Studies on the oyster pathogen Perkinsus marinus (Apicomplexa): Interactions with host defenses of Crassostrea virginica and Crassostrea gigas, and in vitro propagation

The disease caused by the protozoan Perkinsus marinus has been a major source of mortality in the eastern oyster, Crassostrea virginica. Variations in susceptibility to P. marinus infection among eastern oysters collected from the Chesapeake Bay and Gulf of Mexico, as well as between eastern and Pacific (Crassostrea gigas) oysters were determined. Since oyster host defense may play a role in determining susceptibility to pathogen infection, cellular and humoral defense activities of the oyster and their interactions with P. marinus were investigated. Procedures also had to be established to isolate, purify, and propagate in vitro, P. marinus. Eastern oysters from all sites were found to be highly susceptible to the pathogen. Cellular and humoral activities were significantly affected by heavy intensity of P. marinus infection. Prevalence and intensity of P. marinus infection were lower in Pacific oysters than in eastern oysters. Pacific oysters may offer a less favorable environment for the development of P. marinus compared to eastern oysters for at least two possible reasons: the elevated cellular and humoral activities may degrade the parasite more effectively, and lower plasma protein levels may limit parasite growth. Incubation of merozoites with hemocytes of eastern and Pacific oysters in vitro suggested that limited intracellular killing of P. marinus occurred but that killing was not mediated by oxygen metabolites. Perkinsus marinus was successfully propagated in vitro in a culture medium containing most of the known constituents of cell-free oyster hemolymph. Cultures of the parasite were initiated from heart fragments of infected oysters. The cultured protozoan was similar in morphology to P. marinus, enlarged in fluid thioglycollate medium, reacted with polyclonal antibodies raised against hypnospores and was infective. Continuous cultures of P. marinus could also be initiated from hypnospores. Two types of division, progressive cleavage and successive bipartition of the mother cell protoplast, were observed

Predicting the impacts of Mississippi River diversions and sea-level rise on spatial patterns of eastern oyster growth rate and production

© 2017 There remains much debate regarding the perceived tradeoffs of using freshwater and sediment diversions for coastal restoration in terms of balancing the need for wetland restoration versus preserving eastern oyster (Crassostrea virginica) production. Further complicating the issue, climate change-induced sea-level rise (SLR) and land subsidence are also expected to affect estuarine water quality. In this study, we developed a process-based numerical modeling system that couples hydrodynamic, water quality, and oyster population dynamics. We selected Breton Sound Estuary (BSE) (∼2740 km2) in the eastern Mississippi River Deltaic Plain since it is home to several of the largest public oyster seed grounds and private leases for the Gulf coast. The coupled oyster population model was calibrated and validated against field observed oyster growth data. We predicted the responses of oyster population in BSE to small- (142 m3 s−1) and large-scale (7080 m3 s−1) river diversions at the Caernarvon Freshwater Diversion structure planned in the 2012 Coastal Master Plan (Louisiana) under low (0.38 m) and high (1.44 m) relative sea-level rise (RSLR = eustatic SLR + subsidence) compared to a baseline condition (Year 2009). Model results showed that the large-scale diversion had a stronger negative impact on oyster population dynamics via freshening of the entire estuary, resulting in reduced oyster growth rate and production than RSLR. Under the large-scale diversion, areas with optimal oyster growth rates (\u3e15 mg ash-free dry weight (AFDW) oyster−1 wk−1) and production (\u3e500 g AFDW m−2 yr−1) would shift seaward to the southeastern edge of the estuary, turning the estuary into a very low oyster production system. RSLR however played a greater role than the small-scale diversion on the magnitude and spatial pattern of oyster growth rate and production. RSLR would result in an overall estuary-wide decrease in oyster growth rate and production as a consequence of decreased salinities in the middle and lower estuary because rising sea level likely causes increased stage and overbank flow downstream along the lower Mississippi River

