Woods Hole, Massachusetts were made during February, March, April, June, and December, 1953.2. The activity records of these clams maintained under constant laboratory conditions show a persisting diurnal rhythm with maxima and minima occurring in the afternoon and early morning, respectively. There is some evidence that the pattern of the diurnal cycle varies with the time of year.3. A persisting tidal rhythm is also evident. In the group of animals for which correlations with actual tidal phases could be made, it was seen that the times of minimal activity in this cycle correspond closely with the times of low tide in the area from which the animals were collected.4. The summation of these two cycles, diurnal and tidal, is responsible for an observed lunar cycle. The phases of the lunar cycle are probably set directly by the tides to which the animals ...

The inorganic composition of the extrapallial fluids of Mercenaria mercenaria, Mytilus edulis and Crassostrea virginica was significantly different from sea water. Calcium was the principal ion bound in the extrapallial fluids. This binding was accomplished by a non-dialyzable component that appeared to be a glycoprotein.

The literature on Pelecypoda mollusks contains but few accounts of systematic studies of the changes occurring in the gonads of an animal throughout the entire year. This phase of investigation of sexual phenomena in mollusks, though neglected so far, is, however, of considerable importance and interest because, if properly carried out, it could assist greatly in the understanding of the phenomena of change of sex in some forms or add to the knowledge of the alternation of sexual phases in others. Amemiya (1929) reported a very interesting case of sex change in the Japanese oyster, 0. gigas, which was previously considered dioecious. His method consisted of making a hole in the shell of an oyster and removing through it a small piece of gonad for determination of sex. Following this the oysters were placed in cages and returned to the water for a period of one year, at the end of which they were re-examined. Meanwhile, no samples were taken for histological studies to determine what cellular changes precede the reversal of sex. Nelson (1928) gave a more or less detailed description of changes occurring in the gonads of the adult 0. virginica from April until June, but did not continue his studies through the post-spawning time and season of hibernation. In other studies dealing with sexual phenomena of the genus Ostrea, a systematic description of changes occurring in the gonads of adult animals throughout the year is also lacking. This paper deals with the changes occurring in the gonads of adult clams (Venus mercenaria) at different seasons of the annual cycle. The animals were studied for three successive years. Samples for this study were collected at bi-weekly intervals from the clam beds located near Charles Island, Long Island Sound. The depth of the water over the beds was 8-12 feet at mean low water. Since the calendar's new year, occurring in the middle of winter, coincides with the prolonged period of the animal's inactivity, it is thought convenient in this study to consider the beginning of a new year from the ecological point of view, i.e., after the completion of

1. A new apparatus, by means of which the shell activities of many bivalve mollusks can be measured and recorded, is described.2. The analysis of 399 daily records of shell activities of 47 clams, subjected to temperatures ranging from — 1.0° to 28.0° C., showed that the length of time which the animals remain open partly depends upon the temperature of the surrounding water.3. For the majority of clams hibernation begins soon after the water temperature decreases to 5.0° and 6.0° C. At lower temperatures the clams may remain completely closed for very long periods. No shell movements were exhibited, and no disposal of ejecta occurred.4. Within the temperature range of 3.9° to 10.9° C., the average period of openness increased from 4 to 88 per cent of the total time, showing a correlation with the rise of temperature.5. There was no correlation between the duration of openness of the clam shells and the temperature increase from 11.0° to 27.9° C. Within this temperature range the shells were open from 69 ...

1. A method of preparing and employing the isolated heart of the quahog, Venus mercenaria, for the bio-assay of acetylcholine (Ach) is described.2. The activities of choline and certain choline esters; of betaine and its ethyl ester; and of triethylcholine and triethyl-acetylcholine on the isolated Venus heart are compared. In further understanding the fundamental mode of action of Ach, the most significant observation was that the substitution of ethyl groups for methyl on the nitrogen of choline and Ach resulted in a complete loss of activity determined by observation on the amplitude of heart beat.

The growth and reproductive biology of the adult female stone crab, Menippe mercenaria, an economically important species in Florida, have never been investigated in the laboratory. These morphogenetic processes are influenced both by hormones and by seasonal changes in the environment. This study seeks to survey the relation between these processes, their endocrine control, and the changing environmental conditions provided in nature. Special attention has been paid to the interrelationship between growth and reproduction. This aspect has been studied by Bauchau (1961), Cheung (1966) and DA©meusy(1964, 1965a, 1965b and 1965c) in Carcinus mcienas, a boreal species. The present study employs adult female specimens of M. mercenaria since they continue to grow after reach ing sexual maturity, unlike Callinectes in which growth ceases at maturity (Truitt, 1939). The relationship between growth and reproduction was defined by (1) observ ing the occurrence of molting and spawning in a sample of the wild population; and (2) removing the eyestalks at different stages of the molting cycles and during intermolt periods occurring at different times of the year, because the eyestalks contained endocrine factors regulating these processes. Results of this study also yielded information on the life history of this species. MATERIALS AND METHODS

The heart of Venus is sensitive to acetylcholine in dilutions of 10–12 during the spring and 10–9 during the fall. It is useful as a test material for acetylcholine assay.Acetylcholine appears to leave the contracting mechanism intact and to act on the pacemaker and conducting mechanisms of this myogenic heart.Stimulation of the visceral ganglion causes inhibition in diastole resembling the effect of acetylcholine.Atropine is very toxic to the heart. In non-toxic concentrations it antagonizes neither the effect of acetylcholine nor of nerve inhibition.Fluid from a heart inhibited by visceral ganglion stimulation often depresses the beat of an eserinized test heart.Eserine prolongs the inhibition due to acetylcholine and that due to nerve stimulation. It appears likely that acetylcholine is liberated as the normal cardiac inhibitory agent in Venus.

