A comparative study of the basic nuclear proteins from sperm of bivalve molluscs

• 1.1. Basic nuclear proteins from spermatozoa of the three mollusc species belonging to the class Bivalvia have been analyzed using one- and two-dimensional electrophoresis.
• 2.2. Four nuclear basic proteins have been purified and their amino acid compositions determined.
• 3.3. In these spermatozoa histone-type proteins coexist with protamine-like proteins.
• 4.4. The protamine-like proteins that have been studied show different electrophoretic behavior but in general are similar, with a high content of lysine, arginine, alanine and serine.
• 5.5. Interspecific variability has been found for the H1-like histone.

     

The effects of sodium chloride concentration on electrophoretic patterns of adductor muscle proteins from bivalve molluscs

• 1.1. An extraction medium was sought that would produce the highest quality electrophoretic patterns of proteins from bivalve molluscan adductor muscles.
• 2.2. Cellulose acetate electrophoresis was conducted on adductor muscle proteins extracted with distilled water or various concentrations of saline, with and without dialysis, from three bivalve molluscan species: Pacific oysters, Crassostrea gigas; Japanese littleneck clams, Prototheca semidecussata; and Hawaiian mussels (Mahawele), Isognomon constellateum.
• 3.3. The protein patterns obtained by electrophoresis revealed that muscle extracted and dialyzed in 0.030 g% NaCl solution produced superior patterns.

     

Biotransformations of paralytic shellfish toxins by bacteria isolated from bivalve molluscs

Due to the possibility that bacteria could be involved in the clearance of paralytic shellfish toxins (PST) from bivalve molluscs, investigations into which, if any, bacteria were able to grow at the expense of PST focused on several common shellfish species. These species were blue mussels, oysters, razor fish, cockles, and queen and king scallops. Bacteria associated with these shellfish were isolated on marine agar 2216 and characterized by their carbon utilization profiles (BIOLOG). Selected isolates from groups demonstrating 90% similarity were screened for their ability to metabolize a range of PST (gonyautoxins 1 and 4 [GTX 1/4], GTX 2/3, GTX 5, saxitoxin, and neosaxitoxin) using a novel screening method and confirming its results by high-performance liquid chromatography. Results suggest that molluscan bacteria have different capacities to utilize and transform PST analogues. For example, isolates M12 and R65 were able to reductively transform GTX 1/4 with concomitant production of GTX 2/3, while isolate Q5 apparently degraded GTX 1/4 without the appearance of other GTXs. Other observed possible mechanisms of PST transformations include decarbamoylation by isolate M12 and sulfation of GTXs by isolates Q5, R65, M12, and C3. These findings raise questions as to the possible role of bacteria resident in the shellfish food transport system. Some researchers have suggested that the microflora play a role in supplying nutritional requirements of the host. This study demonstrates that bacteria may also be involved in PST transformation and elimination in molluscan species.     

Flow cytometric comparison of haemocytes from three species of bivalve molluscs

Haemocyte subpopulations from three bivalve species (the clams Ruditapes philippinarum and Mercenaria mercenaria and the oyster, Crassostrea virginica) were characterised using light-scatter flow cytometry and a standard set of methods. Two parameter (forward and side scatter) plots for the three species were very similar and resembled plots for mammalian white blood cells. Two haemocyte groups (granulocytes and agranulocytes) were found in both the haemolymph and the extrapallial fluid of the clams while those two groups and an additional third group were found in the haemolymph of the oyster. All subpopulations were sorted on to glass slides, identified, photographed, and measured microscopically. Sorting of the bivalve granulocyte and agranulocyte groups indicated varying degrees of heterogeneity within each population in terms of either size or granularity, or both. However, subsorting of selected regions within the major groupings produced highly pure haemocyte populations. The comparison showed both similarities and differences among species. For instance, a distinct subpopulation of small granulocytes was present only in oysters and a subpopulation of spindle-shaped haemocytes, only in M. mercenaria. The haemocyte subpopulations delineated by light-scatter flow cytometry underscore questions about cell lineages, but the instrument also offers a powerful technique for answering them.     

Hox and paraHox genes in bivalve molluscs

In this study, we sought the presence and analysed the sequences of the Hox and ParaHox genes in bivalve molluscs. The clustered Hox genes play a central role in anterior-posterior axial patterning in bilaterian metazoa, whereas the ParaHox gene cluster is a paralogue (evolutionary sister) of the Hox cluster.Using polymerase chain reaction (PCR)-based approaches, we isolated nine different sequences in five species belonging to three of the main bivalve subclasses: Ensis ensis and Tapes philippinarum (Heterodonta), Pecten maximus and Mytilus galloprovincialis (Pteriomorphia), and Yoldia eightsi (Protobranchia). Comparison with the Hox and ParaHox genes of other bilaterians, particularly lophotrochozoans, allowed us to attribute six of these sequences to the Hox gene cluster (one to paralog group [PG] 3 class, and five to the central class), two to the ParaHox cluster and one to the Gbx gene family.The results of our investigation seem to indicate that homeotic Hox and ParaHox gene clusters are homogeneous for both presence and characteristics in molluscs.     