Perkinsus marinus susceptibility and defense-related activities in eastern oysters Crassostrea virginica: temperature effects

he relationship of potential defense-related cellular and humoral activities and the sus- ceptibility of eastern oysters Crassostrea virginica to the parasite Perkinsus marinus were examined at 10, 15, 20 and 25 C. Oysters were acclimated at experimental temperatures for 20 d and then chal- lenged with R marinus. Total hemocyte counts (TC) and percentage of granulocytes (PG) 20 d after temperature acclimation were higher in oysters at high than at low acclimation temperature. Higher protein (P) and lysozyme (L) concentrations were found in oysters at 10 and 15 C. No significant differ- ences in hemagglutination (H) titers due to temperature acclimation were observed. Infection preva- lence 46 d after challenge by R marinus was 100, 91, 46 and 23 % respectively, for oysters at 25, 20, 15 and 10 C. Disease intensity increased with temperature. Oysters at higher temperatures had greater PG and TC and hemocyte phagocytic activity. No difference was found in TC and PG between control and challenged oysters within each temperature treatment. Bleeding may to some extent reduce TC and PG in oysters. P did not vary much among temperatures. No reduction of P in oysters was found due to P. marinuschallenge and infection. L tended to be higher in oysters at lower than at higher treat- ment temperatures. The oysters at 10 C had the highest L concentration and lowest P marinus infec- tion. But, it is not known whether the high extracellular L in oysters at 10 DC is attributable to the low R marinus susceptibility in these oysters. There was no significant difference in condition index (Cl) between control and challenged oysters and between infected and uninfected oysters. However, C1 de- creased with increasing temperatures. The H titers were not associated with any measured variables. The greater TC, PG, and phagocytic capability in oysters at higher temperatures did not result in fewer or less intense P marinus infections

Characterizing the Epigenetic and Transcriptomic Responses to Perkinsus marinus Infection in the Eastern Oyster Crassostrea virginica

© Copyright © 2020 Johnson, Sirovy, Casas, La Peyre and Kelly. Eastern oysters in the northern Gulf of Mexico are routinely infected with the protistan parasite Perkinsus marinus, the cause of the disease commonly known as dermo. Recent experimental challenges among Atlantic coast populations have identified both resistant and susceptible genotypes using comparative transcriptomics. While controlled experimental challenges are essential first assessments, expanding this analysis to field reared individuals provides an opportunity to identify key genomic signatures of infection that appear both in the laboratory and in the field. In this study we combined reduced representation bisulfite sequencing with 3′ RNA sequencing (Tag-seq) to describe two molecular phenotypes associated with infection in oysters outplanted at a common garden field site. These combined approaches allowed us to examine changes in DNA methylation and gene expression for a large number of individuals (n = 40) that developed infections during the course of a common garden outplant experiment. Our epigenetic analysis of DNA methylation identified significant changes in gene body methylation associated with increasing infection intensity, across genes associated with immune responses. There was a smaller transcriptomic response to increasing infection intensities with 32 genes showing differential expression; however, only 40% of these genes were found to also be differentially methylated. While there was no clear pattern between direction of differential methylation and gene expression, there was a significant effect of percent methylation on gene-by-gene expression levels and the coefficient of variation in gene body methylation between treatments. These results show that in C. virginica, heavily methylated genes have high levels of gene expression with low levels of variation. Comparing our differential expression results with previously published experimental P. marinus challenges identified overlapping expression patterns for genes associated with C1q-domain-containing and V-type proton ATPase proteins. Through our comparative transcriptomic approach using field reared individuals and co-expression network analysis we have also been able to identify a network of genes that change in expression in response to infection. These combined analyses provide evidence for a conserved response to P. marinus infections across infection intensities and suggest that DNA methylation may not be a reliable predictor of differential gene expression in long-term infections

A new lysozyme from the eastern oyster, Crassostrea virginica, and a possible evolutionary pathway for i-type lysozymes in bivalves from host defense to digestion

Background. Lysozymes are enzymes that lyse bacterial cell walls, an activity widely used for host defense but also modified in some instances for digestion. The biochemical and evolutionary changes between these different functional forms has been well-studied in the c-type lysozymes of vertebrates, but less so in the i-type lysozymes prevalent in most invertebrate animals. Some bivalve molluscs possess both defensive and digestive lysozymes. Results. We report a third lysozyme from the oyster Crassostrea virginica, cv-lysozyme 3. The chemical properties of cv-lysozyme 3 (including molecular weight, isoelectric point, basic amino acid residue number, and predicted protease cutting sites) suggest it represents a transitional form between lysozymes used for digestion and immunity. The cv-lysozyme 3 protein inhibited the growth of bacteria (consistent with a defensive function), but semi-quantitative RT-PCR suggested the gene was expressed mainly in digestive glands. Purified cv-lysozyme 3 expressed maximum muramidase activity within a range of pH (7.0 and 8.0) and ionic strength (I = 0.005-0.01) unfavorable for either cv-lysozyme 1 or cv-lysozyme 2 activities. The topology of a phylogenetic analysis of cv-lysozyme 3 cDNA (full length 663 bp, encoding an open reading frame of 187 amino acids) is also consistent with a transitional condition, as cv-lysozyme 3 falls at the base of a monophyletic clade of bivalve lysozymes identified from digestive glands. Rates of nonsynonymous substitution are significantly high at the base of this clade, consistent with an episode of positive selection associated with the functional transition from defense to digestion. Conclusion. The pattern of molecular evolution accompanying the shift from defensive to digestive function in the i-type lysozymes of bivalves parallels those seen for c-type lysozymes in mammals and suggests that the lysozyme paralogs that enhance the range of physiological conditions for lysozyme activity may provide stepping stones between defensive and digestive forms. © 2010 Xue et al; licensee BioMed Central Ltd

