Search

Eastern oysters in the northern Gulf of Mexico are facing rapid environmental changes and can respond to this change via plasticity or evolution. Plasticity can act as an immediate buffer against environmental change, but this buffering could impact the organism's ability to evolve in subsequent generations. While plasticity and evolution are not mutually exclusive, the relative contribution and interaction between them remains unclear. In this study, we investigate the roles of plastic and evolved responses to environmental variation and Perkinsus marinus infection in Crassostrea virginica by using a common garden experiment with 80 oysters from six families outplanted at two field sites naturally differing in salinity. We use growth data, P. marinus infection intensities, 3' RNA sequencing (TagSeq) and low-coverage whole-genome sequencing to identify the effect of genotype, environment and genotype-by-environment interaction on the oyster's response to site. As one of first studies to characterize the joint effects of genotype and environment on transcriptomic and morphological profiles in a natural setting, we demonstrate that C. virginica has a highly plastic response to environment and that this response is parallel among genotypes. We also find that genes responding to genotype have distinct and opposing profiles compared to genes responding to environment with regard to expression levels, Ka/Ks ratios and nucleotide diversity. Our findings suggest that C. virginica may be able to buffer the immediate impacts of future environmental changes by altering gene expression and physiology, but the lack of genetic variation in plasticity suggests limited capacity for evolved responses.

Since the discovery of Perkinsus marinus as the cause of dermo disease in Crassostrea virginica, salinity and temperature have been identified as the main environmental drivers of parasite prevalence. However, little is known about how these variables affect the movement of the parasite from host to water column. In order to elucidate how environmental factors can influence the abundance of this parasite in the water column, we conducted a series of experiments testing the effects of time of day, temperature and salinity on the release of P. marinus cells from infected oysters. We found that P. marinus cells were released on a diurnal cycle, with most cells released during the hottest and brightest period of the day (12:00–18:00). Temperature also had a strong and immediate effect on the number of cells released, but salinity did not, only influencing the intensity of infection over the course of several months. Taken together, our results demonstrate that (1) the number of parasites in the water column fluctuates according to a diurnal cycle, (2) temperature and salinity act on different timescales to influence parasite abundance, and (3) live infected oysters may substantially contribute to the abundance of transmissive parasites in the water column under particular environmental conditions.

After unusually high mortalities of eastern oysters (Crassostrea virginica) were reported at commercial farms in Alabama during the summer of 2016, this study was conducted to determine whether the mortalities were reproducible, site-specific, and linked to ploidy. Seed of triploid and diploid oysters were deployed in November 2016 at four sites adjacent to commercial oyster farms in Alabama: Mobile Bay, Navy Cove, Grand Bay, and Dauphin Island. Mortality, growth, condition index, gametogenesis, and Perkinsus marinus infection intensity were monitored until October 2017. Mortalities were significantly greater in triploids by the end of the study, but the magnitude and timing of mortalities differed among sites. Triploids and diploids at Mobile Bay experienced severe mortalities in summer (100% in about two months), and the start of this mortality event was delayed in diploids. Elevated triploid mortalities were observed at Navy Cove (77%) and Dauphin Island (60%) in late spring through fall, while oysters at Grand Bay suffered the lowest triploid mortalities (34%). Possible causes of the high mortalities included environmental conditions, such as prolonged periods of low salinity and high temperature in summer at Mobile Bay, as well as, heavy P. marinus infection intensities in fall at Grand Bay. Despite elevated triploid mortalities, both triploids and diploids showed faster shell height growth at Navy Cove and Dauphin Island. Conversely, the site with minimal triploid mortality had the slowest shell height growth overall, but the greatest difference in shell height between triploids and diploids. Analysis of gametogenic stages and gonad-to-body ratios over the summer revealed diploids primarily in the spawning stage and triploids showing delayed and diminished gametogenesis, with lower gonad-to-body ratios than diploids.

