Search

Many factors affect an organism's microbiome including its environment, proximity to other organisms, and physiological condition. As filter feeders, bivalves have highly plastic microbiomes that are especially influenced by the surrounding seawater, yet they also maintain a unique core set of microbes. Using 16S ribosomal RNA sequencing, we characterized the bacterial microbiomes of four species of bivalves native to the Mid-Atlantic East Coast of North America: Crassostrea virginica, Macoma balthica, Ameritella mitchelli, and Ischadium recurvum and assessed the impact of their external environment, internal parasites, and size on their microbial communities. We found significant differences in bacterial amplicon sequence variants (ASVs) across species, with each species harboring a core ASV present across all individuals. We further found that some C. virginica co-cultured with I. recurvum had high abundances of the I. recurvum core ASV. We identified ASVs associated with infection by the parasites Perkinsus marinus and Zaops ostreum as well others associated with bivalve size. Several of these ASV are candidates for further investigation as potential probiotics, as they were found positively correlated with bivalve size and health. This research represents the first description of the microbiomes of A. mitchelli, I. recurvum, and M. balthica. We document that all four species have highly plastic microbiomes, while maintaining certain core bacteria, with important implications for growth, health, and adaptation to new environments. [ABSTRACT FROM AUTHOR]

Determining the efficacy of restoration practices is critical for enhancing and rebuilding degraded ecosystems. A fundamental question in marine restoration design is whether to employ active (e.g. using living plants or oysters that form the biogenic habitat) or passive (e.g. restoring the hydrology and sediment materials at the site) restoration strategies. Furthermore, the identity of sources used in restoring biogenic habitats could influence the effectiveness of restoration efforts. We present results from two independent experimental restoration efforts designed to explore the efficacy of these different approaches for the recovery of Crassostrea virginica (eastern oyster) reef habitat. Conducted from 2015–2020 in Rhode Island, USA, the restoration experiments compared 1) passive (unseeded shell) vs. active (seeded spat on shell) restoration methods in Ninigret Pond, and 2) restored reefs seeded with hatchery sourced vs. wild oyster lineages in Quonochontaug Pond. In Quonochontaug Pond, the hatchery-sourced line exhibited greater mortality than the wild lines. Restored reefs in both projects remained viable all four years post-restoration; however, low recruitment continues to limit the long-term sustainability of restored reefs in these Rhode Island systems. Natural mortality varied spatially within each coastal pond, ranging from 33–81% per year. Across both experiments, oyster mortality increased with rising temperature, salinity, and disease prevalence. Our results suggest that utilizing active restoration with spat spawned from multiple sources, including wild populations, may improve the success of oyster restoration. Future habitat restoration efforts should consider the environmental variability associated with restoration sites and the source of restoration species during project planning. [ABSTRACT FROM AUTHOR]

Many factors affect an organism’s microbiome including its environment, proximity to other organisms, and physiological condition. As filter feeders, bivalves have highly plastic microbiomes that are especially influenced by the surrounding seawater, yet they also maintain a unique core set of microbes. Using 16S ribosomal RNA sequencing, we characterized the bacterial microbiomes of four species of bivalves native to the Mid-Atlantic East Coast of North America: Crassostrea virginica, Macoma balthica, Ameritella mitchelli, and Ischadium recurvum and assessed the impact of their external environment, internal parasites, and size on their microbial communities. We found significant differences in bacterial amplicon sequence variants (ASVs) across species, with each species harboring a core ASV present across all individuals. We further found that some C. virginica co-cultured with I. recurvum had high abundances of the I. recurvum core ASV. We identified ASVs associated with infection by the parasites Perkinsus marinus and Zaops ostreum as well others associated with bivalve size. Several of these ASV are candidates for further investigation as potential probiotics, as they were found positively correlated with bivalve size and health. This research represents the first description of the microbiomes of A. mitchelli, I. recurvum, and M. balthica. We document that all four species have highly plastic microbiomes, while maintaining certain core bacteria, with important implications for growth, health, and adaptation to new environments.

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 host–parasite 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.

