Bivalve mollusc circadian clock genes can run at tidal frequency.

Marine coastal habitats are complex cyclic environments as a result of sun and moon interactions. In contrast with the well-known circadian orchestration of the terrestrial animal rhythmicity (approx. 24 h), the mechanism responsible for the circatidal rhythm (approx. 12.4 h) remains largely elusive in marine organisms. We revealed in subtidal field conditions that the oyster Crassostrea gigas exhibits tidal rhythmicity of circadian clock genes and clock-associated genes. A free-running (FR) experiment showed an endogenous circatidal rhythm. In parallel, we showed in the field that oysters' valve behaviour exhibited a strong tidal rhythm combined with a daily rhythm. In the FR experiment, all behavioural rhythms were circatidal, and half of them were also circadian. Our results fuel the debate on endogenous circatidal mechanisms. In contrast with the current hypothesis on the existence of an independent tidal clock, we suggest that a single 'circadian/circatidal' clock in bivalves is sufficient to entrain behavioural patterns at tidal and daily frequencies.

Rhythms during the polar night: evidence of clock-gene oscillations in the Arctic scallop Chlamys islandica.

Arctic regions are highly impacted by climate change and are characterized by drastic seasonal changes in light intensity and duration with extended periods of permanent light or darkness. Organisms use cyclic variations in light to synchronize daily and seasonal biological rhythms to anticipate cyclic variations in the environment, to control phenology and to maintain fitness. In this study, we investigated the diel biological rhythms of the Arctic scallop, Chlamys islandica, during the autumnal equinox and polar night. Putative circadian clock genes and putative light perception genes were identified in the Arctic scallop. Clock gene expression oscillated in the three tissues studied (gills, muscle, mantle edge). The oscillation of some genes in some tissues shifted from daily to tidal periodicity between the equinox and polar night periods and was associated with valve behaviour. These results are the first evidence of the persistence of clock gene expression oscillations during the polar night and might suggest that functional clockwork could entrain rhythmic behaviours in polar environments.

Molecular Characterization of Voltage-Gated Sodium Channels and Their Relations with Paralytic Shellfish Toxin Bioaccumulation in the Pacific Oyster Crassostrea gigas.

Paralytic shellfish toxins (PST) bind to voltage-gated sodium channels (Nav) and block conduction of action potential in excitable cells. This study aimed to (i) characterize Nav sequences in Crassostrea gigas and (ii) investigate a putative relation between Nav and PST-bioaccumulation in oysters. The phylogenetic analysis highlighted two types of Nav in C. gigas: a Nav1 (CgNav1) and a Nav2 (CgNav2) with sequence properties of sodium-selective and sodium/calcium-selective channels, respectively. Three alternative splice transcripts of CgNav1 named A, B and C, were characterized. The expression of CgNav1, analyzed by in situ hybridization, is specific to nervous cells and to structures corresponding to neuromuscular junctions. Real-time PCR analyses showed a strong expression of CgNav1A in the striated muscle while CgNav1B is mainly expressed in visceral ganglia. CgNav1C expression is ubiquitous. The PST binding site (domain II) of CgNav1 variants possess an amino acid Q that could potentially confer a partial saxitoxin (STX)-resistance to the channel. The CgNav1 genotype or alternative splicing would not be the key point determining PST bioaccumulation level in oysters.

Characterization of the transcriptome and temperature-induced differential gene expression in QPX, the thraustochytrid parasite of hard clams.

BACKGROUND: The hard clam or northern quahog, Mercenaria mercenaria, is one of the most valuable seafood products in the United States representing the first marine resource in some Northeastern states. Severe episodes of hard clam mortality have been consistently associated with infections caused by a thraustochytrid parasite called Quahog Parasite Unknown (QPX). QPX is considered as a cold/temperate water organism since the disease occurs only in the coastal waters of the northwestern Atlantic Ocean from Maritime Canada to Virginia. High disease development at cold temperatures was also confirmed in laboratory studies and is thought to be caused predominantly by immunosuppression of the clam host even though the effect of temperature on QPX virulence has not been fully investigated. In this study, the QPX transcriptome was sequenced using Roche 454 technology to better characterize this microbe and initiate research on the molecular basis of QPX virulence towards hard clams. RESULTS: Close to 18,000 transcriptomic sequences were generated and functionally annotated. Results revealed a wide array of QPX putative virulence factors including a variety of peptidases, antioxidant enzymes, and proteins involved in extracellular mucus production and other secretory proteins potentially involved in interactions with the clam host. Furthermore, a 15 K oligonucleotide array was constructed and used to investigate the effect of temperature on QPX fitness and virulence factors. Results identified a set of QPX molecular chaperones that could explain its adaptation to cold temperatures. Finally, several virulence-related factors were up-regulated at low temperature providing molecular targets for further investigations of increased QPX pathogenicity in cold water conditions. CONCLUSIONS: This is one of the first studies to characterize the transcriptome of a parasitic labyrinthulid, offering new insights into the molecular bases of the pathogenicity of members of this group. Results from the oligoarray study demonstrated the ability of QPX to cope with a wide range of environmental temperatures, including those considered to be suboptimal for clam immunity (low temperature) providing a mechanistic scenario for disease distribution in the field and for high disease prevalence and intensity at low temperature. These results will serve as basis for studies aimed at a better characterization of specific putative virulence factors.