A Shell-Neutral Modeling Approach Yields Sustainable Oyster Harvest Estimates: A Retrospective Analysis of the Louisiana State Primary Seed Grounds

A numerical model is presented that defines a sustainability criterion as no net loss of shell, and calculates a sustainable harvest of seed (\u3c75 mm) and sack or market oysters (\u3e= 75 mm). Stock assessments of the Primary State Seed Grounds conducted east of the Mississippi from 2009 to 2011 show a general trend toward decreasing abundance of sack and seed oysters. Retrospective simulations provide estimates of annual sustainable harvests. Comparisons of simulated sustainable harvests with actual harvests show a trend toward unsustainable harvests toward the end of the time series. Stock assessments combined with shell-neutral models can be used to estimate sustainable harvest and manage cultch through shell planting when actual harvest exceeds sustainable harvest. For exclusive restoration efforts (no fishing allowed), the model provides a metric for restoration success namely, shell accretion. Oyster fisheries that remove shell versus reef restorations that promote shell accretion, although divergent in their goals, are convergent in their management; both require vigilant attention to shell budgets

Defining optimal freshwater flow for Oyster production: Effects of freshet rate and magnitude of change and duration on eastern oysters and perkinsus marinus infection

In coastal Louisiana, the development of large-scale freshwater diversion projects has led to controversy over their effects on oyster resources. Using controlled laboratory experiments in combination with a field study, we examined the effects of pulsed freshwater events (freshet) of different magnitude, duration, and rate of change on oyster resources. Laboratory and field evidence indicate that low salinity events (\u3c5 psu) decreased Perkinsus marinus infection intensities. Furthermore, when salinity was low (\u3c5 psu), parasite infection intensities continued to decrease even as temperatures exceeded 20°C. At the same time, oyster growth was positively correlated with salinity. To maximize oyster production, data indicate that both low and high salinity events will be necessary. © 2009 Coastal and Estuarine Research Federation

Improved method for the initiation of continuous cultures of the oyster pathogen perkinsus marinus (apicomplexa)

A simple method for initiating continuous cultures of Perkinsus marinus with hypnospores is described. The visceral mass of an infected eastern oyster Crassostrea virginica was first incubated in Ray’s fluid thioglycollate medium in order to produce large hypnospores from the smaller histozoic stages of the protozoan. Hypnospores were then purified and transferred into the eulture medium JL-ODRP-1 where, within a relatively short period of time, they further divided by either progressive cleavage or successive bipartition. These divisions produced large numbers of merozoites and flagellated cells. This two-step procedure surpassed the previously described method of initiating cultures from hearts of infected eastern oysters because a much higher number of purified, larger P. marinus cells could be obtained from a single infected eastern oyster. © 1995 Taylor & Francis Group, LLC

Identifying factors inducing trophozoite differentiation into hypnospores in Perkinsus species

Trophozoites of species of Perkinsus in host tissues readily differentiate into hypnospores when incubated in Ray\u27s fluid thioglycollate medium (RFTM). In contrast, hypnospores have rarely been observed in vivo, and when reported they have been associated with dying hosts. The objective of this study was to determine what altered environmental conditions trigger the differentiation of Perkinsus trophozoites into hypnospores. In the first part of the study, cultured P. chesapeaki trophozoites were exposed to lowered oxygen, acidic pH, increased nutrient levels, heat shock, or osmotic shock conditions, and hypnospore density was measured. Acidic pH, lowered oxygen, or increased nutrient levels significantly increased P. chesapeaki hypnospore formation. In the second part of the study, P. olseni and P. marinus trophozoites were exposed to acidic pH, lowered oxygen, or increased nutrient levels resulting in hypnospore formation in P. olseni but not P. marinus. This study demonstrated that changes in environmental conditions consistent with changes expected in decaying tissues or with RFTM incubation induce trophozoite differentiation. The response of the cultured trophozoites varied between species and between isolates of the same species. © 2012 Elsevier GmbH