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 host–parasite 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. [ABSTRACT FROM AUTHOR]

Vector or reservoir species of five mollusc diseases listed in the Animal Health Law were identified, based on evidence generated through an extensive literature review, to support a possible updating of Regulation (EU) 2018/1882. Mollusc species on or in which Mikrocytos mackini, Perkinsus marinus, Bonamia exitiosa, Bonamia ostreae and Marteilia refringens were detected, in the field or during experiments, were classified as reservoir species with different levels of certainty depending on the diagnostic tests used. Where experimental evidence indicated transmission of the pathogen from a studied species to another known susceptible species, this studied species was classified as a vector species. Although the quantification of the risk of spread of the pathogens by the vectors or reservoir species was not part of the terms of reference, such risks do exist for the vector species, since transmission from infected vector species to susceptible species was proven. Where evidence for transmission from infected molluscs was not found, these were defined as reservoir. Nonetheless, the risk of the spread of the pathogens from infected reservoir species cannot be excluded. Evidence identifying conditions that may prevent transmission by vectors or reservoir mollusc species during transport was collected from scientific literature. It was concluded that it is very likely to almost certain (90–100%) that M. mackini, P. marinus, B. exitiosaB. ostreae and M. refringens will remain infective at any possible transport condition. Therefore, vector or reservoir species that may have been exposed to these pathogens in an affected area in the wild or at aquaculture establishments or through contaminated water supply can possibly transmit these pathogens. For transmission of M. refringens, the presence of an intermediate host, a copepod, is necessary. This publication is linked to the following EFSA Journal articles: http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2023.8172/full, http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2023.8174/full This publication is linked to the following EFSA Supporting Publications articles: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8122/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8123/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8124/full [ABSTRACT FROM AUTHOR]

Low salinity can negatively affect reproduction in estuarine bivalves. The spatial and temporal extents of these effects are important to inform models of population dynamics, environmental risk assessments, restoration efforts, and predictions of climate change effects. A hypothesis of delayed gametogenesis for oysters at low salinity sites was tested relative to their higher salinity counterparts in downstream experimental cages. In 2018, the timing of gametogenesis and spawning was observed June–August for 2-year-old oysters from three distinct ancestries (native, hatchery, aquaculture), outplanted at age 1 month along the salinity gradient (3–30 psu) of a temperate estuary. A second season of data was collected in 2019 from a 3-year-old aquaculture line and mixed-age native adult oysters dredged and transplanted 1 year prior. Dermo was tested in 2019 and prevalence was 1.3% (n = 240). Gametogenesis and spawning were retarded for all ancestries at low salinity relative to higher salinity sites during July and August. The reverse pattern was found in June, with low salinity sites having more advanced gonad index than at a high salinity site. This difference in average gonad index was 2.65 vs 1.46, respectively, for the native line and 2.62 vs 2.08 for aquaculture. Low salinity seemed to not only induce earlier gametogenesis in June, but also extended the reproductive season relative to higher salinity sites. Among oyster ancestries, the aquaculture line stood out as having 30–48% lower gametogenic synchrony within sites, but only in 2018. Because the native oysters used in this study have been restricted to low salinity conditions for many generations, demonstration of their reproductive plasticity across salinities is notable and broadens the range of potential future restoration strategies. [ABSTRACT FROM AUTHOR]

Aims: To investigate the relationships between individual health status of oysters, particularly with regard to parasitic infection, and variability in abundance of human‐pathogenic Vibrio species. Methods and Results: Aquacultured eastern oysters, Crassostrea virginica, were analysed individually for infection by the protozoan parasite Perkinsus marinus through quantitative PCR, and total Vibrio vulnificus and total and pathogenic Vibrio parahaemolyticus abundance was assessed using a most probable number (MPN)‐qPCR approach. Additionally, perspective on general oyster health and other parasitic infections was obtained through histopathology. Perkinsus marinus infection and human‐pathogenic Vibrio species levels were not correlated, but through histology, analyses revealed that oysters infected by Haplosporidium nelsoni harboured more V. vulnificus. Conclusions: The highly prevalent parasite P. marinus had little influence on human‐pathogenic Vibrio species levels in eastern oysters, but the less prevalent parasite, H. nelsoni, may influence V. vulnificus levels, highlighting the potential nuances of within‐oyster dynamics of Vibrio species. Significance and Impact of the Study: Human‐pathogenic bacteria continue to be a concern to the oyster industry and causes for individual oyster variation in bacterial levels remain unknown. The major oyster pathogen P. marinus does not appear to affect levels of these bacteria within oysters, suggesting that other factors may influence Vibrio spp. levels in oysters. [ABSTRACT FROM AUTHOR]

