Summer Mortality Events Research Articles

Lipid metabolism changes in clam Meretrix petechialis in response to Vibrio infection and the identification of Vibrio-resistance markers

Mass summer mortality events in the clam Meretrix petechialis resulting from Vibrio infection have occurred frequently in recent years, and seriously threaten the clam aquaculture industry. Metabolic processes are known to support host defense and survival in stressful conditions by regulating the allocation of metabolic resources. However, changes in lipid metabolism of clams in response to Vibrio infection are largely undescribed. In the present research, we characterized changes in key gene expression and enzyme activities associated with triglyceride-related metabolism, fatty acid de novo synthesis and fatty acid β-oxidation in M. petechialis during Vibrio infection. By mining dynamic transcriptome data before the increase in mortality, we found Vibrio infection caused significant changes in the metabolic regulations of carbohydrates, lipids, and amino acids. Our data show that levels of triglycerides (TG) decreased and free fatty acids (FFA) increased in the infected clams. Correspondingly, the synthesis activities of TG were significantly reduced relative to TG lipolysis activities after Vibrio infection. Additionally, FFA de novo synthesis was strengthened, especially before massive mortality occurred, but key genes of the fatty acid β-oxidation pathway showed no significant changes after Vibrio infection. We detected the expression of key genes related to lipid metabolism in resistant and susceptible clams under uninfected and infected conditions and found that differences in the basal expression of seven key genes (ATGL, MDY, SREBP, FAS, CPT-1, ACS, and ECH) could be used as potential biomarkers to predict different Vibrio resistance in clams. Overall, our results provide a starting point for investigating the relationship between energy metabolism changes and the emergence of massive mortality induced by bacterial infection in clams.

Effect of farming practices on growth and mortality rates in triploid and diploid eastern oysters Crassostrea virginica

Commercial off-bottom aquaculture of the eastern oyster Crassostrea virginica is challenged by repeated summer mortality events that appear to affect triploid oysters disproportionally. Many farmers believe common farming practices, especially when performed during hot summer months, may increase triploid mortality. The goal of this study was to investigate how diploid and triploid oysters react to common stressors imposed by farmers: tumbling during size grading and desiccation to prevent biofouling. Triploid and diploid oysters were deployed in floating cages at 3 farm sites along the US Gulf of Mexico coast. In May and July, oysters in 7 cages were subjected to 1 of 6 stress treatments: 1 of 3 levels of desiccation (18, 24, or 48 h) mixed with 1 of 2 levels of tumbling (tumbled or not tumbled), along with a never handled, submersed control. The mortality and growth rate of oysters were assessed in June, August, and September. Growth rates of both ploidies were affected by compounding stressors; that is, treatments with both tumbling and the longest desiccation period exhibited slower growth. Triploid oysters exhibited a greater increase in mortality in response to farm stress than diploid oysters, and mortality increased in all oysters subjected to extreme stress treatments when compared to the control. Based on this study, farmers should limit the desiccation time of oysters (particularly triploids) in the summer months to avoid any mortality or reduced growth resulting from compounding stressor effects.

Metabolomics investigation of summer mortality in New Zealand Greenshell™ mussels (Perna canaliculus)

Increasing water temperatures due to climate change have resulted in more frequent high mortality events of New Zealand Greenshell™ mussels (Perna canaliculus Gmelin 1791). These events have significant impacts within mussel farms which support a major shellfish industry for New Zealand. The present study investigates metabolic responses of farmed mussels during a summer mortality event in order to identify health impacts and elucidate mechanistic effects of external stressors on mussels. A gas chromatography–mass spectrometry (GC–MS)-based metabolomics approach was used to identify metabolic perturbations and flow cytometry assays were used to assess viability, oxidative stress and apoptosis of haemocytes from healthy and unhealthy mussels during a summer mortality event. The results showed significantly higher mortality and apoptosis of haemocytes in unhealthy mussels compared to healthy mussels. Reactive oxygen species (ROS) production, which is an indicator of oxidative stress was very high in both mussel groups, but no differences were observed between the two mussel groups. Metabolomics revealed alterations of many metabolites in both haemolymph and hepatopancreas (digestive gland) of unhealthy mussels compared to healthy mussels, reflecting perturbations in several molecular pathways, including energy metabolism, amino acid metabolism, protein degradation/tissue damage and oxidative stress. An increased level of itaconic acid which is an antimicrobial metabolite and biomarker of pathogen infection was observed in haemolymph, but not in hepatopancreas samples. This investigation provides the first detailed metabolic characterization of mussel immune responses to a summer mortality event and illustrates the benefits of using an integrated metabolomics and flow cytometry workflow for mussel health assessment and biomarker identification for summer mortality early detection.

