Effect of temperature on defense parameters in manila clam Ruditapes philippinarum challenged with Vibrio tapetis

Brown Ring Disease (BRD), a vibriosis affecting the clam Ruditapes philippinarum, is present on the Atlantic coasts of Western Europe and is considered to be a cold water disease. The present work investigated the effect of temperature on immune response and its relationships with BRD development. Clams maintained at different temperatures (8, 14 and 21 degrees C) were experimentally challenged with the pathogen Vibrio tapetis, the etiologic agent of BRD. Results demonstrated significant effects of temperature on disease development and on hemolymph immune parameters including total and viable hemocyte counts, lysozyme and leucine aminopeptidase activities. Thirty days after challenge, clams maintained at 21 degrees C displayed significantly higher values for all the measured immune parameters in comparison to specimens incubated at 14 degrees C. Improved performance of the immune system was associated with a low BRD prevalence. The recovery process, which occured mainly at 21 degrees C, was associated with high percentages of viable hemocytes and high activities of leucine amino-peptidase and lysozyme. This laboratory study clearly demonstrates that temperature strongly affects BRD development and clam immune response during infection. Favourable immune status at higher temperature may confer upon the clam a better capacity to fight the disease agent, and therefore to recover more easily.     

Involvement of nitric oxide in the in vitro interaction between Manila clam, Ruditapes philippinarum, hemocytes and the bacterium Vibrio tapetis

The Manila clam, Ruditapes philippinarum can become infected by the bacterium Vibrio tapetis which causing the Brown Ring Disease along North European Atlantic coasts. Variations in clam immune parameters have been reported in clam challenged with V. tapetis but no studies have been done on Nitric Oxide (NO) production. NO is a toxic agent to pathogens produced mostly by immune cells such as hemocytes in invertebrates. In this study, we demonstrated that NO production in hemolymph and extrapallial fluid of clams is dose dependent and increases with incubation time with V. tapetis. Moreover, the augmentation of NO production seems to be directly correlated to cell rounding and to the loss of pseudopods-forming capacity of hemocytes during the infection process.     

First evidence of immunomodulation in bivalves under seawater acidification and increased temperature

Water acidification, temperature increases and changes in seawater salinity are predicted to occur in the near future. In such a global climate change (GCC) scenario, there is growing concern for the health status of both wild and farmed organisms. Bivalve molluscs, an important component of coastal marine ecosystems, are at risk. At the immunological level, the ability of an organism to maintain its immunosurveillance unaltered under adverse environmental conditions may enhance its survival capability. To our knowledge, only a few studies have investigated the effects of changing environmental parameters (as predicted in a GCC scenario) on the immune responses of bivalves. In the present study, the effects of both decreased pH values and increased temperature on the important immune parameters of two bivalve species were evaluated for the first time. The clam Chamelea gallina and the mussel Mytilus galloprovincialis, widespread along the coast of the Northwestern Adriatic Sea, were chosen as model organisms. Bivalves were exposed for 7 days to three pH values (8.1, 7.7 and 7.4) at two temperatures (22 and 28°C). Three independent experiments were carried out at salinities of 28, 34 and 40 PSU. The total haemocyte count, Neutral Red uptake, haemolymph lysozyme activity and total protein levels were measured. The results obtained demonstrated that tested experimental conditions affected significantly most of the immune parameters measured in bivalves, even if the variation pattern of haemocyte responses was not always linear. Between the two species, C. gallina appeared more vulnerable to changing pH and temperature than M. galloprovincialis. Overall, this study demonstrated that climate changes can strongly affect haemocyte functionality in bivalves. However, further studies are needed to clarify better the mechanisms of action of changing environmental parameters, both individually and in combination, on bivalve haemocytes.     