Abstract Background Antioxidative enzymes contribute to a parasite’s ability to counteract the host’s intracellular killing mechanisms. The facultative intracellular oyster parasite, Perkinsus marinus, a sister taxon to dinoflagellates and apicomplexans, is responsible for mortalities of oysters along the Atlantic coast of North America. Parasite trophozoites enter molluscan hemocytes by subverting the phagocytic response while inhibiting the typical respiratory burst. Because P. marinus lacks catalase, the mechanism(s) by which the parasite evade the toxic effects of hydrogen peroxide had remained unclear. We previously found that P. marinus displays an ascorbate-dependent peroxidase (APX) activity typical of photosynthetic eukaryotes. Like other alveolates, the evolutionary history of P. marinus includes multiple endosymbiotic events. The discovery of APX in P. marinus raised the questions: From which ancestral lineage is this APX derived, and what role does it play in the parasite’s life history? Results Purification of P. marinus cytosolic APX activity identified a 32 kDa protein. Amplification of parasite cDNA with oligonucleotides corresponding to peptides of the purified protein revealed two putative APX-encoding genes, designated PmAPX1 and PmAPX2. The predicted proteins are 93% identical, and PmAPX2 carries a 30 amino acid N-terminal extension relative to PmAPX1. The P. marinus APX proteins are similar to predicted APX proteins of dinoflagellates, and they more closely resemble chloroplastic than cytosolic APX enzymes of plants. Immunofluorescence for PmAPX1 and PmAPX2 shows that PmAPX1 is cytoplasmic, while PmAPX2 is localized to the periphery of the central vacuole. Three-dimensional modeling of the predicted proteins shows pronounced differences in surface charge of PmAPX1 and PmAPX2 in the vicinity of the aperture that provides access to the heme and active site. Conclusions PmAPX1 and PmAPX2 phylogenetic analysis suggests that they are derived from a plant ancestor. Plant ancestry is further supported by the presence of ascorbate synthesis genes in the P. marinus genome that are similar to those in plants. The localizations and 3D structures of the two APX isoforms suggest that APX fulfills multiple functions in P. marinus within two compartments. The possible role of APX in free-living and parasitic stages of the life history of P. marinus is discussed.

Perkinsus marinus (Perkinsozoa), a close relative of apicomplexans, is an osmotrophic facultative intracellular marine protozoan parasite responsible for “Dermo” disease in oysters and clams. Although there is no clinical evidence of this parasite infecting humans, HLA-DR40 transgenic mice studies strongly suggest the parasite as a natural adjuvant in oral vaccines. P. marinus is being developed as a heterologous gene expression platform for pathogens of medical and veterinary relevance and a novel platform for delivering vaccines. We previously reported the transient expression of two rodent malaria genes Plasmodium berghei HAP2 and MSP8. In this study, we optimized the original electroporation-based protocol to establish a stable heterologous expression method. Using 20 μg of pPmMOE[MOE1]:GFP and 25.0 × 106P. marinus cells resulted in 98% GFP-positive cells. Furthermore, using the optimized protocol, we report for the first time the successful knock-in of GFP at the C-terminus of the PmMOE1 using ribonucleoprotein (RNP)-based CRISPR/Cas9 gene editing methodology. The GFP was expressed 18 h post-transfection, and expression was observed for 8 months post-transfection, making it a robust and stable knock-in system.