Effects of Environmental Factors and Husbandry Practices on Summer Mortality Events in the Cultivated Pacific Oyster Crassostrea gigas in the North of Ireland

The Pacific oyster Crassostrea gigas (Thunberg, 1793) is an important part of the aquaculture industry in Europe; however, since 2008, oyster mortality events (>20%) have become a regular summer occurrence. This study monitored and recorded C. gigas mortality events during a 3-y period at a commercial oyster site in Lough Foyle, North of Ireland, and related these to environmental conditions, oyster physiology, and husbandry practices. Early summer temperature rises above 16°C were consistently correlated with large mortality events, but warmer temperatures in late summer did not cause mortalities, including in 1 y when there were no early summer mortalities. Mortalities were also correlated with a sharp early summer decrease in salinity from 22.2 to 16.9. In the laboratory, the combined effects of various temperatures and salinities on oyster stress levels were investigated using total hemocyte count and neutral red retention techniques. The cell membrane integrity and hemocyte density in C. gigas are sensitive to temperatures of 20°C or greater and at salinities of 20 or lower. This suggests that high temperature and low salinity combinations can negatively impact oyster stress levels and potentially increase oyster vulnerability to infections, including oyster herpes OsHV-1 µvar. Comparing survivorship in experimental bags simulating commercial growing conditions, these results equivocally support previous studies in which higher shore position was correlated with higher survivorship. Bags of oysters at lower density in lower shore positions were equally successful and may be more protected from climate-related stressors. Focusing on a single stressor is insufficient to predict future oyster mortalities, but balancing effects and relieving stress on oyster stocks through husbandry could limit the impacts of summer mortalities.

Characterisation of the Pacific Oyster Microbiome During a Summer Mortality Event.

The Pacific oyster, Crassostrea gigas, is a key commercial species that is cultivated globally. In recent years, disease outbreaks have heavily impacted C. gigas stocks worldwide, with many losses incurred during summer. A number of infectious agents have been associated with these summer mortality events, including viruses (particularly Ostreid herpesvirus 1, OsHV-1) and bacteria; however, cases where no known aetiological agent can be identified are common. In this study, we examined the microbiome of disease-affected and disease-unaffected C. gigas during a 2013-2014 summer mortality event in Port Stephens (Australia) where known oyster pathogens including OsHV-1 were not detected. The adductor muscle microbiomes of 70 C. gigas samples across 12 study sites in the Port Stephens estuary were characterised using 16S rRNA (V1-V3 region) amplicon sequencing, with the aim of comparing the influence of spatial location and disease state on the oyster microbiome. Spatial location was found to be a significant determinant of the disease-affected oyster microbiome. Furthermore, microbiome comparisons between disease states identified a significant increase in rare operational taxonomic units (OTUs) belonging to Vibrio harveyi and an unidentified member of the Vibrio genus in the disease-affected microbiome. This is indicative of a potential role of Vibrio species in oyster disease and supportive of previous culture-based examination of this mortality event.

Histological Alterations in Pacific Oysters Crassostrea gigas that Survived a Summer Mortality Event in Baja California, Mexico.