Differential immune response in the hard clam (mercenaria mercenaria) against bacteria and the protistan pathogen QPX (quahog parasite unknown).

The immune response of the hard clam (quahog) Mercenaria mercenaria following challenge with live bacteria (Vibrio alginolyticus) and the protist QPX (Quahog Parasite Unknown) was investigated. The study also compared immune responses following QPX challenge in two different hard clam broodstocks exhibiting different degrees of susceptibility toward this parasite. Different immune and stress-related cellular and humoral factors were assessed including general hemocyte parameters (total and differential hemocyte counts, percentage of dead cells, reactive oxygen production, phagocytosis), parameters geared toward QPX (anti-QPX activity in plasma and hemocyte resistance to the cytotoxicity of QPX extracellular products). Two genes (ferritin and metallothionein) previously shown to be modulated following QPX exposure were molecularly characterized by rapid amplification of cDNA ends (RACE) and their transcription levels were determined in resistant and susceptible clams in response to QPX and bacterial challenge. Results indicated that both V. alginolyticus and QPX challenge triggered significant immune responses in clams with similar trends for most measured parameters. However, specific responses were observed for anti-QPX activity in plasma and hemocyte resistance to QPX products as well as ferritin and metallothionein expression according to each inoculum. Similarly, different response patterns were detected following QPX challenge in susceptible and resistant clam stocks. Resistant clams were able to elicit effective response against the parasite leading to the elimination of QPX and the restoration of constitutive immune status whereas QPX-susceptible clams triggered a strong immune modulation characterized by an acute phase response and associated acute phase protein but appeared to be less active in eliminating the parasite. These results suggest that different signaling pathways are triggered during V. alginolyticus and QPX challenge. Moreover, differences in the immune response toward QPX might be linked to the susceptibility or resistance of different clam stocks to the infection by this parasite.

Effects of salinity on hard clam (Mercenaria mercenaria) defense parameters and QPX disease dynamics.

QPX (Quahog Parasite Unknown) is a protistan parasite affecting hard clams (Mercenaria mercenaria) along the Northeast coast of the United States. The fact that QPX disease epizootics are usually observed in field sites with high salinities led to the general assumption that salinity represents an important factor for disease distribution. This study was designed to investigate the effect of salinity on QPX disease development as well as constitutive and QPX-induced defense factors in M. mercenaria. Naïve and QPX-infected (both experimentally and naturally) clams were submitted to 17 and 30 psu for 4 months. Standard and QPX-specific cellular and humoral defense parameters were assessed after 2 and 4 months. These included total and differential hemocyte counts, reactive oxygen species production, phagocytic activity of hemocytes, lysozyme concentration in plasma, anti-QPX activity in plasma and resistance of hemocytes to cytotoxic QPX extracellular products. Results demonstrated higher QPX-associated mortality in naturally infected clams maintained at high salinity compared to those held at 17 psu. Our findings also showed an increase in mortality following experimental challenge with QPX in clams submitted to 30 psu but not in those held at 17 psu. Constitutive clam defense factors and the response to QPX challenge were also affected by salinity. QPX challenge caused significant but transitory changes in hemolymph parameters that were obvious at 2 months but disappeared at 4 months. Overall, our results show that salinity modulates clam immunity and the progress of QPX disease although its impact appears secondary as compared to findings we reported earlier for temperature.

Identification, molecular characterization and expression analysis of a mucosal C-type lectin in the eastern oyster, Crassostrea virginica.

Lectins are well known to actively participate in the defense functions of vertebrates and invertebrates where they play an important role in the recognition of foreign particles. They have also been reported to be involved in other processes requiring carbohydrate-lectin interactions such as symbiosis or fertilization. In this study, we report a novel putative C-type lectin (CvML) from the eastern oyster Crassostrea virginica and we investigated its involvement in oyster physiology. The cDNA of this lectin is 610 bp long encoding for a 161-residue protein. CvML presents a signal peptide and a single carbohydrate recognition domain (CRD) which contains a YPD motif and two putative conserved sites, WID and DCM, for calcium binding. CvML transcripts were expressed in mucocytes lining the epithelium of the digestive gland and the pallial organs (mantle, gills, and labial palps) but were not detected in other tissues including hemocytes. Its expression was significantly up-regulated following starvation or bacterial bath exposure but not after injection of bacteria into oyster's adductor muscle. These results highlight the potential role of CvML in the interactions between oyster and waterborne microorganisms at the pallial interfaces with possible involvement in physiological functions such as particle capture or mucosal immunity.

