Co-phylogeographic structure in a disease-causing parasite and its oyster host.

With the increasing affordability of next-generation sequencing technologies, genotype-by-sequencing has become a cost-effective tool for ecologists and conservation biologists to describe a species' evolutionary history. For host­parasite interactions, genotype-by-sequencing can allow the simultaneous examination of host and parasite genomes and can yield insight into co-evolutionary processes. The eastern oyster, Crassostrea virginica, is among the most important aquacultured species in the United States. Natural and farmed oyster populations can be heavily impacted by 'dermo' disease caused by an alveolate protist, Perkinsus marinus. Here, we used restricted site-associated DNA sequencing (RADseq) to simultaneously examine spatial population genetic structure of host and parasite. We analysed 393 single-nucleotide polymorphisms (SNPs) for P. marinus and 52,100 SNPs for C. virginica from 36 individual oysters from the Gulf of Mexico (GOM) and mid-Atlantic coastline. All analyses revealed statistically significant genetic differentiation between the GOM and mid-Atlantic coast populations for both C. virginica and P. marinus, and genetic divergence between Chesapeake Bay and the outer coast of Virginia for C. virginica, but not for P. marinus. A co-phylogenetic analysis confirmed significant coupled evolutionary change between host and parasite across large spatial scales. The strong genetic divergence between marine basins raises the possibility that oysters from either basin would not be well adapted to parasite genotypes and phenotypes from the other, which would argue for caution with regard to both oyster and parasite transfers between the Atlantic and GOM regions. More broadly, our results demonstrate the potential of RADseq to describe spatial patterns of genetic divergence consistent with coupled evolution.

First report of Perkinsus marinus occurrence associated with wild Pacific oysters Crassostrea gigas from the west coast of Korea.

This study reports the occurrence of Perkinsus marinus associated with wild Pacific oyster (Crassostrea gigas) specimens collected along the west coast of Korea. Confirmation of P. marinus presence was achieved by conventional PCR using World Organization of Animal Health (WOAH)-recommended primers that specifically targeted regions of the rDNA locus (ITS1, 5.8S, and ITS2). Sequencing of 10 samples revealed two distinct sequences differing by a single base pair, indicating potential haplotype variability. One sequence closely resembled the P. marinus strain found in Maryland, USA, whereas the other exhibited divergence, indicative of species diversity in the Korean strain, as was evident from the haplotype network analysis. Further validation involved the Ray's Fluid Thioglycollate Medium (RFTM) assay, which initially yielded inconclusive results, possibly due to low infection intensity. Subsequently, RFTM and 2 M NaOH assays conducted on the isolates in the present study, cultured P. marinus cells in standard DMEM/F12 medium, and a positive P. marinus strain (ATCC 50509), revealed characteristic hypnospores of P. marinus upon Lugol's iodine staining. These comprehensive investigations underscore the conclusive confirmation of P. marinus in Korean waters and mark a significant milestone in our understanding of the distribution and characteristics of this parasite in previously unreported regions.

Haplosporidium nelsoni and Perkinsus marinus occurrence in waters of Great Bay Estuary, New Hampshire.

In Great Bay Estuary, New Hampshire, USA, Haplosporidium nelsoni and Perkinsus marinus are 2 active pathogens of the eastern oyster Crassostrea virginica (Gmelin), that cause MSX (multinucleated sphere with unknown affinity 'X') and dermo mortalities, respectively. Whereas studies have quantified infection intensities in oyster populations and determined whether these parasites exist in certain planktonic organisms, no studies thus far have examined both infectious agents simultaneously in water associated with areas that do and do not have oyster populations. As in other estuaries, both organisms are present in estuarine waters throughout the Bay, especially during June through November, when oysters are most active. Waters associated with oyster habitats had higher, more variable DNA concentrations from these pathogenic organisms than waters at a non-oyster site. This finding allows for enhanced understanding of disease-causing organisms in New England estuaries, where oyster restoration is a priority.

Detection of the protistan parasite, Haplosporidium costale in Crassostrea gigas oysters from the French coast: A retrospective study.