Insights into the Establishment of the Manila Clam on a Tidal Flat at the Southern End of an Introduced Range in Southern California,USA

Coastal ecosystem modifications have contributed to the spread of introduced species through alterations of historic disturbance regimes and resource availability, and increased propagule pressure. Frequency of occurrence of the Manila clam (Venerupis phillipinarum, Veneridae) in Southern California estuaries has increased from absent or sparse to common since the mid-1990s. Potential invasion vectors include seafood sales and aquaculture, and spread from established northern populations over decades. The clam’s post-settlement habitat preferences are, however, uncertain in this region. Our project aimed to identify factors associated with established patches of the clam within a bay toward the southern end of this introduced range. During summer 2013, we sampled 10 tidal flat sites in Mission Bay, San Diego; each containing an area with and without hard structure (e.g., riprap, boulders). We measured likely environmental influences (e.g., sediment variables, distance to ocean). Manila clam densities across the bay were most strongly associated with site, where highest densities were located in the northern and/or back halves of the bay; and weakly correlated with lower porewater salinities. Within sites, Manila clam density was enhanced in the presence of hard structure in most sites. Prevailing currents and salinity regimes likely contribute to bay wide distributions, while hard structures may provide suitable microhabitats (refuge from predators and physical stress) and larval entrapment within sites. Results provide insights into decisions about future shoreline management efforts. Finally, we identify directions for future study to better understand and therefore predict patterns of establishment of the Manila clam in the southern portion of its introduced range.     

Mislabeled: eco-labeling an invasive alien shellfish fishery

The invasive alien Manila clam fishery from Ria Arousa, Spain, was recommended for a Marine Stewardship Council certificate which implies the fishery maintains the integrity of the ecosystem. By certifying an environmentally harmful invasive alien clam fishery, the MSC harms the environment and risks its credibility, at the time when environmental responsibility and social responsibility are increasingly important to policymakers, industry, and consumers, particularly in Europe. We call on the MSC to reassess their evaluation of the Ruditapes philippinarum fishery in Ria Arousa, Spain.     

Alteration of sensory abilities regulates the spatial scale of nonlethal predator effects

Many studies have shown that nonlethal predator effects such as trait-mediated interactions (TMIs) can have significant impacts on the structure and function of communities, but the role that environmental conditions play in modulating the scale and magnitude of these effects has not been carefully investigated. TMIs occur when prey exhibit behavioral or physiological responses to predators and may be more prevalent when abiotic conditions increase prey reactions to consumers. The purpose of this study was to determine if turbulence would alter the distance over which prey in aquatic systems respond to chemical cues emitted by predators in nature, thus changing the scales over which nonlethal predator effects occur. Using hard clams and blue crabs as a model predator–prey system, we investigated the effects of turbulence on clam reactive distance to predatory blue crabs in the field. Results suggest that turbulence diminishes clam reactions to predators and that the environmental context must be considered when predicting the extent of indirect predator effects in natural systems.     

Megapitaria squalida (Bivalvia: Veneridae) fishery landings and temperature relationship in Bahía de la Paz, México

The clam Megapitaria squalida is a fishing resource with increasing importance in Northwestern Mexico. Nevertheless, this fishery has shown important variations that could be related to environmental factors. To assess this, monthly landings of M. squalida were analyzed during 2002-2005 for Bahía de La Paz, B.C.S., and were related with monthly time series of sea surface temperature, derived from MODIS-Aqua Sensor. The results showed a positive and significant relationship between sea surface temperature and clam landings. The likely impact of anomalous conditions of sea temperature on this resource is discussed.     

An aryl hydrocarbon receptor (AHR) homologue from the soft-shell clam, Mya arenaria: evidence that invertebrate AHR homologues lack 2,3,7,8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone binding.