Effects of temperature on hard clam (Mercenaria mercenaria) immunity and QPX (Quahog Parasite Unknown) disease development: I. Dynamics of QPX disease.

Quahog Parasite Unknown (QPX) causes disease and mortality in hard clams, Mercenaria mercenaria. Seasonality of QPX disease prevalence in the field and changes in QPX growth and survival in vitro suggest a role of temperature in the hard clam-QPX interaction and disease development. This study specifically examined the effect of temperature on QPX disease development and dynamics. Naturally and experimentally infected clams were separately maintained in the laboratory at 13°C, 21°C, or 27°C for 4 months. Following this initial treatment, temperature was adjusted to 21°C for 5 additional months to simulate seasonal changes of temperature in the field and to investigate the effect of temperature variations on QPX disease dynamics. Mortality was continuously monitored during the experiment and clams were sampled at 2, 4 and 9 months for the assessment of QPX disease prevalence and intensity using our standard histological and quantitative PCR techniques. Results demonstrated significantly higher QPX disease prevalence and intensity, as well as higher mortality, in naturally-infected clams maintained at 13°C as compared to those held at 21°C or 27°C. Similarly, disease development was significantly higher in experimentally infected clams maintained at the colder temperature (70% prevalence after 4 months) as compared to those maintained under warmer conditions (<10%). Additionally, our results demonstrated an improvement in the condition of clams initially maintained at 13°C for 4 months after transfer to 21°C for 5 additional months, with a significant reduction of QPX prevalence (down to 19%). Interestingly, disease development or healing in clams maintained at different temperatures exhibited a strong relationship with clam defense status (jointly submitted paper) and highlighted the impact of temperature on clam activity and QPX disease dynamics. These findings should be taken into account for the timing of activities involving the monitoring, movement (e.g. relays, transplants) or grow out (e.g. commercial culture, municipal enhancement) of hard clams in enzootic areas.

Effects of temperature on hard clam (Mercenaria mercenaria) immunity and QPX (Quahog Parasite Unknown) disease development: II. Defense parameters.

Quahog Parasite Unknown (QPX) is a protistan parasite affecting hard clams Mercenaria mercenaria along the Northeastern coast of the United States. The geographic distribution and occurrence of disease epizootics suggests a primary role of temperature in disease development. This study was designed to investigate the effect of temperature on constitutive and QPX-induced defense factors in M. mercenaria. Control and QPX-challenged (both experimentally and naturally) clams were maintained at 13, 21 and 27°C for 4 months. Control and experimentally-infected clams originated from a southern broodstock (Florida, no prior reports of disease outbreak) while naturally-infected clams originated from a northern broodstock (Massachusetts, enzootic area). Standard and QPX-specific cellular and humoral defense parameters were assessed after 2 and 4 months. Measured parameters included total and differential hemocyte counts, reactive oxygen species production, phagocytic activity of hemocytes, lysozyme concentration in plasma, anti-QPX activity in plasma and resistance of hemocytes to cytotoxic QPX extracellular products. Results demonstrated a strong influence of temperature on constitutive clam defense factors with significant modulation of cellular and humoral parameters of control clams maintained at 13°C compared to 21 and 27°C. Similarly, clam response to QPX challenge was also affected by temperature. Challenged clams exhibited no difference from controls at 27°C whereas different responses were observed at 21°C and 13°C compared to controls. Despite differences in infection mode (experimentally or naturally infected) and clam origin (northern and southern broodstocks), similarities were observed at 13°C and 21°C between QPX infected clams from Florida and Massachusetts. Clam response to temperature and to QPX exhibited interesting relationship with QPX disease development highlighting major influence of temperature on disease development.

Effect of environmental factors on survival and growth of quahog parasite unknown (QPX) in vitro.

Quahog parasite unknown (QPX) is a protistan microorganism associated with mass mortalities of hard clams (Mercenaria mercenaria) along the northeastern coasts of the United States and maritime Canada. Because several studies indicate modulatory effects of prevailing environmental parameters on disease outbreaks, this study tested the effect of major environmental parameters (temperature, salinity and oxygen concentration; individually or combined) on QPX survival in artificial seawater and parasite growth in culture media in vitro. Three QPX isolates from two different geographic locations were compared. Results indicated that in vitro growth of QPX was optimal in standard culture medium at 34ppt between 20 degrees C and 23 degrees C. Additionally, significant differences in temperature optima were observed for geographically distinct QPX isolates (p<0.001) confirming previous studies suggesting the existence of different QPX strains (or ecotypes). When tested in seawater, QPX exhibited opposite trends with higher survival at 15 degrees C and 15ppt. Results also demonstrated limited survival and growth of QPX under anoxic conditions. Additionally, results showed that the parasite is able to survive extreme temperatures (-12 degrees C to 32 degrees C) suggesting that QPX could overcome short periods of extreme conditions in the field. These results contribute to a better understanding of interactions between QPX and its environment, but potential impacts of environmental conditions on QPX disease development need further work as it also involves clam response to these factors.