The parasite Haplosporidium costale is known to infect and cause mortality in the oyster Crassostrea virginica in the USA. Decades after its first description in the 1960s, this parasite was detected in Crassostrea gigas in the USA and China. However, it presented a low prevalence and no mortality was associated with it. More recently, in 2019, H. costale was detected in France in a batch of moribund oysters. In order to observe how long this parasite has been present on French coasts, from Normandy to Thau lagoon, a retrospective investigation was conducted on 871 adult and spat oyster batches from 2004 to 2020. To allow rapid detection on a large panel of samples, a real-time PCR for the H. costale actin gene was developed. This method allowed the detection of H. costale DNA in adults from 2005 and in spat from 2008. The H. costale prevalence in spat appeared higher than in adults over the years studied, 14.59 % compared to 6.50 %, respectively. All samples presenting positive results were then sequenced on two targets, H. costale rRNA and actin genes. The actin gene sequencing highlighted the presence of two H. costale strains. Adult C. gigas as well as spat batches coming from hatcheries and DNA controls from C. virginica all presented with the Profile 1 H. costale strain. The Profile 2 H. costale strain was detected only in C. gigas spat coming from natural sources. These observations suggest a correlation between the origin of oysters and H. costale strains which may have been caused by commercial imports between Japan, USA and France back to the 1970s. Over the positive samples studied, only few batches (n = 3) suffered mortalities which could be hypothesized to be caused by H. costale, all presenting the Profile 1 H. costale strain.

Development of a multiplex qPCR for the quantification of three protozoan parasites of the eastern oyster Crassostrea virginica.

A multiplex quantitative PCR (qPCR) assay for the simultaneous detection of 3 eastern oyster Crassostrea virginica parasites, Perkinsus marinus, Haplosporidium nelsoni, and H. costale, was developed using 3 different fluorescently labeled hydrolysis probes. The primers and probe from a previously validated singleplex qPCR for P. marinus detection were combined with newly designed primers and probes specific for H. nelsoni and H. costale. The functionality of the multiplex assay was demonstrated on 2 different platforms by the linear relationship of the standard curves and similar cycle threshold (CT) values between parasites. Efficiency of the multiplex qPCR assay on the Roche and BioRad platforms ranged between 93 and 101%. The sensitivity of detection ranged between 10 and 100 copies of plasmid DNA for P. marinus and Haplosporidium spp., respectively. The concordance between the Roche and BioRad platforms in the identification of the parasites P. marinus, H. nelsoni, and H. costale was 91, 97, and 97%, respectively, with a 10-fold increase in the sensitivity of detection of Haplosporidium spp. on the BioRad thermocycler. The concordance between multiplex qPCR and histology for P. marinus, H. nelsoni, and H. costale was 54, 57, and 87%, respectively. Discordances between detection methods were largely related to localized or low levels of infections in oyster tissues, and qPCR was the more sensitive diagnostic. The multiplex qPCR developed here is a sensitive diagnostic tool for the quantification and surveillance of single and mixed infections in the eastern oyster.

Environmental factors drive the release of Perkinsus marinus from infected oysters.

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.

Unveiling protist diversity associated with the Pacific oyster Crassostrea gigas using blocking and excluding primers.

BACKGROUND: Microbiome of macroorganisms might directly or indirectly influence host development and homeostasis. Many studies focused on the diversity and distribution of prokaryotes within these assemblages, but the eukaryotic microbial compartment remains underexplored so far. RESULTS: To tackle this issue, we compared blocking and excluding primers to analyze microeukaryotic communities associated with Crassostrea gigas oysters. High-throughput sequencing of 18S rRNA genes variable loops revealed that excluding primers performed better by not amplifying oyster DNA, whereas the blocking primer did not totally prevent host contaminations. However, blocking and excluding primers showed similar pattern of alpha and beta diversities when protist communities were sequenced using metabarcoding. Alveolata, Stramenopiles and Archaeplastida were the main protist phyla associated with oysters. In particular, Codonellopsis, Cyclotella, Gymnodinium, Polarella, Trichodina, and Woloszynskia were the dominant genera. The potential pathogen Alexandrium was also found in high abundances within some samples. CONCLUSIONS: Our study revealed the main protist taxa within oysters as well as the occurrence of potential oyster pathogens. These new primer sets are promising tools to better understand oyster homeostasis and disease development, such as the Pacific Oyster Mortality Syndrome (POMS) targeting juveniles.