The protozoan parasite Perkinsus marinus, which causes dermo disease in Crassostrea virginica, is one of the most ecologically important and economically destructive marine pathogens. The rapid and persistent intensification of dermo in the USA in the 1980s has long been enigmatic. Attributed originally to the effects of multi-year drought, climatic factors fail to fully explain the geographic extent of dermo’s intensification or the persistence of its intensified activity. Here we show that emergence of a unique, hypervirulent P. marinus phenotype was associated with the increase in prevalence and intensity of this disease and associated mortality. Retrospective histopathology of 8355 archival oysters from 1960 to 2018 spanning Chesapeake Bay, South Carolina, and New Jersey revealed that a new parasite phenotype emerged between 1983 and 1990, concurrent with major historical dermo disease outbreaks. Phenotypic changes included a shortening of the parasite’s life cycle and a tropism shift from deeper connective tissues to digestive epithelia. The changes are likely adaptive with regard to the reduced oyster abundance and longevity faced by P. marinus after rapid establishment of exotic pathogen Haplosporidium nelsoni in 1959. Our findings, we hypothesize, illustrate a novel ecosystem response to a marine parasite invasion: an increase in virulence in a native parasite.

The eastern oyster, Crassostrea virginica, is a foundation species within US Gulf of Mexico (GoM) estuaries that has experienced substantial population declines. As changes from management and climate are expected to continue to impact estuarine salinity, understanding how local oyster populations might respond and identifying populations with adaptations to more extreme changes in salinity could inform resource management, including restoration and aquaculture programs. Wild oysters were collected from four estuarine sites from Texas [Packery Channel (PC): 35.5, annual mean salinity, Aransas Bay (AB): 23.0] and Louisiana [Calcasieu Lake (CL): 16.2, Vermilion Bay (VB): 7.4] and spawned. The progeny were compared in field and laboratory studies under different salinity regimes. For the field study, F1 oysters were deployed at low (6.4) and intermediate (16.5) salinity sites in Alabama. Growth and mortality were measured monthly. Condition index and Perkinsus marinus infection intensity were measured quarterly. For the laboratory studies, mortality was recorded in F1 oysters that were exposed to salinities of 2.0, 4.0, 20.0/22.0, 38.0 and 44.0 with and without acclimation. The results of the field study and laboratory study with acclimation indicated that PC oysters are adapted to high-salinity conditions and do not tolerate very low salinities. The AB stock had the highest plasticity as it performed as well as the PC stock at high salinities and as well as Louisiana stocks at the lowest salinity. Louisiana stocks did not perform as well as the Texas stocks at high salinities. Results from the laboratory studies without salinity acclimation showed that all F1 stocks experiencing rapid mortality at low salinities when 3-month oysters collected at a salinity of 24 were used and at both low and high salinities when 7-month oysters collected at a salinity of 14.5 were used.

Since the discovery of Perkinsus marinus as the cause of dermo disease in Crassostrea virginica, salinity and temperature have been identified as the main environmental drivers of parasite prevalence. However, little is known about how these variables affect the movement of parasites from host to water column. In order to elucidate how environmental factors can influence the abundance of this parasite in the water column, we conducted a series of experiments testing the effects of time of day, temperature, and salinity on release of P. marinus cells from infected oysters. We found that P. marinus cells were released on a diurnal cycle, with most cells released during the hottest and brightest period of the day (12:00-18:00). Temperature also had a strong and immediate effect on number of cells released, but salinity did not, only influencing the intensity of infection over the course of several months. Taken together, our results demonstrate that 1) the number of parasites in the water column fluctuates according to a diurnal cycle, 2) temperature and salinity act on different timescales to influence parasite abundance, and 3) live infected oysters may substantially contribute to the abundance of transmissive parasites in the water column under particular environmental conditions.

The alveolate Perkinsus marinus is the most devastating parasite of the eastern oyster Crassostrea virginica. The parasite is readily phagocytosed by oyster hemocytes, but instead of intracellular killing and digestion, P. marinus can survive phagocytosis and divide in host cells. This intracellular parasitism is accompanied by a regulation of host cell apoptosis. This study was designed to gain a better understanding of the molecular mechanisms of apoptosis regulation in oyster hemocytes following exposure to P. marinus. Regulation of apoptosis-related genes in C. virginica, and apoptosis-regulatory genes in P. marinus, were investigated via qPCR to assess the possible pathways involved during these interactions. In vitro experiments were also carried out to evaluate the effect of chemical inhibitors of P. marinus antioxidant processes on hemocyte apoptosis. Results indicate the involvement of the mitochondrial pathway (Bcl-2, anamorsin) of apoptosis in C. virginica exposed to P. marinus. In parallel, the antioxidants peroxiredoxin and superoxide dismutase were regulated in P. marinus exposed to C. virginica hemocytes suggesting that apoptosis regulation in infected oysters may be mediated by anti-oxidative processes. Chemical inhibition of P. marinus superoxide dismutase resulted in a marked increase of reactive oxygen species production and apoptosis in infected hemocytes. The implication of oxygen-dependent apoptosis during P. marinus infection and disease development in C. virginica is discussed.