A mortality episode (>90%) of triploid and diploid Pacific oysters Crassostrea gigas cultured in Baja California Sur occurred during summer 2012, coinciding with a thermal anomaly, an algal bloom, and low oxygen values. To help explain the cause of the mortalities, histological analyses and molecular tests for specific pathogens (ostreid herpesvirus 1 [OsHV-1] and Perkinsus marinus) were performed on oysters surviving at the end of the episode. Triploid oysters showed a high percentage of males (43%) and hermaphrodites (30%); 93% of these oysters were in the gonadic reabsorption stage, and in some cases, hemocytes completely filled the lumen of the gonadic follicles. Oysters presented large areas with severe hemocyte infiltration that extended toward the digestive gland. Diploid oysters showed similar gonad alterations. None of samples showed histological or molecular evidence of OsHV-1 or P. marinus. Histological alterations can be related to physiological disorders caused by the mechanism driving summer mortality. This is the first case history of a summer mortality episode among Pacific oysters in Mexico.

Epipodial Tentacle Gene Expression and Predetermined Resilience to Summer Mortality in the Commercially Important Greenlip Abalone, Haliotis laevigata

“Summer mortality” is a phenomenon that occurs during warm water temperature spikes that results in the mass mortality of many ecologically and economically important mollusks such as abalone. This study aimed to determine whether the baseline gene expression of abalone before a laboratory-induced summer mortality event was associated with resilience to summer mortality. Tentacle transcriptomes of 35 greenlip abalone (Haliotis laevigata) were sequenced prior to the animals being exposed to an increase in water temperature—simulating conditions which have previously resulted in summer mortality. Abalone derived from three source locations with different environmental conditions were categorized as susceptible or resistant to summer mortality depending on whether they died or survived after the water temperature was increased. We detected two genes showing significantly higher expression in resilient abalone relative to susceptible abalone prior to the laboratory-induced summer mortality event. One of these genes was annotated through the NCBI non-redundant protein database using BLASTX to an anemone (Exaiptasia pallida) Transposon Ty3-G Gag Pol polyprotein. Distinct gene expression signatures were also found between resilient and susceptible abalone depending on the population origin, which may suggest divergence in local adaptation mechanisms for resilience. Many of these genes have been suggested to be involved in antioxidant and immune-related functions. The identification of these genes and their functional roles have enhanced our understanding of processes that may contribute to summer mortality in abalone. Our study supports the hypothesis that prestress gene expression signatures are indicative of the likelihood of summer mortality.

Persistence, seasonal dynamics and pathogenic potential of Vibrio communities from Pacific oyster hemolymph.

Bacteria of the genus Vibrio occur at a continuum from free-living to symbiotic life forms, including opportunists and pathogens, that can contribute to severe diseases, for instance summer mortality events of Pacific oysters Crassostrea gigas. While most studies focused on Vibrio isolated from moribund oysters during mortality outbreaks, investigations of the Vibrio community in healthy oysters are rare. Therefore, we characterized the persistence, diversity, seasonal dynamics, and pathogenicity of the Vibrio community isolated from healthy Pacific oysters. In a reciprocal transplant experiment we repeatedly sampled hemolymph from adult Pacific oysters to differentiate population from site-specific effects during six months of in situ incubation in the field. We characterized virulence phenotypes and genomic diversity based on multilocus sequence typing in a total of 70 Vibrio strains. Based on controlled infection experiments we could show that strains with the ability to colonize healthy adult oysters can also have the potential to induce high mortality rates on larvae. Diversity and abundance of Vibrio varied significantly over time with highest values during and after spawning season. Vibrio communities from transplanted and stationary oysters converged over time, indicating that communities were not population specific, but rather assemble from the surrounding environment forming communities, some of which can persist over longer periods.

Effects of glyphosate-based herbicides on embryo-larval development and metamorphosis in the Pacific oyster, Crassostrea gigas