Variation in global transcriptomic response to Perkinsus marinus infection among eastern oyster families highlights potential mechanisms of disease resistance.

Dermo disease, caused by the protozoan parasite Perkinsus marinus, negatively impacts wild and cultured Eastern oyster populations, yet our knowledge of the mechanistic bases for parasite pathogenicity and the Eastern oyster's response to it is limited. To better understand host responses to the parasite and identify molecular mechanisms underlying disease-resistance phenotypes, we experimentally challenged two families exhibiting divergent Dermo-resistance phenotypes with the parasite, generated global expression profiles using RNAseq and identified differentially expressed transcripts between control and challenged oysters from each family at multiple time points post-parasite injection. The susceptible and resistant families exhibited strikingly different transcriptomic responses to the parasite over a 28-day time period. The resistant family exhibited a strong, focused, early response to P. marinus infection, where many significantly upregulated transcripts were associated with the biological processes "regulation of proteolysis" and "oxidation-reduction process." P. marinus virulence factors are mainly comprised of proteases that facilitate parasite invasion and weaken host humoral defenses, thus host upregulation of transcripts associated with negative regulation of proteolysis is consistent with a Dermo-resistant phenotype. In contrast, the susceptible family mounted a very weak, disorganized, initial response to the parasite. Few transcripts were differentially expressed between control and injected oysters, and no functional enrichment was detected among them. At the final 28 d time point 2450 differentially expressed transcripts were identified and were associated with either "G-protein coupled receptor activity" (upregulated) or "microtubule-based process" (downregulated). A handful of protease inhibitors were differentially expressed between control and injected susceptible oysters, but this function was not enriched in the susceptible data set. The differential expression patterns observed in this study provide valuable insight into the functional basis of Dermo resistance and suggest that the timing of expression is just as important as the transcripts being expressed.

Haplosporidian host:parasite interactions.

The host:parasite interactions of the 3 serious haplosporidian pathogens of oysters, on which most information exists, are reviewed. They are Bonamia ostreae in Ostrea spp. and Crassostrea gigas; Bonamia exitiosa in Ostrea spp.; and Haplosporidium nelsoni in Crassostrea spp. Understanding the haemocytic response to pathogens is constrained by lack of information on haematopoiesis, haemocyte identity and development. Basal haplospridians in spot prawns are probably facultative parasites. H. nelsoni and a species infecting Haliotis iris in New Zealand (NZAP), which have large extracellular plasmodia that eject haplosporosomes or their contents, lyse surrounding cells and are essentially extracellular parasites. Bonamia spp. have small plasmodia that are phagocytosed, haplosporosomes are not ejected and they are intracellular obligate parasites. Phagocytosis by haemocytes is followed by formation of a parasitophorous vacuole, blocking of haemocyte lysosomal enzymes and the endolysosomal pathway. Reactive oxygen species (ROS) are blocked by antioxidants, and host cell apoptosis may occur. Unlike susceptible O. edulis, the destruction of B. ostreae by C. gigas may be due to higher haemolymph proteins, higher rates of granulocyte binding and phagocytosis, production of ROS, the presence of plasma ß-glucosidase, antimicrobial peptides and higher levels of haemolymph and haemocyte enzymes. In B.exitiosa infection of Ostrea chilensis, cytoplasmic lipid bodies (LBs) containing lysosomal enzymes accumulate in host granulocytes and in B. exitiosa following phagocytosis. Their genesis and role in innate immunity and inflammation appears to be the same as in vertebrate granulocytes and macrophages, and other invertebrates. If so, they are probably the site of eicosanoid synthesis from arachidonic acid, and elevated numbers of LBs are probably indicative of haemocyte activation. It is probable that the molecular interaction, and role of LBs in the synthesis and storage of eicosanoids from arachidonic acid, is conserved in innate immunity in vertebrates and invertebrates. However, it seems likely that haplosporidians are more diverse than realized, and that there are many variations in host parasite interactions and life cycles.

Detection of haplosporidian protistan parasites supports an increase to their known diversity, geographic range and bivalve host specificity.