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. [ABSTRACT FROM AUTHOR]

Perkinsus marinus, a protozoan and the causative agent of perkinsosis (dermo disease) is a prevalent parasite found within the eastern oyster (Crassostrea virginica). In this study, we explore metabolic processes of P. marinus cells under lipid-depleted medium conditions to elucidate the interchanging flux of lipid and carbohydrate metabolism. Although P. marinus can synthesize their own lipids from available nutrients, they display a slower growth in medium not supplemented with lipids as opposed to medium with lipids. Under these conditions, using transcriptomics, we surprisingly observed evidence of stimulated lipid degradation through increased transcription of two core β-oxidation pathway enzymes. Simultaneously, phospholipid biosynthetic pathways were downregulated. Metabolomic analysis supported the transcriptomic results. Most fatty acids were decreased in lipid-deplete medium as opposed to lipid-replete medium, and available glucose was fermented to lactate. A significant increase in the cholesterol derivative zymosterol further supported a downregulation of membrane synthesis under the experimental conditions. A robust tricarboxylic acid (TCA) cycle was apparent by enhanced citrate synthase transcription, and a simultaneous reduction in branched chain amino acids. It is concluded that although P. marinus has the capacity for synthesizing its own lipids, it can respond to lipid deprivation in medium by oxidizing readily available stores, and likely transitioning into a resting stage. [ABSTRACT FROM AUTHOR]

The protozoan Perkinsus marinus has been associated with high mortality episodes of the eastern oyster Crassostrea virginica in the USA. The presence of P. marinus on the pleasure oyster Crassostrea corteziensis cultivated in two estuaries on the southeast coast of the Gulf of California was evaluated. Oysters were collected monthly (September 2016 to September 2017) and analyzed using Ray's fluid thioglycollate medium (RFTM) and polymerase chain reaction (PCR). Water parameters and oyster biometrics were also recorded. Pathogen prevalence increased over time from 0 to 100% in oysters from La Pitahaya, and from 0 to 83.33% in those from Bacorehuis. At both oyster farms, infection intensity was light (<1×104 parasites g-1 wet tissue), pathogen prevalence and infection intensity were correlated with oyster size and weight, and there was a strong correlation between P. marinus prevalence and intensity (La Pitahaya r = 0.91; Bacorehuis r = 0.82). The oysters that resulted positive for P. marinus by RFTM also were assayed using PCR. P. marinus presence was confirmed in 98.27% (114/116) of the oysters from La Pitahaya, and 95.83% (46/48) of those from Bacorehuis. The detection of P. marinus confirms that this pathogen is well established in the area with high expression during the warmer season. Despite the light infection intensity of this parasite at both sites, health surveillance of this bivalve in the region is highly advisable. [ABSTRACT FROM AUTHOR]

[Display omitted] • Perkinsus marinus was found for the first time associated with wild Crassostrea gigas collected in the intertidal zone along the west coast of Korea. • P. marinus was confirmed by molecular diagnosis and validated via RFTM using cultured cells. • Korean P. marinus showed potential haplotype variability with P. marinus in the US. • Further study of the distribution and characteristics of this parasite is needed. 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. [ABSTRACT FROM AUTHOR]

Through the National Sea Grant’s Oyster Disease Research Program, scientists from around the country are being funded to find approaches to restoring the ecological and commercial viability of oyster beds.This one-page publication highlights the efforts of Dr. Jane Burns of the Theodore Gildred Cancer Center at UC San Diego’s School of Medicine to create a genetically engineered,disease-resistant version of the commercially important oyster, Crassostrea virginica. Conceptually, this could be achieved by inserting appropriate genes from a horseshoe crab—the crab produces antimicrobial proteins that make it immune to disease—into an oyster genome. Though simple in theory, in practice it requires the development of many intermediary techniques and tools.