Environmental contaminants influencing immunefunction in marine bivalve molluscs

The increased observation of pollution induced disease conditions in marine organisms has led to a growing interest on the effects of environmental contaminants on the immune system. Most studies on modulation of the immune system in bivalves by pollutants have concentrated on the effects of heavy metals and polycyclic aromatic hydrocarbons (PAHs). The current literature on contaminant effects on specific components of the bivalve immune system is reviewed together with the effects on susceptibility to infection. Data are presented showing the effects on immune parameters of exposure to Vibrio tubiashi following pre-exposure to copper or cadmium. Mussels exposed to cadmium for 7 days followed by 7 days exposure to V. tubiashi demonstrated significantly higher numbers of circulating haemocytes compared with non-Vibrio-exposed groups. Similar experiments conducted with copper exposure for both 7 days and 7 weeks followed by V. tubiashi for 7 days demonstrated a significant decrease in the percentage of circulating eosinophils compared with basophilic cells for both short and long term exposures. The intracellular release of superoxide (NBT reduction) by haemocytes was stimulated in Vibrio-challenged mussels with no copper pre-exposure but was significantly reduced in mussels pre-exposed to 0·2 ppm of copper for 7 weeks. The mortalities for the copper experiments showed increased levels with increasing copper concentration and were consistently higher in the V. tubiashi challenged mussels which had also been exposed to copper.     

Ciliary activity on the ctenidium of bivalve molluscs

1. Lateral ciliary activity was studied on the ctenidial preparations of several bivalves.2. The cerebral and visceral ganglia exhibit a coordinated role in the control of ciliary beating.3. Exposure of the ctenidia to changes in potassium and magnesium ion concentrations at acclimation salinities were salinity-dependent and probably reflect an effect on the ciliated epithelium.4. The magnitude of cilio-inhibition is directly related to the percentage of lamellar conversions in cytosomes.5. Since sequestered calcium has been shown to be released from lamellar-type cytosomes, it is postulated that lateral cilio-inhibition is due to an increase in the neuronal intracellular calcium concentration with subsequent release of the cilio-inhibitory neurotransmitter, dopamine.6. There is a seasonal effect on cytosomal transformations and decarboxylase activity in neuronal tissues both in the central and peripheral elements and lateral ciliary activity.7. The experimental design and procedures used in our studies will have broad applications for quantitatively assessing the effects of environmental factors on ciliary activity of marine and estuarine organisms.     

Anaerobic metabolism of bivalve molluscs during exposure to air

The anaerobic metabolism of Mytilus edulis, Cardium edule, Scrobicularia plana and Macoma balthica was investigated. On exposure to the atmosphere, all of these species were found to be able to utilise ¹⁴CO₂. This suggests that these species gape during exposure to the atmosphere. A comparative study on the pattern of ¹⁴C incorporation suggests that there is a similarity between M. edulis and S. plana in the extent of the utilization of the succinate pathway during exposure to air. However C. edule and M. balthica were also similar in the extent of utilizing the succinate pathway, even though there were significant species differences between the similar animals. It is suggested that exposure in S. plana represents a stressful situation and that this species might react to this stress by utilizing the succinate pathway. A higher incorporation of radiocarbon into alanine by M. balthica could be due to high activity of the enzymes that control the reactions leading to production of radioactive pyruvate.     

The adenosine triphosphate content of some marine bivalve molluscs

The total ATP-content of representatives of 23 bivalve families was found to vary from 1.26–0.26 % of the dry tissue weight, with the dry tissue containing about 40 % carbon. Representatives from certain families consistently had more than average and others less than average ATP values, and there were greater differences between species from different families than between closely related species. Variations in the oxygen uptake of whole animals accounted for little of the observed variation in ATP-content. The ATP-content of individual tissues from the same individual showed wide variation, as did that of the same tissue from different species. The highest values of ATP were found in muscle tissues. High maintained levels of ATP were associated with the ability of the species to use energy rapidly for short periods, for example, in escape responses.     

Biotic interactions revealed by macroborings in arctic bivalve molluscs

The presence of organisms whose bodies have low preservation potential may be deduced by searching for the traces produced by them. The addition of predatory gastropods and soft-bodied epizoans to Quaternary marine faunas dominated by bivalves was facilitated by an examination of borings in bivalve shells. Borings attributed to predatory gastropods (ichnogenus Oichnus ) were observed in shells of Astarte spp., Hiatella arctica and Macoma calcarea. Astarte, Hiatella and Macoma were preyed upon in preference to other members of a diverse suspension-feeding bivalve community. Borings attributed to epizoans (ichnogenus Cautostrepsis ) were observed in bivalve shells (Astarte spp. Hiatella arctica ), calcareous algae and limestone clasts. Biotic interactions revealed by trace fossils are employed, for the first time, to reconstruct the trophic structure of arctic Quaternary marine benthic faunas. ▭ Arctic molluscs, palaeoecology, Oichnus, Caulostrepsis.     