Haplosporidian protist parasites are a major concern for aquatic animal health, as they have been responsible for some of the most significant marine epizootics on record. Despite their impact on food security, aquaculture and ecosystem health, characterizing haplosporidian diversity, distributions and host range remains challenging. In this study, water filtering bivalve species, cockles Cerastoderma edule, mussels Mytilus spp. and Pacific oysters Crassostrea gigas, were screened using molecular genetic assays using deoxyribonucleic acid (DNA) markers for the Haplosporidia small subunit ribosomal deoxyribonucleic acid region. Two Haplosporidia species, both belonging to the Minchinia clade, were detected in C. edule and in the blue mussel Mytilus edulis in a new geographic range for the first time. No haplosporidians were detected in the C. gigas, Mediterranean mussel Mytilus galloprovincialis or Mytilus hybrids. These findings indicate that host selection and partitioning are occurring amongst cohabiting bivalve species. The detection of these Haplosporidia spp. raises questions as to whether they were always present, were introduced unintentionally via aquaculture and or shipping or were naturally introduced via water currents. These findings support an increase in the known diversity of a significant parasite group and highlight that parasite species may be present in marine environments but remain undetected, even in well-studied host species.

Lethal effects of silver nanoparticles on Perkinsus marinus, a protozoan oyster parasite.

Perkinsus marinus, a World Organisation for Animal Health (OIE) notifiable parasite, infects several species of oyster, including Crassostrea virginica and Crassostrea corteziensis. There is little information on possible treatments for this parasite, but the biocidal properties of silver nanoparticles (AgNP) suggest their potential use. The lethal effects of the Argovit™ formulation of AgNP was evaluated for the first time against hypnospores of P. marinus, a particularly resistant stage of the parasite that persists in the environment until favorable conditions occur for zoosporulation to be induced. Hypnospores were exposed to 1, 10 and 100 µg/mL of silver compounded in Argovit™ (corresponding to 0.009, 0.093 and 0.927 mM of Ag), to 157.47 µg/mL (0.927 mM) of silver nitrate (AgNO3) used as a positive control, and to polyvinylpyrrolidone (PVP, 1570 µg/mL) used as a vehicle control. Hypnospores in culture medium without treatment served as a negative control. Dose-dependence after 24 h of exposure to AgNP was observed. A concentration of 0.093 mM AgNP resulted in 50% mortality of P. marinus. Treatment with 0.927 mM of silver, as AgNP or AgNO3, was highly lethal, with greater than 90% mortality. Silver nanoparticles were implicated in the deformation of hypnospores. Transmission electron microscopy (TEM) revealed AgNP within the hypnospore wall and involved in the degradation of lipid droplets in the cytoplasm. AgNP were effective in a saline medium, suggesting the utility of detailed studies of the physicochemical interactions of AgNP under these conditions. These results suggest investigations of possible effect of Argovit™ formulation of AgNP against stages of the parasite like trophozoites and tomonts that develop in tissues or hemolymph of infected oysters as well as studies on its effects in the host and environment.

Two epizootic Perkinsus spp. events in commercial oyster farms at Santa Catarina, Brazil.

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 São Francisco do Sul. This pathology was also detected in C. gasar from Balneário Barra do Sul both, by RFTM and histology. Perkinsus marinus was identified in C. gigas and C. gasar from São Francisco do Sul and Perkinsus beihaiensis in C. gasar from Balneário 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.

Regulation of oyster (Crassostrea virginica) hemocyte motility by the intracellular parasite Perkinsus marinus: A possible mechanism for host infection.

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.

Regulation of apoptosis-related genes during interactions between oyster hemocytes and the alveolate parasite Perkinsus marinus.

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.

Transepithelial migration of mucosal hemocytes in Crassostrea virginica and potential role in Perkinsus marinus pathogenesis.