Abstract Background The protozoan Perkinsus marinus (Mackin, Owen & Collier) Levine, 1978 causes perkinsosis in the American oyster Crassostrea virginica Gmelin, 1791. This pathogen is present in cultured C. virginica from the Gulf of Mexico and has been reported recently in Saccostrea palmula (Carpenter, 1857), Crassostrea corteziensis (Hertlein, 1951) and Crassostrea gigas (Thunberg, 1793) from the Mexican Pacific coast. Transportation of fresh oysters for human consumption and repopulation could be implicated in the transmission and dissemination of this parasite across the Mexican Pacific coast. The aim of this study was two-fold. First, we evaluated the P. marinus infection parameters by PCR and RFTM (Ray’s fluid thioglycollate medium) in C. virginica from four major lagoons (Términos Lagoon, Campeche; Carmen-Pajonal-Machona Lagoon complex, Tabasco; Mandinga Lagoon, Veracruz; and La Pesca Lagoon, Tamaulipas) from the Gulf of Mexico. Secondly, we used DNA sequence analyses of the ribosomal non-transcribed spacer (rNTS) region of P. marinus to determine the possible translocation of this species from the Gulf of Mexico to the Mexican Pacific coast. Results Perkinsus marinus prevalence by PCR was 57.7% (338 out of 586 oysters) and 38.2% (224 out of 586 oysters) by RFTM. The highest prevalence was observed in the Carmen-Pajonal-Machona Lagoon complex in the state of Tabasco (73% by PCR and 58% by RFTM) and the estimated weighted prevalence (WP) was less than 1.0 in the four lagoons. Ten unique rDNA-NTS sequences of P. marinus [termed herein the “P. marinus (Pm) haplotype”] were identified in the Gulf of Mexico sample. They shared 96–100% similarity with 18 rDNA-NTS sequences from the GenBank database which were derived from 16 Mexican Pacific coast infections and two sequences from the USA. The phylogenetic tree and the haplotype network showed that the P. marinus rDNA-NTS sequences from Mexico were distant from the rDNA-NTS sequences of P. marinus reported from the USA. The ten rDNA-NTS sequences described herein were restricted to specific locations displaying different geographical connections within the Gulf of Mexico; the Carmen-Pajonal-Machona Pm1 haplotype from the state of Tabasco shared a cluster with P. marinus isolates reported from the Mexican Pacific coast. Conclusions The rDNA-NTS sequences of P. marinus from the state of Tabasco shared high similarity with the reference rDNA-NTS sequences from the Mexican Pacific coast. The high similarity suggests a transfer of oysters infected with P. marinus from the Mexican part of the Gulf of Mexico into the Mexican Pacific coast.

Abstract Perkinsus marinus is a marine protozoan parasite that infects natural and farmed oysters, attracting attention from researchers in both fisheries and evolutionary biology. The functions of almost all cellular components and organelles are, however, poorly understood even though a draft genome sequence of P. marinus is publicly available. One of the major obstacles for a functional study of the parasite is limited experimental means for genetic manipulation: a transfection method was established in 2008, and the first drug selection system with bleomycin was reported in 2016. We here introduce the second drug-selectable marker for selection of P. marinus transfectants. The parasite growth is efficiently inhibited by puromycin (IC 50 = 4.96 μg/mL), and transfection of its resistance gene, puromycin- N -acetyl-transferase (pac), confers resistance to the drug on the parasite. Stable transfectants can be obtained within 2 months by treating with puromycin at 100 μg/mL. Furthermore, combining puromycin and bleomycin treatment can select transfectants co-expressing two marker genes. This dual-transfection method raises the possibility of using co-localization to identify the cellular localization of novel proteins in P. marinus , thereby contributing to the understanding of cellular functions and pathogenesis. Graphical abstract Unlabelled Image Highlights • Perkinsus marinus is sensitive to puromycin (IC 50 = 4.96 μg/mL). • Cells transfected with puromycin- N -acetyl-tranferase gain puromycin resistance. • Untransfected parasites can be removed by 100 μg/mL puromycin treatment. • Combining puromycin/bleomycin can select cells co-expressing two marker genes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: The Down syndrome cell adhesion molecule (Dscam) gene in invertebrates encodes numerous isoforms derived by alternative splicing as a pathogen‐specific response. The role of Dscam in immune system responses has been established in fruit fly, mosquito, shrimp, crab, and crayfish, among other arthropods. In molluscs, this gene has not yet been clearly identified, and more importantly, its function has not been elucidated. The present study examined Dscam expression in Crassostrea gigas (CgDscam) and Crassostrea corteziensis (CcDscam) infected with Ostreid herpesvirus type 1 (OsHV‐1) and Perkinsus marinus, respectively, through in situ hybridization and quantitative PCR. The consensus coding sequences for CgDscam and CcDscam showed the same organization of domains identified in other taxonomic groups. Furthermore, conserved regions from the signal peptide to Ig1 and from FNIII6 to the cytoplasmic tail were identified. The Dscam transcript was detected in gills and mantles of both species. The hybridization signals were remarkable in infected tissues. QPCR analysis showed higher Dscam expression levels in the infected gills of both species, but in the mantle, no difference in CgDscam expression was observed between control and infected tissues. Moreover, CcDscam expression was inhibited in infected tissue. These results suggest that Dscam could be directly involved in bivalve immune responses. [ABSTRACT FROM AUTHOR]