Hemocytes associated with the mucus lining of pallial (mantle, gill) surfaces of the oyster Crassostrea virginica have been recently suggested to facilitate infection by the Alveolate parasite Perkinsus marinus by mediating the uptake and dispersion of parasite cells. These "pallial hemocytes", which are directly exposed to microbes present in surrounding seawater, are able to migrate bi-directionally between mucosal surfaces and the circulatory system, potentially playing a sentinel role. Interestingly, P. marinus was shown to increase trans-epithelial migration of hemocytes suggesting it may regulate cell motility to favor infection establishment. The purpose of this study was to investigate the effect of P. marinus on hemocyte motility and identify specific molecular mechanisms potentially used by the parasite to regulate hemocyte migration. In a first series of experiments, various components of P. marinus (live P. marinus cells, extracellular products, fragments of P. marinus cell membrane, membrane-modified live P. marinus cells, heat-killed P. marinus) along with components of the opportunistic bacterial pathogen Vibrio alginolyticus (bacterial cells and extracellular products) were investigated for their effects on hemocyte motility. In a second series of experiments, inhibitors of specific molecular pathways involved in motility regulation (Y-27632: inhibitor of Rho-associated protein kinase, RGDS: integrin inhibitor, CK-666: Arp2/3 inhibitor) were used in conjunction with qPCR gene expression experiments to identify pathways regulated by P. marinus exposure. Results showed a specific increase in hemocyte motility following exposure to live P. marinus cells. The increase in motility induced by P. marinus was suppressed by RGDS and CK-666 implicating the involvement of integrins and Arp2/3 in cell activation. Gene expression data suggest that Arp2/3 is possibly regulated directly by an effector produced by P. marinus. The implications of increased hemocyte motility prompted by P. marinus during the early stage of the infection process are discussed.

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.

Risk of infection by parasites can be driven by environmental heterogeneity, often at small scales. We quantified the effect of tidal elevation on infection patterns of two lethal parasites, Perkinsus marinus and Haplosporidium nelsoni , in an important coastal species, the eastern oyster, Crassostrea virginica . Within the southeastern US, oysters in Georgia and South Carolina are rarely found in the subtidal zone. Historically, it has been hypothesized that this pattern could be due to the increased exposure of hosts to waterborne parasites. We manipulated oysters at two tidal elevations (intertidal and subtidal) in a Georgia estuary to test if P. marinus , H. nelsoni , and co-infection by both parasites were different between tidal heights. We found that though P. marinus prevalence and co-infection prevalence of both parasites were not significantly different between tidal elevations, as has been found previously, P. marinus intensity and H. nelsoni prevalence were significantly higher intertidally than subtidally. These findings show that parasite infections can be higher in the host's natural (preferred) tidal height, and suggest that longer exposure to parasites in the subtidal is not a likely reason for the paucity of oysters at that tidal elevation in certain regions of the southeastern US. More broadly, our results provide further evidence that environmental effects on host-parasite interactions can vary by parasite species and across small (meter-long) spatial scales.

A 2007 study of Suminoe (Crassostrea ariakensis) and eastern (Crassostrea virginica) oysters exposed to Indian River Lagoon (IRL), Florida waters revealed the presence of Bonamia spp. in exposed oysters. In 2010, a survey of oysters and other bivalves (n = 721, 18 species) was conducted in the southern IRL (10 sites) and in the more southerly Lake Worth Lagoon (LWL, 3 sites). Polymerase chain reaction (PCR) was used to assess the prevalence of three oyster parasites, Bonamia spp., Perkinsus marinus, and Haplosporidium nelsoni, in collected samples. Histology, fluorescent in situ hybridization (FISH) and standard H&E staining, was used to confirm PCR-positive Bonamia results. Bonamia spp. was detected in eight species (Brachidontes exustus, Crassostrea virginica, Geukensia demissa, Ischadium recurvum, Isognomon alatus, Isognomon bicolor, Mercenaria campechiensis, and Ostrea stentina). The prevalence was 17.3% in the IRL and 9.9% in LWL with 66% of PCR-positive samples subsequently identified as either Bonamia exitiosa or Bonamia perspora. The presence of Bonamia spp. was confirmed by FISH in C. virginica, O. stentina, I. alatus, I. bicolor, and G. demissa, although only C. virginica and O. stentina showed evidence of infection. The presence of P. marinus was detected in eight species (Atrina rigida, B. exustus, C. virginica, G. demissa, I. recurvum, I. alatus, I. bicolor, and O. stentina). The prevalence was 12.4% in the IRL and 7.7% in LWL. The presence of MSX was not detected in any of the collected bivalves. This study confirms the presence of Bonamia spp. and P. marinus in Florida east coast waters and widens the described geographic range for Bonamia spp., and provides further evidence that MSX is unlikely to be found in Florida waters