Transfection of spermatozoa in bivalve molluscs using naked DNA

Spermatozoa from the bivalve molluscs Mytilus galloprovincialis, Mytilus chilensis and Chamelea gallina were transfected in vitro using the p-GeneGrip construct, which encodes green fluorescent protein. The efficiency of transfection after brief incubation was assessed by fluorescence and confocal laser microscopy, and was about 58.5-70.01% in the species used. The foreign gene was principally located in the sperm nuclei, as demonstrated by laser confocal serial sections. In some spermatozoa, mitochondria, which are grouped in the base of the nucleus, also appeared to be transfected. Polymerase chain reaction and Southern blot analyses suggested that the foreign DNA had been integrated into the nuclear genome in Mytilus galloprovincialis spermatozoa. This simple method for spermatozoon transfection in molluscs of commercial interest could have biotechnological applications.     

Podocytes in bivalve molluscs: Morphological evidence for ultrafiltration

Summary In representatives from a survey of three major taxa of bivalves the pericardial glands were found in two distinct positions. In protobranches (Acila castrensis) and filibranch bivalves (Glycymeris subobsoleta, Chlamys hastata, Pecten caurinus, Placopecten magellanicus, Mytilus edulis andMytilus californianus) the pericardial glands are located on the auricular surface. In heterodonts (Mercenaria mercenaria, Clinocardium nuttallii andMya arenaria) the pericardial glands are found in an anterodorsal position to the pericardial cavity.The sites of ultrafiltration are described. They consist of podocytes with basally extending pedicels forming an interdigitating network apposed to a basal lamina. Other characteristics of this ultrafiltration barrier described are anionic sites on the basal lamina and presence of substructural components within the ultrafiltration slits between pedicels.The pathway for the ultrafiltrate in protobranchs and filibranchs is from the hemocoel through the basal lamina, through the ultrafiltration slits of the pedicel network, into the urinary spaces between the podocyte cell bodies and into the pericardial cavity. The pathway for the ultrafiltrate in heterodonts is from the hemocoel through the basal lamina, through the ultrafiltration slits of the pedicel network, into urinary spaces between the podocyte cell bodies, into the lumen of the pericardial gland tubules and into the pericardial cavity.All podocyte cells have electron dense granules, Golgi apparatus and vacuoles associated with their cytoplasm. Heterodont species have microvilli on the cell surfaces of the podocytes apposed to urinary spaces.In all cases the morphological sites of ultrafiltration were associated with the pericardial glands of the heart-pericardial complex.     

The ecology of Bonamia and decline of bivalve molluscs

Bonamia is a protozoan parasite of the haemocytes of oysters (Tiostrea chilensis), in which it has an annual developmental cycle between November and August each year. The parasite transmits directly, oyster to oyster, and therefore disease spread is related to host stock density. The Foveaux Strait oyster population experiences large mortalities every 20-30 years, and these may be attributable to Bonamia. The parasite appears to become less pathogenic at the end of, and probably between, mass mortalities, and some oysters appear more tolerant of infection than others. On the basis of these observations, and considering other protist pathogen:oyster models, the apparently reduced pathogenicity of Bonamia is discussed in terms of parasite kinetics. The population dynamics and selection of parasite tolerant host stocks, and kinetics of parasite transmission, may explain the cyclic nature of large-scale mortalities in Foveaux Strait, without change in parasite pathogenicity.     

Multilocus genetic analysis of Cryptosporidium in naturally contaminated bivalve molluscs

AIMS: To evaluate the application of discriminatory multilocus PCR procedures for the characterization of Cryptosporidium in samples of naturally contaminated bivalve molluscan shellfish. METHODS AND RESULTS: Nucleic acid was extracted from 22 shellfish previously identified as contaminated with Cryptosporidium spp. and subjected to PCR-based analysis for two independent fragments of the Cryptosporidium oocyst wall protein (COWP) gene, three microsatellite markers (ML 1, GP 15 and MS 5) and an extra-chromosomal small double-stranded RNA (dsRNA). Overall, at least one COWP gene fragment was amplified from all 22 samples, 21 amplified the dsRNA and 14 at least one of the three microsatellite loci. More than one dsRNA or microsatellite allele was detected in 50% of samples. The majority of samples were contaminated with Cryptosporidium parvum types circulating in both humans and livestock. A novel dsRNA element was identified in one sample, which did not amplify any of the three microsatellite loci investigated. CONCLUSIONS: Multilocus analysis of Cryptosporidium can be applied to DNA extracted from naturally contaminated shellfish. SIGNIFICANCE AND IMPACT OF STUDY: This multilocus genetic analysis highlights that filter feeder molluscs are a potential source of cryptosporidial oocysts, which may be infectious to humans.