Abstract 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. Graphical abstract Image 1 Highlights • Perkinsus marinus induces an increase of hemocyte migration in oyster. • Hemocytes are chemoattracted to P. marinus. • Enhanced motility is suppressed by RGDS and CK-666. • Findings support the role of integrins and Arp2/3 pathways in motility regulation. [ABSTRACT FROM AUTHOR]

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

The benefits of exposing eastern oysters to air during commercial culture have not been well-characterized. An adjustable longline system ( ALS) designed in Australia and recently adopted by the nascent aquaculture industry in the northern Gulf of Mexico, allows growing oysters at any position in the water column and is perfectly suited to study the benefits of air exposure. Four-month old diploid oysters were deployed in an ALS and divided into three groups: 1) oysters exposed to air daily for 8-12 hr during low tide, 2) oysters exposed to air weekly (~24 hr once a week), and 3) oysters kept subtidally. Oyster mortality and growth rates, Perkinsus marinus load and condition index were then determined every 3 months over 2 years, while refrigerated shelf life and Vibrio vulnificus load were determined in summer and early fall of the second year. Summer mortalities were delayed, P. marinus infection intensities tended to be lower and condition index was significantly higher in oysters exposed to air daily compared with oysters exposed to air weekly or held subtidally. Shell heights of oysters exposed to air daily were lower for most of the study due to a lower growth rate during the initial sampling interval following deployment. No consistent differences were found in V. vulnificus loads or refrigerated shelf lives between the groups. It is recommended that ALS be set so that oysters are kept subtidally fall through spring to promote growth, and exposed to air daily during summer to delay P. marinus proliferation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Infectious diseases have been a major limiting factor for large scale production of oyster farming. Several factors have contributed to the emergence and spread of infectious diseases in all cultivation sites around the world. Therefore, the prevention and control of diseases has become a priority for the sustainability of global aquaculture. Crassostrea corteziensis is a native species that contributes to the Mexican Pacific oyster production and used in this study to assess seasonal simultaneous infective events between distant areas in the Gulf of California. The results of the molecular analysis showed a higher prevalence of Perkinsus marinus in the north area and Marteilia refringens in the south. OsHV-1 was only present in summer and autumn with low prevalence in the two areas. The histological analysis of the PCR-positive organisms presented alterations characteristic of infections. The presence of M. refringens in a new location on the Gulf of California suggests that this pathogen is already well established in the area and the dual presence of pathogens in C. corteziensis is reported for the first time. [ABSTRACT FROM AUTHOR]

Background: The protozoan Perkinsus marinus (Mackin, Owen & Collier) Levine, 1978 causes perkinsosis in the American oyster Crassostrea virginica Gmelin, 1791. This pathogen is present in cultured C. virginica from the Gulf of Mexico and has been reported recently in Saccostrea palmula (Carpenter, 1857), Crassostrea corteziensis (Hertlein, 1951) and Crassostrea gigas (Thunberg, 1793) from the Mexican Pacific coast. Transportation of fresh oysters for human consumption and repopulation could be implicated in the transmission and dissemination of this parasite across the Mexican Pacific coast. The aim of this study was two-fold. First, we evaluated the P. marinus infection parameters by PCR and RFTM (Ray's fluid thioglycollate medium) in C. virginica from four major lagoons (Términos Lagoon, Campeche; Carmen-Pajonal-Machona Lagoon complex, Tabasco; Mandinga Lagoon, Veracruz; and La Pesca Lagoon, Tamaulipas) from the Gulf of Mexico. Secondly, we used DNA sequence analyses of the ribosomal nontranscribed spacer (rNTS) region of P. marinus to determine the possible translocation of this species from the Gulf of Mexico to the Mexican Pacific coast. Results: Perkinsus marinus prevalence by PCR was 57.7% (338 out of 586 oysters) and 38.2% (224 out of 586 oysters) by RFTM. The highest prevalence was observed in the Carmen-Pajonal-Machona Lagoon complex in the state of Tabasco (73% by PCR and 58% by RFTM) and the estimated weighted prevalence (WP) was less than 1.0 in the four lagoons. Ten unique rDNA-NTS sequences of P. marinus [termed herein the "P. marinus (Pm) haplotype"] were identified in the Gulf of Mexico sample. They shared 96-100% similarity with 18 rDNA-NTS sequences from the GenBank database which were derived from 16 Mexican Pacific coast infections and two sequences from the USA. The phylogenetic tree and the haplotype network showed that the P. marinus rDNA-NTS sequences from Mexico were distant from the rDNA-NTS sequences of P. marinus reported from the USA. The ten rDNA-NTS sequences described herein were restricted to specific locations displaying different geographical connections within the Gulf of Mexico; the Carmen-Pajonal-Machona Pm1 haplotype from the state of Tabasco shared a cluster with P. marinus isolates reported from the Mexican Pacific coast. Conclusions: The rDNA-NTS sequences of P. marinus from the state of Tabasco shared high similarity with the reference rDNA-NTS sequences from the Mexican Pacific coast. The high similarity suggests a transfer of oysters infected with P. marinus from the Mexican part of the Gulf of Mexico into the Mexican Pacific coast. [ABSTRACT FROM AUTHOR]