Both vertebrates and invertebrates display active innate immune mechanisms for defense against microbial infection, including diversified repertoires of soluble and cell-associated lectins that can effect recognition and binding to potential pathogens, and trigger downstream effector pathways that clear them from the host internal milieu. Galectins are widely distributed and highly conserved lectins that have key regulatory effects on both innate and adaptive immune responses. In addition, galectins can bind to exogenous (“non-self”) carbohydrates on the surface of bacteria, enveloped viruses, parasites, and fungi, and function as recognition receptors and effector factors in innate immunity. Like most invertebrates, eastern oysters (Crassostrea virginica) and softshell clams (Mya arenaria) can effectively respond to most immune challenges through soluble and hemocyte-associated lectins. The protozoan parasite Perkinsus marinus, however, can infect eastern oysters and cause “Dermo” disease, which is highly detrimental to both natural and farmed oyster populations. The sympatric Perkinsus chesapeaki, initially isolated from infected M. arenaria clams, can also be present in oysters, and there is little evidence of pathogenicity in either clams or oysters. In this review, we discuss selected observations from our studies on the mechanisms of Perkinsus recognition that are mediated by galectin-carbohydrate interactions. We identified in the oyster two galectins that we designated CvGal1 and CvGal2, which strongly recognize P. marinus trophozoites. In the clam we also identified galectin sequences, and focused on one (that we named MaGal1) that also recognizes Perkinsus species. Here we describe the biochemical characterization of CvGal1, CvGal2, and MaGal1 with focus on the detailed study of the carbohydrate specificity, and the glycosylated moieties on the surfaces of the oyster hemocytes and the two Perkinsus species (P. marinus and P. chesapeaki). Our goal is to gain further understanding of the biochemical basis for the interactions that lead to recognition and opsonization of the Perkinsus trophozoites by the bivalve hemocytes. These basic studies on the biology of host-parasite interactions may contribute to the development of novel intervention strategies for parasitic diseases of biomedical interest.

The parasite Perkinsus marinus, which causes Dermo disease, affects Eastern oyster (Crassostrea virginica) survival and is responsible for substantial losses to aquaculture production. Controlled laboratory challenges enable definition and measurement of disease response phenotypes as well as characterization of gene expression patterns during the infection process. The method of parasite exposure and severity of infection likely affect response measures and exploring variation among them can enhance our understanding of this host-parasite interaction. Here we compared response traits and global expression patterns over time for a selectively bred Eastern oyster family exposed to P. marinus via either suspension feeding or direct injection to elucidate mechanisms determining survival. All exposed oysters (n = 56 per group) received a dose of 5 × 106 cultured P. marinus cells g−1 wet tissue weight but oysters in the injected group had higher initial parasite load and significantly reduced survival probability compared to the fed group. Using RNAseq, we identified differentially expressed transcripts between fed and control animals 6 h after exposure and between fed and injected groups at 36 h, 7 d, and 28 d post exposure. Immediately after infection, caspase-8 and transcripts associated with mediating cell damage from reactive oxygen species were upregulated in the fed group compared to unchallenged controls. The comparison between fed and injected groups early in the infection process revealed increased innate immune activity and overexpression of transcripts involved in proinflammatory cytokine pathways, including toll-like receptor 4 and TNF receptor-associated factor 6-like protein, in the injected group. Later in the infection process, the fed group expressed transcripts associated with growth, specifically microtubule-based processes, at a higher level than the injected group. This study demonstrates that dosing method influences initial parasite load, survival, and transcriptomic response to Dermo disease and highlights the importance of considering exposure method when interpreting interactions between P. marinus and the Eastern oyster.