The aryl hydrocarbon receptor (AHR) mediates numerous toxic effects following exposure of vertebrate animals to certain aromatic environmental contaminants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To investigate possible effects of TCDD on invertebrates, a cDNA encoding an AHR homologue was cloned from the soft-shell clam, Mya arenaria. The predicted amino acid sequence contains regions characteristic of vertebrate AHRs: basic helix-loop-helix (bHLH) and PER-ARNT-SIM (PAS) domains and a glutamine-rich region. Phylogenetic analysis shows that the clam AHR sequence groups within the AHR subfamily of the bHLH-PAS family, in a clade containing AHR homologues from Drosophila melanogaster and Caenorhabditis elegans. AHR mRNA expression was detected in all tissue types tested: adductor muscle, digestive gland, foot, gill, gonad, mantle, and siphon. The in vitro-expressed clam AHR exhibited sequence-specific interactions with a mammalian xenobiotic response element (XRE). Velocity sedimentation analysis using either in vitro-expressed clam AHR or clam cytosolic proteins showed that this AHR homologue binds neither [(3)H]TCDD nor [(3)H]beta-naphthoflavone (BNF). Similarly, in vitro-expressed D. melanogaster and C. elegans AHR homologues lacked specific binding of these compounds. Thus, the absence of specific, high-affinity binding of the prototypical AHR ligands TCDD and BNF, is a property shared by known invertebrate AHR homologues, distinguishing them from vertebrate AHRs. Comparative studies of phylogenetically diverse organisms may help identify an endogenous ligand(s) and the physiological role(s) for this protein.     

Molecular characterization of a glutathione peroxidase gene and its expression in the selected Vibrio-resistant population of the clam Meretrix meretrix

Glutathione peroxidase (GPx) is an important member of cellular enzymatic antioxidant system, which may be involved in pathogen defense of host. In the present study, a selenium-dependent glutathione peroxidase (MmeGPx) gene from clam Meretrix meretrix was cloned and analyzed. The MmeGPx gene was composed of two introns of 723 bp and 238 bp and an open reading frame (ORF) of 711 bp. The ORF encodes a protein of 237 amino acids with a putative selenocysteine residue encoded by an unusual stop codon. MmeGPx shares a higher level of similarity with human GPx 3 than with other human GPx isozymes. The level of MmeGPx mRNA roughly paralleled GPx enzyme activity in different tissues except in gills, with the highest mRNA expression and enzyme activity occurring in hepatopancreas. MmeGPx mRNA expressions were detected in different larval stages and the results showed that MmeGPx mRNA increased significantly in pediveliger stage, which may be a response to oxidative stress. After challenge of clam with a Vibrio parahaemolyticus-related bacterium (MM21), the expression of MmeGPx was significantly up-regulated at 6 h and 12 h in hepatopancreas, which suggested that MmeGPx may be involved in the immune response to MM21 infection. To better understand its role in the immunity of clam, the expression of MmeGPx in hepatopancreas was compared between a selected Vibrio-resistant population and a control population after immersion challenge with MM21. Early up-regulation of MmeGPx was observed in the resistant population. These results suggested that MmeGPx might be involved in maintaining the redox state of immune system, and the early immune response to pathogen infection may help the clam against pathogen infection.     

Immune response-related gene expression profile of a novel molluscan IκB protein member from Manila clam (Ruditapes philippinarum)

Mollusks lack an adaptive immune system and rely solely on the innate immune response. The nuclear factor-kappa B (NF-κB) signaling pathway is one of the most important components of the innate immune system and its activity is regulated by physical interaction with the inhibitor of NF-κB (IκB) protein. The manila clam, Ruditapes philippinarum (Rp), is a key species of the world’s aquaculture industry, and recent pathogenic threats, such as the Gram-negative lipopolysaccharide (LPS)-expressing Vibrio tapetis bacteria, have produced severe adverse economic impacts. Here, we describe identification, characterization and immune responses of novel IκB (Rp-IκB) in the manila clam. The Rp-IκB cDNA is comprised of a 1,032 bp open reading frame, which encodes 343 amino acid residues and has a predicted molecular mass of 38 kDa. The Rp-IκB protein exhibits typical structural features of IκB family members, including the IκB degradation motif, PEST sequence, and six ankyrin repeats. Phylogenetic analysis showed that manila clam and other known molluscan IκB proteins grouped together in the invertebrate cluster. Analysis of the tissue expression distribution revealed that Rp-IκB was ubiquitously expressed. However, immune challenge with V. tapetis and purified LPS endotoxin induced significant up-regulation of Rp-IκB expression in gill and hemocytes. These results indicated that Rp-IκB may play an important role in manila clam defense against bacterial infection.     