Pesticides may be involved in oyster summer mortality events, not necessarily as a single causative agent but as an additional stressor. In this context, the present study aimed to assess the toxicity of glyphosate, its by-product, aminomethylphosphonic acid (AMPA) and two commercial formulations, Roundup Express® (REX) and Roundup Allées et Terrasses® (RAT), containing glyphosate as the active ingredient, on the early life stages of the Pacific oyster, Crassostrea gigas. The embryotoxicity of these chemicals were quantified by considering both the rates of abnormalities and the arrested development or types of abnormalities in D-shaped larvae after 48h exposure. The success of metamorphosis was examined in pediveliger larvae exposed for 24h. Experiments involving both endpoints included range finding experiments for herbicide concentrations ranging from 0.1 to 100,000μgL−1. This range was then narrowed down in order to determine precise EC50 values. Actual concentrations of the herbicide were determined at the beginning and after 48h (embryotoxicity) and 24h (metamorphosis) to evaluate the potential temporal variation in the concentrations. During embryo-larval development, no mortalities were recorded at any of the concentrations of glyphosate and AMPA, whereas no embryos or D-shaped larvae could be observed after exposure to 10,000μgL−1 of REX or RAT. Compared with the controls, no effects on embryo-larval development were recorded between 0.1 and 1000μgL−1, regardless of the chemical tested. Above a threshold, which varied according to the chemical used, the gradient of herbicide concentrations correlated with a gradient of severity of abnormality ranging from normal larvae to arrested development (an “old embryo” stage). The EC50 values were 28,315 and 40,617μgL−1 for glyphosate and its metabolite, respectively, but much lowered values of 1133 and 1675μgL−1 for REX and RAT, respectively. Metamorphosis tests also revealed a significant difference between molecules, as the EC50 values exceeded 100,000μgL−1 for glyphosate and AMPA but were as low as 6366 and 6060μgL−1 for the commercial formulations, which appeared relatively more toxic. Overall, the embryo-larval development of C. gigas was more sensitive to glyphosate-based herbicides compared to various endpoints studied in regulatory model organisms, and embryos and D-shaped larvae were more sensitive compared to pediveliger larvae.

Detection of early effects of a single herbicide (diuron) and a mix of herbicides and pharmaceuticals (diuron, isoproturon, ibuprofen) on immunological parameters of Pacific oyster (Crassostrea gigas) spat

In the context of massive summer mortality events of the Pacific oyster Crassostrea gigas, the aim of this study was to investigate the early effects on genes, enzymes and haemocyte parameters implicated in immune defence mechanisms in C. gigas oysters exposed to a potentially hostile environment, i.e. to an herbicide alone or within a mixture. Following 2h of exposure to the herbicide diuron at 1μgL−1, the repression of different genes implicated in immune defence mechanisms in the haemocytes and the inhibition of enzyme activities, such as laccase-type phenoloxidase (PO) in the plasma, were observed. The inhibition of superoxide dismutase (SOD) activity in the plasma was also observed after 6 and 24h of exposure. In the mixture with the herbicides diuron and isoproturon, and the pharmaceutical ibuprofen, catecholase-type PO activity in the plasma and the percentage of phagocytosis in the haemocytes were reduced after 6h of exposure. Our results showed that early effects on molecular, biochemical and cellular parameters can be detected in the presence of diuron alone or within a mixture, giving an insight of its potential effect in situations that can be found in natural environments, i.e. relatively high concentrations for short periods of time.

Characterisation and genetic polymorphism of metallothionein gene CgMT4 in experimental families of Pacific oyster Crassostrea gigas displaying summer mortality

Summer mortality events have been observed in Pacific oyster Crassostrea gigas for several decades. This paper examines the selective pressure exerted by summer mortality on the polymorphism of a newly identified oyster metallothionein gene. CgMT4 cDNA and genomic sequences were obtained. CgMT4 was studied in two generations of oysters reared in three sites on the French Atlantic coast, using single strand conformation polymorphism analysis. Four alleles were detected. Individuals carrying genotype MT4-CD seem to have higher susceptibility to summer risk conditions. The MT4 gene could be a potential new genetic marker for susceptibility; further validation studies are recommended.