Dermo disease caused by the protistan Perkinsus marinus in Eastern oysters Crassotrea virginica is an important source of mortality impacting oyster population dynamics resulting in substantial losses in fisheries and aquaculture. The rapid transmission and spread of the disease minimized the importance of transmission models and past models (proliferation-based models) assumed simple density-dependent transmission or rapid infection post-settlement. This approach is a good approximation only for low population densities. A transmission model was developed for P. marinus in Eastern oysters that accounts for the seasonal change in disease dynamics and density-dependent foraging (of suspended particles) interference among hosts. The model, verified and evaluated against field observations, incorporates parasite release to the water column from live and dead individuals, parasite consumption by living oysters, the diffusion of parasites in the water, body burden-based dose-dependent transmission, recruitment, and disease-caused mortality. The model returns a basic reproduction number R 0 for Dermo much greater than unity ( R 0 = 90) in accordance with the current persistence and pandemic nature of this disease in oysters. No population density is obtained that is low enough to suppress R 0 below 1 (i.e. disease extinction). R 0 is also estimated for high oyster densities (>300 individuals m −2 ) and particularly for relatively large oysters (∼90 mm), today rare but once common before generalized overfishing occurred on healthy oyster reefs. In this scenario, R 0 drops below 1, indicating that high oyster density can limit disease invasion through foraging interference and depletion of parasites in the water column. High intensity recruitment events allow the oyster population to attain such densities and limit the development of epizootics. These results provide insight into the transition from past populations, where Dermo is inferred to have been limited in its impact, to the current persistent and pandemic nature of this disease. Further coupling of this model into metapopulation and hydrodynamic models could be a promising tool to support management decision-making for bivalve populations impacted by Dermo disease.

Eastern oyster, Crassostrea virginica , aquaculture has expanded greatly in recent years, but further growth of the industry is constrained by disease-related losses. Oyster breeding programs supporting the oyster aquaculture industry along the east coast of the US have targeted resistance to three prominent diseases: MSX, Dermo, and ROD, caused by Haplosporidium nelsoni , Perkinsus marinus , and Roseovarius crassostreae respectively. Consequently, selected oyster lines possess some level of resistance and/or tolerance but the extent to which these lines, derived from various programs, perform across diverse growing environments used by industry has not been tested. The performance of six selected eastern oyster lines was evaluated at five sites along the east coast of the US (Maine to Virginia) to 1) identify differences in performance among lines at each site, and 2) identify lines that perform well across all sites. Performance measures included growth, mortality, and yield over a 15-month evaluation period. During unusually high mortality events, subsets of oysters were processed for disease diagnosis. Growth trajectories were similar among lines within a site, but varied significantly across sites (78% of random variance explained). Oysters grown in Rhode Island were largest while oysters grown in Maine were smallest at the end of the study. Mortality varied greatly among lines at each site as well as among sites. Line × site interaction explained 61% of the total random variance in the mortality data. In Maine, extensive mortality was observed early in the year for all lines, coincident with increased ROD prevalence. In New Jersey and Virginia, unusually high mortality was evident in the UMFS, Clinton, and NEH-RI lines during the final months of the experiment when the prevalence of both Dermo and MSX were 100% and Statement of relevance First to evaluate multiple oyster lines across diverse sites.

The National Oceanic and Atmospheric Administration (NOAA) Status and Trends Mussel Watch Program sampled the largest oysters in nearly every major US coastal lagoon and estuary in the Gulf of Mexico once during the winter from 1986 to 2010. This contribution examines trends in the principal oyster disease in the Gulf of Mexico, Dermo, caused by Perkinsus marinus, and some related population dynamic characteristics for its host, Crassostrea virginica. During the 1986–2000 period, P. marinus prevalence and infection intensity and oyster population dynamics followed the El Nino-Southern Oscillation (ENSO) cycle, responding to variations in salinity caused by variations in rainfall and freshwater inflow. The ENSO signal in the oyster population effectively ceased circa 2002. Beginning around this time, wintertime P. marinus prevalence and weighted prevalence began a decadal decline, as did the length of the largest oysters and the fraction of these largest animals that were female. The trends in Dermo disease, oyster length, and oyster sex ratio are all consistent with the following hypothesis: increasing temperature during the 2000s resulted in an increase in P. marinus infection intensity sufficient to increase the mortality rate in late summer and fall in the larger animals. This simultaneously reduced Dermo prevalence and infection intensity in the winter at the time of sampling and also resulted in the decline in the length of the largest animals targeted by Mussel Watch. Coincident with the decline in length is the expected decline in the fraction female, such that the percent female in the largest animals dropped to ≤50 % throughout much of the Gulf of Mexico. The decline in length leading to fewer large animals reproducing and the loss of females are predicted to have reduced oyster population reproductive capacity substantially during the 2000s. The early 60 % of the Mussel Watch time series took place during a period of negative Atlantic Multidecadal Oscillation (AMO) indices. The AMO moved into positive territory circa 2000. A positive AMO index is consistent with observed warmer water temperatures, and increased water temperature is consistent with an increase in Dermo-induced mortality.