Helpful habitant or pernicious passenger: interactions between an infaunal bivalve,an epifaunal hydroid and three potential predators

The importance of positive interspecific interactions within physically stressful habitats has received increased attention from community ecologists. The exposed sandy beach is an example of a physically rigorous environment where biological interactions have long been considered insignificant. We examined the interaction between the infaunal clam, Donax variabilis, and the hydroid, Lovenella gracilis, on exposed sandy beaches in North Carolina. Epibiotic occupation of Donax by hydroids has been repeatedly observed on ocean beaches but rarely investigated. By providing a stable substrate for attachment, the clam facilitates the persistence of the hydroid in the intertidal beach; however, benefits or costs experienced by the host as a result of this association are unknown. By exposing clams with and without hydroid colonies to multiple types of clam predators, we tested the effectiveness of the hydroid, which possesses stinging nematocysts, in defending its host. The hydroid defended the clam against one common predator, the Florida pompano ( Trachinotus carolinus). Against speckled crabs ( Arenaeus cribrarius) and ghost crabs ( Ocypode quadrata), however, the hydroid offered no protection for its host and instead facilitated predation. The epibiotic hydroid, which projects above the surface of the sand, allowed the crabs to more readily detect clams below the surface. In the field, we evaluated the effect of the hydroid on the tidally synchronized migrations and burrowing speed of the clam. The hydroid, which can form large colonies on the posterior end of the clam, had no effect on Donax burrowing speed, but did reduce the speed of transport of clams by wave swash. Depending on relative predation pressure, the occupation of D. variabilis by L. gracilis can alternately be characterized as beneficial or detrimental to the host.     

Electrophoretic separation of gill proteins of the clam Codakia orbicularis

Marine organisms inhabiting the coastal environment of the Caribbean islands have attracted the attention of a number of research scientists. These organisms generally live in areas of high environmental stress and may, therefore, contain specialized proteins and enzymes which exhibit valuable biotechnological applications. Among them, a small number of clams have been studied. Our work relates to the clam Codakia orbicularis. This bivalve lives in areas of high hydrogen sulfide concentrations on the Caribbean island of Guadeloupe. Its enzymatic system must, therefore, have evolved to allow its adaptation to this high-stress environment. C. orbicularis also contains endosymbiotic bacteria which are housed in the bacteriocytes of the gills. Its protein content can, therefore, be expected to have an impact on this symbiotic relationship. We have analysed gill protein extracts of this clam by various biochemical techniques: SDS-PAGE, IEF, PAS, and Western blotting using a panel of lectins, in order to establish its protein and glycoprotein profiles. This biochemical analysis, the first of its kind, constitutes an important step in separating and characterizing the proteins involved in the biochemical pathways of this organism whose stock is in decline in Guadeloupe. Our results show the presence of three major proteins whose molecular weights vary between 14,000 and 24,000 Daltons, and some of which are glycoproteins with predominantly alpha-mannose and N-acetylgalactosamine moieties. Their pI values are in the range between 4.5 and 5.6. These protein profiles are different from those observed for Lucina pectinata, a clam which has been the subject of earlier studies in the literature.     

The tropical giant clam <Emphasis Type="Italic">Hippopus hippopus</Emphasis> shell,a new archive of environmental conditions as revealed by sclerochronological and δ<Superscript>18</Superscript>O profiles

The use of the sclerochronology and geochemistry of a New Caledonian (South West Pacific) giant clam Hippopus hippopus shell as markers of environmental changes has been investigated. Growth increment thickness and δ¹⁸O ratios were measured on 4 years of shell growth of a modern specimen. During the last year, this giant clam was placed in a tank equipped for high-frequency environmental monitoring. Because shell is secreted in isotopic equilibrium with the seawater, the palaeo-sea surface temperature (SST) equation obtained faithfully reproduces the seasonal SST amplitudes. Growth increment thickness changes are seasonal and, for more than 50%, governed by the SST changes. The transplantation from the in situ site to the tank and reproduction events reduces the strength of growth and SST relationships. Nevertheless, growth increment thickness measurements can give information on average, minimal and maximal past SST in diagenetically altered shells. A peculiar growth event characterized by a short, drastic and significant decrease has been identified and attributed to an intense upwelling event. This study further highlights the use of giant clam shell δ¹⁸O as a SST proxy but also demonstrates that H. hippopus growth increment thickness changes provide useful information on past environmental settings and on exceptional events, for example, intense upwellings.     