Some organisms have retained plastids even after they have lost the ability to photosynthesize. Several studies of nonphotosynthetic plastids in apicomplexan parasites have shown that the isopentenyl pyrophosphate biosynthesis pathway in the organelle is essential for their survival. A phytohormone, abscisic acid, one of several compounds biosynthesized from isopentenyl pyrophosphate, regulates the parasite cell cycle. Thus, it is possible that the phytohormone is universally crucial, even in nonphotosynthetic plastids. Here, we examined this possibility using the oyster parasite Perkinsus marinus, which is a plastid-harboring cousin of apicomplexan parasites and has independently lost photosynthetic ability. Fluridone, an inhibitor of abscisic acid biosynthesis, blocked parasite growth and induced cell clustering. Nevertheless, abscisic acid and its intermediate carotenoids did not affect parasite growth or rescue the parasite from inhibition. Moreover, abscisic acid was not detected from the parasite using liquid chromatography mass spectrometry. Our findings show that abscisic acid does not play any significant roles in P. marinus. [ABSTRACT FROM AUTHOR]

Perkinsosis is a disease caused by protozoan parasites from the Perkinsus genus. In Brazil, two species, P. beihaiensis and P. marinus, are frequently found infecting native oysters ( Crassostrea gasar and C. rhizophorae ) from cultured and wild populations in several states of the Northeast region. The impacts of this disease in bivalves from Brazil, as well as the interactions with environmental factors, are poorly studied. In the present work, we evaluated the in vitro effects of the cyanobacteria Synechocystis spp. on trophozoites of P. marinus and haemocytes of C. gasar . Four cyanobacteria strains isolated from the Northeast Brazilian coast were used as whole cultures (WCs) and extracellular products (ECPs). Trophozoites of P. marinus were exposed for short (4 h) and long (48 h and 7 days, the latter only for ECPs) periods, while haemocytes were exposed for a short period (4 h). Cellular and immune parameters, i.e. cell viability, cell count, reactive oxygen species production (ROS) and phagocytosis of inert (latex beads) and biological particles (zymosan and trophozoites of P. marinus ) were measured by flow cytometry. The viability of P. marinus trophozoites was improved in response to WCs of Synechocystis spp., which could be a beneficial effect of the cyanobacteria providing nutrients and reducing reactive oxygen species. Long-term exposure of trophozoites to ECPs of cyanobacteria did not modify in vitro cell proliferation nor viability. In contrast, C. gasar haemocytes showed a reduction in cell viability when exposed to WCs, but not to ECPs. However, ROS production was not altered. Haemocyte ability to engulf latex particles was reduced when exposed mainly to ECPs of cyanobacteria; while neither the WCs nor the ECPs modified phagocytosis of the biological particles, zymosan and P. marinus . Our results suggest a negative effect of cyanobacteria from the Synechocystis genus on host immune cells, in contrast to a more beneficial effect on the parasite cell, which could together disrupt the balance of the host-parasite interaction and make oysters more susceptible to P. marinus as well as opportunistic infections. [ABSTRACT FROM AUTHOR]