14 páginas, 1 figura, 3 tablas.-- Simone Sühnel ... et al., The protozoans Perkinsus marinus, Perkinsus olseni, Bonamia ostreae, Bonamia exitiosa, and Marteilia refringens are responsible for some of the most detrimental diseases in the production of cultivated shellfish worldwide and are classified as notifiable diseases by the World Organization for Animal Health (OIE). This study examined the general health status of wild and cultured bivalves from southern Brazil and included diagnostic tests for the presence of Perkinsus sp., Bonamia sp., andMarteilia sp. Cultured bivalves included the mangrove oyster Crassostrea gasar (syn. Crassostrea brasiliana), the brown mussel Perna perna, the lion's paw scallop Nodipecten nodosus, andwing pearl oyster Pteria hirundo. Themangrove oyster Crassostrea rhizophorae and the carib pointed venus clam Anomalocardia brasiliana (syn. Anomalocardia flexuosa) were collected from wild populations. A variety of parasitic or commensal organisms were detected by histology including Ancistrocoma-like and Spenophrya-like organisms; Bucephalus genus, Nematopsis sp., Steinhausia sp., and Tylocephalum sp.; unidentified trematode; unknown protozoan and metazoans; and an amoeba parasite. Hemocytic infiltration was most commonly associated with parasitized animals. Histological, culture and molecular diagnostic tests did not find any evidence of the presence of OIE-listed pathogens or related species in this study. Although the current survey did not identify any pathogens or diseases of concern, it provides baseline health assessment data for these species against which any future disease developments or significant changes in population health can be compared. These data are also valuable with respect to the development and implementation of public policies related to aquatic animal health, We would like to thank to the Ministry of Fisheries and Aquaculture, Brazil, for the research support provided to the Fundaçao de Amparo à Pesquisa do Estado de Santa Catarina (FAPESC), Brazil; the National Council of Technological and ScientificDevelopment, Brazil (CNPq-558222/2008-0), for supporting themacro- andmicroscopical analysis and for providing a grant (CNPq-301072/2007-8); the Coordenaçao de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES), Brazil, for post-doctoral scholarship support for SS; the Pacific Biologic Station and Vancouver Island University (VIU) in Canada for supporting the molecular studies

Disease caused by the protozoan parasite Perkinsus marinus remains a challenge for fisheries and aquaculture of the eastern oyster, Crassostrea virginica. Near infrared reflectance (NIR) spectroscopy is a technology that allows measurements of multiple biochemical components at the same time, providing extensive information for every tested sample using a single, high throughput, rapid assay. Adding another parameter such as determination of P. marinus level of infection may be of great interest to assess, along with other biochemical parameters, oyster's general condition and also characterize oysters that present resistance or tolerance to this problematic parasite. During this study, the NIR spectroscopy technology was used to test the feasibility of an alternative means of quantitative detection of P. marinus in the eastern oyster. In order to accomplish this, two experiments were performed: a P. marinus “spike” experiment and a natural infection study. During the spike experiment, pathogen-free, homogenized C. virginica slurries were spiked with known concentrations of cultured P. marinus cells. NIR spectroscopy spectra were collected on these preparations as is (wet) and after freeze drying (dry). During the natural infection study, oysters from the upper portion of the Rappahannock River were transferred to the York River, where P. marinus is endemic. Oysters were subsampled monthly for 7 months and diagnosed for the presence and intensity of acquired infections. NIR spectroscopy spectra were collected on freeze dried samples and P. marinus concentration was determined using quantitative PCR (qPCR). Calibration models were developed using modified partial least squares regression (PLS) based on cross-validation and tested using a validation set. Comparison of reference values to predicted values showed a high correlation for all models: for the spike experiment reaching a R2val of 0.70 and 0.93 for the wet and dry model respectively; and for the natural infection model reaching a R2val of 0.81 for the dry model. These metrics indicate that P. marinus cells present a quantifiable spectral signature against the background of C. virginica oyster tissues and implies that NIR spectroscopy is a promising technology to detect protozoan infection in animal tissues. This technology seems unlikely to replace qPCR or other classical methods like RFTM, primarily because of the amount of preparation needed to effect NIR sampling. However, NIR spectroscopy is used for assessing other facets of oyster condition simultaneously, such as glycogen and lipid, and may be used to obtain reasonable estimates of P. marinus infection when more traditional methods could not or were not used.