We have recently described the presence of hemocytes associated with mucus covering the pallial organs (mantle, gills, and body wall) 3 of the eastern oyster Crassostrea virginica. These hemocytes, hereby designated "pallial hemocytes" share common general characteristics with circulating hemocytes but also display significant differences particularly in their cell surface epitopes. The specific location of pallial hemocytes as peripheral cells exposed directly to the marine environment confers them a putative sentinel role. The purpose of this study was to gain a better understanding of the source of these pallial hemocytes by evaluating possible exchanges between circulatory and pallial hemocyte populations and whether these exchanges are regulated by pathogen exposure. Bi-directional transepithelial migrations of hemocytes between pallial surfaces and the circulatory system were monitored using standard cell tracking approaches after staining with the vital fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) in conjunction with fluorescent microscopy and flow cytometry. Results showed bi-directional migration of hemocytes between both compartments and suggest that hemocyte migration from the pallial mucus layer to the circulatory system may occur at a greater rate compared to migration from the circulatory system to the pallial mucus layer, further supporting the role of pallial hemocytes as sentinel cells. Subsequently, the effect of the obligate parasite Perkinsus marinus and the opportunistic pathogen Vibrio alginolyticus on transepithelial migration of oyster hemocytes was investigated. Results showed an increase in hemocyte migration in response to P. marinus exposure. Furthermore, P. marinus cells were acquired by pallial hemocytes before being visible in underlying tissues and the circulatory system suggesting that this parasite could use pallial hemocytes as a vehicle facilitating its access to oyster tissues. These results are discussed in light of new evidence highlighting the role of oyster pallial organs as a portal for the initiation of P. marinus infections in oysters.

Perkinsus infection is associated with alterations in the level of global DNA methylation of gills and gastrointestinal tract of the oyster Crassostrea gasar.

Bivalves are filter feeders that obtain food from seawater that may contain infectious agents, such as the protozoan parasites Perkinsus marinus and P. olseni that are associated with massive mortalities responsible for losses in the aquaculture industry. Despite all physical and chemical barriers, microorganisms cross epithelia and infect host tissues to cause pathologies. Epigenetics mechanisms play important roles in a variety of human processes, from embryonic development to cell differentiation and growth. It is currently emerging as crucial mechanism involved in modulation of host-parasite interactions and pathogenesis, promoting discovery of targets for drug treatment. In bivalves, little is known about epigenetic mechanism in host parasite interactions. The objective of the present study was to evaluate the effect of Perkinsus sp. infections on DNA methylation levels in tissues of Crassostrea gasar oysters. Samples were collected in 2015 and 2016 in the Mamanguape River estuary (PB). Oyster gills were removed and used for Perkinsus sp. DIAGNOSIS: Gills (G) and gastrointestinal tract (GT), as well as cultured P. marinus trophozoites were preserved in 95% ethanol for DNA extractions. DNA methylation levels were estimated from G and GT tissues of uninfected (n=60) and infected oysters (n=60), and from P. marinus trophozoites, by ELISA assays. Results showed that the mean prevalence of Perkinsus sp. infections was high (87.3%) in 2015 and moderate (59.6%) in 2016. DNA methylation levels of G and GT tissues were significantly lower in infected oyster than in uninfected oysters, suggesting that infections are associated with hypomethylation. Methylation level was significantly higher in G than in GT tissues, indicating a likely tissue-specific mechanism. P. marinus trophozoites showed 33% methylation. This was the first study that confirms alterations of DNA methylation in two tissues of C. gasar oysters in association with Perkinsus sp. infections.

Seawater detection and biological assessments regarding transmission of the oyster parasite Mikrocytos mackini using qPCR.

Mikrocytos mackini is an intracellular parasite of oysters and causative agent of Denman Island disease in Pacific oysters Crassostrea gigas. Although M. mackini has been investigated for decades, its natural mode of transmission, mechanism for host entry, and environmental stability are largely unknown. We explored these biological characteristics of M. mackini using a recently described quantitative PCR (qPCR) assay. We detected M. mackini in the flow-through tank water of experimentally infected oysters and during disease remission in host tissues following 6 wk of elevated water temperature. Waterborne exposure of oysters to M. mackini further confirmed the potential for extracellular seawater transmission of this parasite and also identified host gill to have the highest early and continued prevalence for M. mackini DNA compared to stomach, mantle, labial palps, or adductor muscle samples. However, infections following waterborne challenge were slow to develop despite a substantial exposure (>106 M. mackini l-1 for 24 h), and further investigation demonstrated that M. mackini occurrence and infectivity severely declined following extracellular seawater incubation of more than 24 h. This study demonstrates a potential for using qPCR to monitor M. mackini in wild or farmed oyster populations during periods of disease remission or from environmental seawater samples. This work also suggests that gill tissues may provide a primary site for waterborne entry and possibly shedding of M. mackini in oysters. Further, although extracellular seawater transmission of M. mackini was possible, poor environmental stability and infection efficiency likely restricts the geographic transmission of M. mackini between oysters in natural environs and may help to explain localized areas of infection.