Coupling of bacterial endosymbiont and host mitochondrial genomes in the hydrothermal vent clam Calyptogena magnifica

The hydrothermal vent clam Calyptogena magnifica (Bivalvia: Vesicomyidae) depends for its nutrition on sulfur-oxidizing symbiotic bacteria housed in its gill tissues. This symbiont is transmitted vertically between generations via the clam's eggs; however, it remains uncertain whether occasionally symbionts are horizontally transmitted or acquired from the environment. If symbionts are transmitted strictly vertically through the egg cytoplasm, inheritance of symbiont lineages should behave as if coupled to the host's maternally inherited mitochondrial DNA. This coupling would be obscured, however, with low rates of horizontal or environmental transfers, the equivalent of recombination between host lineages. Population genetic analyses of C. magnifica clams and associated symbionts from eastern Pacific hydrothermal vents clearly supported the hypothesis of strictly maternal cotransmission. Host mitochondrial and symbiont DNA sequences were coupled in a clam population that was polymorphic for both genetic markers. These markers were not similarly coupled with sequence variation at a nuclear gene locus, as expected for a randomly mating sexual population. Phylogenetic analysis of the two cytoplasmic genes also revealed no evidence for recombination. The tight association between vesicomyid clams and their vertically transmitted bacterial endosymbionts is phylogenetically very young (<50 million years) and may serve as a model for the origin and evolution of eukaryotic organelles.     

Clams (Corbicula fluminea) as bioindicators of fecal contamination with Cryptosporidium and Giardia spp. in freshwater ecosystems in California

This study evaluated clams as bioindicators of fecal protozoan contamination using three approaches: (i) clam tissue spiking experiments to compare several detection techniques; (ii) clam tank exposure experiments to evaluate clams that had filtered Cryptosporidium oocysts from inoculated water under a range of simulated environmental conditions; (iii) sentinel clam outplanting to assess the distribution and magnitude of fecal contamination in three riverine systems in California. Our spiking and tank experiments showed that direct fluorescent antibody (DFA), immunomagnetic separation (IMS) in combination with DFA, and PCR techniques could be used to detect Cryptosporidium in clam tissues. The most analytically sensitive technique was IMS concentration with DFA detection of oocysts in clam digestive gland tissues, which detected 10 oocysts spiked into a clam digestive gland 83% of the time. In the tank experiment, oocyst dose and clam collection time were significant predictors for detecting Cryptosporidium parvum oocysts in clams. In the wild clam study, Cryptosporidium and Giardia were detected in clams from all three study regions by IMS-DFA analysis of clam digestive glands, with significant variation by sampling year and season. The presence of C. parvum DNA in clams from riverine ecosystems was confirmed with PCR and DNA sequence analysis.     

Analysis of differentially expressed genes in response to bacterial stimulation in hemocytes of the carpet-shell clam Ruditapes decussatus: identification of new antimicrobial peptides

Suppression subtractive hybridization was used to identify differentially expressed genes in hemocytes from carpet-shell clam Ruditapes decussatus stimulated with a mixture of dead bacterial strains. Putative function could be assigned to 100 of the 253 sequenced cDNAs. Based on sequence homologies, 3.16% of the total identified genes were possibly related to immune functions. Clam myticin isoforms 1, 2 and 3, and clam mytilin, with similarity with myticins and mytilins previously reported on Mytilus galloprovincialis were identified and characterized for the first time in clams. The analysis of their expression levels by quantitative PCR showed that they were induced by bacterial challenge. The results obtained in this work could be the first step leading to the understanding of molecular mechanisms by which these economically important marine bivalves respond to pathogens.