Effects of age and environment on survival of summer mortality by two selected groups of the Pacific oyster Crassostrea gigas

This study investigated the effects of age (first or second summer of life) and environment (three study sites on French coasts) on summer mortality in Crassostrea gigas, using three generations of oysters produced during the Morest project. For each generation, two groups of oysters, selected either as ‘resistant’ (R) or ‘susceptible’ (S) to summer mortality at the age of 6 months in Rivière d'Auray (RA, South Brittany), were evaluated. In Ronce (Marennes-Oléron Bay) and RA, R oysters had low mortality during their first and second summers, while mortality was higher during the second summer in Baie des Veys (BDV, Normandy). In RA, S oysters exhibited higher mortality during their first summer than during their second, while the opposite result was found in BDV, indicating a significant environment by age interaction. Some oysters were also protected from mortality risk factors during their first year by keeping them in a nursery; they were then deployed at the two sites the following spring. Mortality in R oysters during their second year was lower than in S oysters in RA and BDV. Interestingly, mortality of the S oysters deployed in RA during their second year was much lower than the mortality observed in S oysters deployed during their first year, suggesting that S juveniles are more susceptible to summer mortality than S adults. As a consequence, the difference in mortality between the two selected groups in RA decreased from 40–45% to 5–9% when oysters were protected from summer mortality at the juvenile stage. In BDV, the difference in mortality between the two selected groups was similar whether oysters were deployed during their first or second year. The first reproductive event in oysters, combined with genetic differences between the groups interacting with local environmental conditions can go some way to explaining summer mortality events in our study. Cumulative mortality for each generation and site was higher in the S group than in the R group, indicating a positive response to selection in oysters up to 18 months old. Mortality was split into three categories: low background mortality, genetic effects from the selection and unknown mortality on which no investigation has yet concentrated. Finally, culture strategy on oyster farms is discussed with respect to oyster age and environment.

Microarray-Based Identification of Gonad Transcripts Differentially Expressed Between Lines of Pacific Oyster Selected to Be Resistant or Susceptible to Summer Mortality

Summer mortality of the Pacific oyster Crassostrea gigas is the result of a complex interaction between oysters, their environment, and pathogens. Heredity appears to be a major factor determining the sensitivity of oysters to summer mortality, allowing resistant (R) and susceptible (S) lines to be produced. We conducted genome-wide expression profiling of R and S gonads during the 3-month period preceding a summer mortality event, using a cDNA microarray that we designed. ANOVA analysis revealed that 34 genes were differentially expressed between R and S lines on four dates preceding the mortality event. Annotation of some of these genes highlights reproduction and its allocation and antioxidant defenses as the main pathways that operate differentially between R and S lines. This transcriptional analysis provides new indications to define markers for quantitative trait loci searches and functional studies and evaluate the potential role of each gene in the resistance to summer mortality.

Level of contamination and impact of pesticides in cupped oyster, Crassostrea gigas, reared in a shellfish production area in Normandy (France)

Over the last decade, oysters in the Bay of Veys (Northwest France) have sporadically experienced significant summer mortality events which appear to be due to a combination of extrinsic and intrinsic factors. In the present work, the involvement of pesticides (as additional stressors) was investigated using both artificial exposure and field studies (at two sites). Six herbicides were detected in seawater following tests for a total of 15 herbicides. The most estuarine site was the most contaminated, showing relatively high values compared with those recorded in a neighboring river. No pesticides were detected in the flesh of oysters in the field but exposure experiments led to an accumulation of two substituted ureas (diuron and isoproturon, from 0.5 μ g/L and 1 μ gL− 1 respectively). Some physiological effects were observed in terms of reproduction (partial spawning) and histopathology (atrophy of the digestive tubule epithelium) but results related to other tissue alterations and to neutral red retention (NRR) assays were not conclusive and require further investigation.

Hemocyte characteristics in families of oysters, Crassostrea gigas, selected for differential survival during summer and reared in three sites