Mass mortalities of cultured triploid Crassostrea virginica in late spring, or “triploid mortality,” have been reported on farms in the Chesapeake Bay since 2012. Typical causes, such as disease or poor husbandry, were not responsible, and mortalities occurred without clear signs of biological or physical stressors. Previous comparisons of the effects of genetic origin on triploid mortality have been uncontrolled, initiating this investigation of the effect of brood stock source and ploidy on triploid mortality. Four triploid and four diploid crosses, produced by crossing different combinations of brood stock of Virginia, Louisiana, and Maine origin in early 2015, were tested at four commercial oyster farms in Virginia throughout 2016. From February to November, oysters from all crosses and sites were regularly sampled, and growth (shell height), condition (meat weight), and pathology were monitored, as were environmental conditions. Compared to diploids made from Virginia brood stock, diploids with Maine genetic origin had high mortality and Virginia-Maine diploid hybrids exhibited mid-parent heterosis. A triploid mortality event occurred in late spring at only one site and only affected the triploid crosses. Evidence for substantial disease pressure from Haplosporidium nelsoni or Perkinsus marinus or of especially stressful environmental conditions based on temperature, salinity, pH, and dissolved oxygen was absent during the triploid mortality event. At 18 months, shell height was similar in the diploids and triploids with the most similar genetic origin. Triploids maintained meat weight through the summer, while meat weight in diploids dropped sharply. Triploids may be especially susceptible to late spring mortality events in the Chesapeake Bay, which justifies their classification as “triploid mortality,” and warrants further investigation on traits in triploids that may affect susceptibility.

Perkinsus spp. have been detected in various bivalve species from north-east Brazil. Santa Catarina is a South Brasil state with the highest national oyster production. Considering the pathogenicity of some Perkinsus spp., a study was carried out to survey perkinsosis in two oyster species cultured in this State, the mangrove oyster Crassostrea gasar and the Pacific oyster Crassostrea gigas. Sampling involved eight sites along the state coast, and oyster sampling was collected during the period between January 2013 and December 2014. For the detection of Perkinsus, Ray's fluid thioglycollate medium (RFTM) and histology were used, and for the identification of the species, PCR and DNA sequencing were used. Perkinsus spp. was found by RFTM in C. gigas and C. gasar from Sao Francisco do Sul. This pathology was also detected in C. gasar from Balneario Barra do Sul both, by RFTM and histology. Perkinsus marinus was identified in C. gigas and C. gasar from Sao Francisco do Sul and Perkinsus beihaiensis in C. gasar from Balneario Barra do Sul. This is the first report of P. marinus in C. gigas from South America. Results of this preliminary study suggest that both oyster species tolerate the species of Perkinsus identified, without suffering heavy lesions.

Protozoan pathogens represent a serious threat to oyster aquaculture, since they can lead to significant production loses. Moreover, oysters can concentrate human pathogens through filter feeding, thus putting at risk raw oyster consumers' health. Using PCR-based assays in oysters (Crassostrea virginica) from Maine, we expand the Northeast range in the USA for the protozoans Perkinsus marinus, Perkinsus chesapeaki, and Haplosporidium nelsoni, and report for the first time the detection of the human pathogens Toxoplasma gondii and Cryptosporidium parvum. Oysters hosting both P. marinus and P. chesapeaki were more than three times as likely to be infected by a non-Perkinsus than those free of Perkinsus infections.

The Mussel Watch Program sampled bays on the East, Gulf, and West coasts of the United States over a period of 16 y. Analytical protocols included the recording of parasites and pathologies. Oysters (Crassostrea virginica) harbored significantly more parasitic taxa than mussels (Mytilidae). Cases where body burden was higher in mytilids were exclusively eukaryotic parasites, trematode metacercariae and trematode sporocysts. Oysters had higher body burdens of Nematopsis, alimentary tract ciliates, prokaryotic inclusions, and a number of unique taxa including haplosporidians, Perkinsus marinus, cestodes, and nematodes. Major pathologies were much more common in mytilids. For oysters, many parasitic taxa were more common in the Gulf of Mexico, including Nematopsis, P. marinus, trematode sporocysts, and nematodes. For mytilids, most parasites and pathologies were more common on the East Coast. Most parasite distributions were clinal on the East and West coasts, with clear relationships to well-known ...