Extreme ocean acidification reduces the susceptibility of eastern oyster shells to a polydorid parasite.

Ocean acidification poses a threat to marine organisms. While the physiological and behavioural effects of ocean acidification have received much attention, the effects of acidification on the susceptibility of farmed shellfish to parasitic infections are poorly understood. Here we describe the effects of moderate (pH 7.5) and extreme (pH 7.0) ocean acidification on the susceptibility of Crassostrea virginica shells to infection by a parasitic polydorid, Polydora websteri. Under laboratory conditions, shells were exposed to three pH treatments (7.0, 7.5 and 8.0) for 3- and 5-week periods. Treated shells were subsequently transferred to an oyster aquaculture site (which had recently reported an outbreak of P. websteri) for 50 days to test for effects of pH and exposure time on P. websteri recruitment to oyster shells. Results indicated that pH and exposure time did not affect the length, width or weight of the shells. Interestingly, P. websteri counts were significantly lower under extreme (pH 7.0; ~50% reduction), but not moderate (pH 7.5; ~20% reduction) acidification levels; exposure time had no effect. This study suggests that extreme levels - but not current and projected near-future levels - of acidification (∆pH ~1 unit) can reduce the susceptibility of eastern oyster shells to P. websteri infections.

Parasites and pathological conditions in the edible oyster, Crassostrea madrasensis (Preston), from the east and west coasts of India.

A detailed pathological survey was carried out on the commercially important edible oyster, Crassostrea madrasensis (Preston), from two distinct coastal/brackish water ecosystems of south India. Samples were collected twice a year during wet and dry seasons from 2009 to 2012. Bacterial colonies in the form of prokaryotic inclusions, protozoans (Perkinsus beihaiensis, Nematopsis sp. and ciliates Sphenophrya sp. and Stegotricha sp.), metazoans (trematodes, turbellaria, cestodes and crustaceans) and shell parasites (Polydora spp. and Cliona spp.) along with various pathological conditions (digestive tubule atrophy, ceroid bodies, haemocytic infiltration, tissue necrosis and neoplastic disorders) were observed in C. madrasensis collected from two sites. Intensity, spatial and seasonal variations in infection prevalence and pathological effects on the host were studied. The protozoan parasite, P. beihaiensis; shell parasite, Polydora spp. and pathological condition, digestive gland atrophy were most prevalent in occurrence. High-intensity infections with P. beihaiensis, larval trematodes and Polydora spp. were found to cause significant impact on host physiology. All other parasites were observed with low mean prevalence and intensity. Karapad in Tuticorin bay, the site reported with marked pollution levels, exhibited higher number of parasitic taxa and high mean prevalence and intensity for pathological conditions.

Effects of salinity and temperature on in vitro cell cycle and proliferation of Perkinsus marinus from Brazil.

Field and in vitro studies have shown that high salinities and temperatures promote the proliferation and dissemination of Perkinsus marinus in several environments. In Brazil, the parasite infects native oysters Crassostrea gasar and Crassostrea rhizophorae in the Northeast (NE), where the temperature is high throughout the year. Despite the high prevalence of Perkinsus spp. infection in oysters from the NE of Brazil, no mortality events were reported by oyster farmers to date. The present study evaluated the effects of salinity (5, 20 and 35 psu) and temperature (15, 25 and 35 °C) on in vitro proliferation of P. marinus isolated from a host (C. rhizophorae) in Brazil, for a period of up to 15 days and after the return to the control conditions (22 days; recovery). Different cellular parameters (changes of cell phase's composition, cell density, viability and production of reactive oxygen species) were analysed using flow cytometry. The results indicate that the P. marinus isolate was sensitive to the extreme salinities and temperatures analysed. Only the highest temperature caused lasting cell damage under prolonged exposure, impairing P. marinus recovery, which is likely to be associated with oxidative stress. These findings will contribute to the understanding of the dynamics of perkinsiosis in tropical regions.