High variability among individuals is often encountered when hemocyte characteristics are measured in bivalves. Such variability is suspected to result partly from genetic factors. In this study, hemocyte characteristics of six families of Crassostrea gigas were compared by flow cytometry at one sampling date in October 2001. These families were obtained from a nested, half-sibling cross design, and reared from July to October 2001 at three sites distributed along the French Atlantic coast from north to south: Baie des Veys (Normandy), Rivière d'Auray (Brittany) and Ronce (Marennes-oléron Basin, Poitou Charentes). Among the 15 measured hemocyte characteristics, production of reactive oxygen species (ROS) of untreated hemocytes (maintained in filtered sterile seawater) and treated hemocytes (zymosan at 20 particles per hemocyte, and with Vibrio sp. S322 at 50 bacteria per hemocyte) was the most notable differences between families. This supports the existence of a genetic basis, at least partly, for the hemocyte characteristics of oysters, and especially for ROS production. Among the six families analyzed, three have shown high survival during summer (named as “resistant”, mean mortality 5.2%) and three experienced high mortality during summer (named as “susceptible”, 30.6% mean mortality). Families showing high or low survival to summer mortality had similar hemocyte characteristics, regardless of the environmental conditions or reproductive state. Resistant families were observed to have higher total hemocyte counts and lower production of ROS than susceptible families. Moreover, ROS production of hemocytes from susceptible families was diminished significantly more by pathogenic Vibrio than that of resistant families. However, this study demonstrates also that rearing site strongly affected the hemocyte characteristics of all families of oysters, most notably hemocyte concentration and morphology (size and granularity), production of reactive oxygen species (ROS), and susceptibility to the cytotoxic activity of the pathogenic Vibrio sp. S322 (50 bacteria/hemocyte). Food availability and reproductive state are the most probable explanations for the site differences observed. Finally, it appeared difficult to link oyster survival during summer mortality to hemocyte profiles evaluated at one sampling date; other relevant indicators would probably help explaining oyster survival during summer mortality events.

SUMMER MORTALITY OF CRASSOSTREA GIGAS (THUNBERG) IN RELATION TO ENVIRONMENTAL REARING CONDITIONS

The purpose of this study is to investigate summer mortality of the cupped oyster, Crassostrea gigas, in relation to culture practices in the traditional oyster production region of Marennes-Oléron (France). Four oyster rearing conditions, varying culture location (“on-“ or “off-bottom”), and site depth (65% to 80% daily immersion termed “deep” or 45% to 65%, termed “shallow”) were studied to compare biologic performance and maturation status of oysters, in relation to sediment and water column parameters. The most severe mortality occurred in June to July in “on-bottom” reared oysters (25%), as compared with 10% mortality in “off-bottom” cultured oysters. Oysters (shell and meat) grew significantly better when reared “off-bottom” than “on-bottom.” Reproductive effort was almost double in “off-bottom” reared oysters, compared with those “on-bottom”; thus, reproduction cannot be directly related to mortality in this summer mortality event. Low glycogen content recorded for both “on” and “off-bottom” reared oysters in summer, confirmed the probable lack of food and/or the overstocking in the Marennes-Oléron Bay, but did not discriminated among culture conditions. Whatever the immersion depth (“deep” or “shallow” conditions), “on-bottom” cultured oysters were adversely affected in growth, reproductive effort, and survival suggesting a direct effect of the mud (the so called “mud effect”) on the biologic performance of oysters cultured on the bottom. Data from monitoring of sediment redox potential, organic content, and ammonium release did not support hypotheses that these parameters were alone responsible for the observed differences in mortality events.

Growth and mortality rates of the zebra mussel,Dreissena polymorpha, in the Lower Mississippi River

We investigated seasonal growth and mortality of a population of zebra mussels, Dreissena polymorpha, near the extreme southern limit of their current North American distribution. Dreissena in the Lower Mississippi River experience seasonal regimes of temperature and suspended sediment that are distinctly different from those found in northern lentic environments. Shell growth and mortality were determined biweekly using marked animals in caged environments. Tissue condition was also estimated biweekly using mussels from caged and wild environments. Our results demonstrate that, although annual growth is comparable with that reported for more temperate environments, the seasonal pattern of increase is different. Shell growth and tissue condition were highest during the early spring. Tissue condition declined and shell growth ceased during the summer, when temperatures remained at 29-30°C for 3 months. This midsummer depression was accompanied by increased mortality, particularly for mussels greater than 15 mm. Condition improved for all sizes in fall and winter. These patterns of growth and mortality appear to be primarily driven by fluctuations in seasonal temperature and spawning condition with some influence of suspended sediment. Our observations indicate that periodic summer mortality events will occur, and the extent of this mortality will depend primarily on temperature, population size structure, and spring tissue condition.