An immunoprecipitation assay was developed for measuring instantaneous reproductive effort in female American oysters, Crassostrea virginica. Oysters were injected with 14C-leucine and incubated in situ for 1 to 30 h periodically throughout the annual gametogenic cycle. Gonadal protein labeled with 14C-leucine was precipitated from an oyster homogenate with rabbit anti-oyster egg IgG as the primary antibody. Antibody-oyster egg protein complex was further purified by immunoadsorption with staphylococcal protein A cell suspension. The quantity of oyster eggs was determined by single-ring immunodiffusion. A mathematical model was developed to calculate the instantaneous reproductive rate of oysters and to estimate the number of days required from the initiation of gonadal development to spawning. The oyster population was lightly to moderately infected with a protozoan parasite, Perkinsus marinus. A negative correlation between the intensity of infection and the rate of gonadal production suggests that P. marinus retards the rate of gamete development. The seasonal cycle of gamete production determined by direct measurements of egg protein production was not equivalent to that determined by standard gonadal-somatic index (GSI), except at the most basic level. GSI was highest during the spring spawning peak, but the rate of gamete production was highest in the fall. Accordingly, the two measurements, rate versus standing crop (volume of gonad), reveal a substantially different picture about the details of the spawning season. Estimates of the time required to reach spawning condition ranged from several weeks to 1 or 2 months; these values agree with published estimates derived from less direct methods. Direct rate measurements thus seem to accurately reflect the true rate at which gametic tissue is produced in the field. A positive correlation between oyster size and the estimated days to spawn suggests that larger oysters require longer to prepare to spawn. Furthermore, the range in observed somatic and gametic growth emphasizes the conservatism of somatic growth and the volatility of gonadal growth that is borne out by the results of population dynamics models of oysters.

Crassostrea rhizophorae and C. gasar oysters are cultivated in the northeast region. Perkinsus parasites infect bivalves, and their effects on oysters from tropical regions are poorly understood. This study evaluated the impact of Perkinsus infection on the productive traits of native oysters. Oysters were sampled bimonthly during 7 months, from July 2010 to February 2011, to evaluate growth rate, mortality and shell color patterns (white and dark-gray) (n = 500), and to determine the prevalence and intensity of Perkinsus (n = 152). Perkinsus and Crassostrea species were determined using molecular tools. Results showed that most dark-gray (90%, n = 20) and white (67%, n = 18) oysters were C. gasar and C. rhizophorae, respectively. Oysters showed a high growth rate and moderate cumulative mortality (44%). C. gasar oysters grew better and showed lower mortality and lower incidence of Perkinsus compared to C. rhizophorae. The mean prevalence of Perkinsus was moderate (48%), but the infection intensity was light (2.2). Perkinsosis affected very small oysters (19.4 mm). In conclusion, native oysters, especially C. gasar, have a great potential for culture, mortality is not associated with perkinsosis, and the shell color of oysters can be used to improve selection for spats with better performance. Resumo Crassostrea rhizophorae e C. gasar são cultivadas na região Nordeste. Parasitas Perkinsus infectam bivalves e seus efeitos em ostras de regiões tropicais são pouco compreendidos. Este estudo avaliou o impacto da infecção por Perkinsus em parâmetros de produção de ostras nativas. Ostras foram coletadas bimestralmente durante 7 meses, de julho de 2010 a fevereiro de 2011, para avaliar crescimento, mortalidade e padrão de coloração da concha (branca e cinza-escura) (n = 500); além da presença e intensidade de Perkinsus (n = 152). Perkinsus e Crassostrea foram identificados por abordagem molecular. Os resultados mostraram que as ostras cinza-escuras (90%, n = 20) e brancas (67%, n = 18) eram C. gasar e C. rhizophorae, respectivamente. As ostras mostraram uma boa taxa de crescimento e mortalidade acumulada moderada (44%). C. gasar cresceu melhor com menor mortalidade e menor incidência de Perkinsus que C. rhizophorae. A prevalência média de Perkinsus foi moderada (48%), mas a intensidade de infecção foi leve (2,2). A perkinsiose afetou ostras pequenas (19,4 mm). Em conclusão, ostras nativas, especialmente C. gasar, têm grande potencial de produção; sem mortalidade associada à perkinsiose; e, a cor da concha pode ser usada para melhorar a seleção de sementes com melhor desempenho.