1. Examination of the developing gonads of young clams from Long Island Sound, at two-week intervals during the first year of life, shows that a primary bisexual gonad is formed in each individual when the animal reaches the size of 4-6 mm.2. The primary gonads form from associations of germinal cells in the connective tissue, not immediately beneath the body walls but at some distance from them, at the level of the heart or slightly below it. A lumen soon appears in each gonad follicle and the germinal cells begin to proliferate rapidly.3. The primary gonad contains the antecedent cells of both sexes but the protandric nature of the primary gonad becomes manifested by the rapid proliferation of the spermatogenic cells.4. This species is not strictly protandric because few individuals develop into females without passing through a functional male phase.5. Functional sperniatozoa have been observed in the gonads of young clams, collected in October and November when only 5-7 mm. long. In some cases dischar...

1. Twelve strains of bacteria were isolated from a moribund Venus mercenaria larva in a laboratory culture. These, ten other clones, and mixed bacteria from sea water were assayed by adding broth culture yielding 106-107 cells/ml. to beaker cultures of healthy clam larvae. Only the mixed bacterial culture from the moribund larva and two of the 12 strains isolated from it caused extensive mortality. One of the virulent clones (6-) is a species of Vibrio,the other (13-1) is a Pseudomonas species.2. Larvae exposed to virulent bacteria and simultaneously treated with antibiotics were as healthy as controls, showing that active bacteria were necessary to destroy larvae and that metabolites in the bacterial inoculum were not harmful to larvae.3. Larvae were grown free of contaminating micro-organisms by allowing washed eggs to develop in antibiotic solutions and then isolating straight-hinge larvae by pipette. Either virulent clone (106-107/ml.) destroyed 10-100% of such larvae. However, exposing the animals to...

Chromosome numbers of n = 19, 2n = 38 are reported for Mercenaria mercenaria, M. campechiensis and their F1 hybrids. Meiosis is normal in the hybrids and yields no evidence of chromosome nonhomology or structural rearrangements between the two parents. The hybrids produce functional eggs and sperm which result in normal fertilization and early embryonic divisions in reciprocal backcrosses and at least some F2' s. No gross chromosomal barrier to gene exchange appears to exist between the two species.

1. Some clam eggs developed normally in concentrations of 4.0 g./l. of clay, precipitated chalk or finely ground Fuller's earth, although the percentage developing normally decreased as the concentration of these suspended materials increased.2. In silt concentrations below 0.75 g./l. the percentage of clam eggs developing normally was not significantly different from that in control cultures but decreased progressively in successively higher concentrations.3. None of the clam eggs developed normally in silt concentrations of 3.0 or 4.0 g./l.4. Larvae resulting from clam eggs developing in high concentrations of each of the suspended materials were reared to metamorphosis after being returned to normal sea water at 48 hours.5. Clam larvae were unable to grow in concentrations of clay, chalk or Fuller's earth as high as those at which some eggs developed.6. The highest concentration of chalk was 0.25 g./l. and 0.5 g./l. was the highest concentration of clay and Fuller's earth at which clam larvae showed an...

1. Whole Venus heart homogenates hydrolyze acetylcholine and benzoylcholine at a low rate.2. The enzymatic activity of such homogenates is the sum of that contributed by amoebocytes, intestinal epithelium and heart muscle.3. Isolated amoebocytes hydrolyze acetylcholine and the enzyme responsible is inhibited by 10-4 M physostigmine, thus indicating the presence of cholinesterase.4. Recently developed histochemical procedures demonstrate serum cholinesterase and lipase in amoebocytes, intestinal epithelium and heart muscle.5. Greatest lipase activity is present in the amoebocytes and intestinal epithelium, while serum cholinesterase activity is greatest in the ventricular muscle.6. The potentiation of the action of acetylcholine on the isolated Venus heart by physostigmine is due to inhibition of serum cholinesterase in the amoebocytes and especially in the heart muscle.7. The presence of varying levels of lipase in the intestinal epithelium of Venus lends support to the suggested presence of extracellular...

1. An energy budget was constructed for Menippe mercenaria from hatching to first juvenile crab when fed newly-hatched Artemia nauplii.2. Larvae of M. mercenaria grow exponentially through the zoeal stages. Growth rate decreases during megalopa.3. Consumption reaches its peak during megalopa where one individual may consume 91 Artemia nauplii/day. Feeding rate decreases prior to molting to first crab stage.4. The caloric content per unit dry weight increases from 2.503 cal/mg at hatching to 3.746 cal/mg at first juvenile crab. Molts contain 1.296 cal/mg.5. Respiration by zoea is proportional to the two thirds power of body weight and is described by the function R = 0.0017W0.67.6. Over the period of larval development, 7.329 calories are consumed, 2.207 calories are used for growth, 0.502 calories are lost to production of exuvia, 1.933 calories are expended for maintenance, and 2.687 calories are lost as rejecta.

Four gene loci were characterized by polyacrylamide disc-gel electrophoresis of four enzymes in the hard clams, M. mercenaria and M. campechiensis. Four populations of M. mercenaria and two populations of M. campechiensis were selected from a transect between Prince Edward Island, Canada and Tampa Bay, Florida. Three of the four enzymes were polymorphic in all six populations. M. mercenaria averaged 2.6 alleles per locus for four populations while M. campechiensis averaged 3.5 alleles per locus for two populations. Malate dehydrogenase—NAD form (MDH) had a single allele predominating in all populations. A second rare allele appeared in the Maine and South Carolina populations of M. mercenaria. Polymorphism was pronounced at an esterase (EST) locus but the data showed no patterned shift along the transect. North to south clines were observed at both the tetrazolium oxidase (TO) and the lactate dehydrogenase (LDH) loci. The different patterns observed for each locus suggest a complex of selective processes ...