• First record of Perkinsus marinus infecting oysters Crassostrea sp. in the estuaries of the Rio Grande do Norte, Brazil. • ITS primers recommended for official diagnosis of P. marinus by PCR were efficient in the qPCR performed. • New reports of P. marinus infecting oysters in Brazilian northeastern put producers and animal defense agencies on alert. A pathogen with high virulence potential in some host species, Perkinsus marinus remains a challenge for the ecological integrity of marine ecosystems and the health of bivalve molluscs. This study investigates the occurrence of P. marinus in Crassostrea sp. in estuaries of the Potengi River and the Guaraíras lagoon in Rio Grande do Norte, Brazil. A total of 203 oyster samples that tested positive for Perkinsus sp. in Ray's fluid thioglycollate medium (RFTM) were subjected to species-specific quantitiative PCR, where 61 animals (30.05 %) presented amplification graphs with a melting temperature of 80.1 ± 0.6 °C matching the positive control. This was the first record of P. marinus in oysters in these estuaries using qPCR as a diagnostic tool. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Perkinsus marinus is a protozoan parasite of molluscs that can be propagated in vitro in a defined culture medium, in the absence of host cells. We previously reported that P. marinus trophozoites can be transfected with high efficiency by electroporation using a plasmid based on MOE, a highly expressed gene, and proposed its potential use as a 'pseudoparasite.' This is a novel gene expression platform for parasites of medical relevance for which the choice of the surrogate organism is based on phylogenetic affinity to the parasite of interest, while taking advantage of the whole engineered surrogate organism as a vaccination adjuvant. Here we improved the original transfection plasmid by incorporating a multicloning site, an enterokinase recognition sequence upstream of GFP, and a His-tag and demonstrate its potential suitability for the heterologous expression of Plasmodium sp. genes relevant to the development of anti-malarial vaccines. Plasmodium berghei HAP2 and MSP8, currently considered candidate genes for a malaria vaccine, were cloned into p[MOE]:GFP, and the constructs were used to transfect P. marinus trophozoites. Within 48 hr of transfection we observed fluorescent cells indicating that the P. berghei genes fused to GFP were expressed. The expression appeared to be transient for both P. berghei genes, as florescence of the transfectants diminished gradually over time. Although this heterologous expression system will require optimization for integration and constitutive expression of Plasmodium genes, our results represent attainment of proof for the 'pseudoparasite' concept we previously proposed, as we show that the engineered P. marinus system has the potential to become a surrogate system suitable for expression of Plasmodium spp. genes of interest, which could eventually be used as a malaria vaccine delivery platform. The aim of the present study was to test the ability of marine protozoan parasite P. marinus to express genes of P. berghei. [ABSTRACT FROM AUTHOR]

There is little information on the performance of oyster populations from Louisiana estuaries limiting the ability to choose stocks for hatchery seed production and field grow-out. The objectives of this study were therefore to compare the mortality, growth, dermo ( Perkinsus marinus ) infection intensity and condition index of the progeny of wild oysters collected from three Louisiana estuaries differing in salinity regime and oysters specifically selected for dermo resistance. Progeny were deployed in cages in the field, along a salinity gradient in coastal Louisiana. Overall, salinity and temperature had major impacts on the mortality, growth, dermo infection intensity and condition index of oysters of all four stocks and a few differences between stocks could be shown at some sites. At the lowest salinity site, the progeny of wild oysters from Sister Lake, a low salinity estuarine lake, had the lowest mortality suggesting enhanced tolerance to low salinity conditions compared to the other stocks. At the highest salinity site, the progeny of wild oysters from Lake Calcasieu, a high salinity estuarine lake, had the lowest mortality during summer concomitant with increasing dermo infection intensities suggesting a better resistance to dermo disease compared to the other wild stocks and confirming an earlier finding. This initial result suggests that the stocks used are genetically differentiated with respect to low salinity tolerance as well as dermo-related mortality at high salinity and that stock selection for aquaculture grow-out or restoration effort will benefit from being site-specific and dependent on the dominant environmental conditions. Statement of relevance Assessment of eastern oyster stocks in Louisiana or other Gulf of Mexico estuaries for seed production and field grow-out is lacking. [ABSTRACT FROM AUTHOR]

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 Niño-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. [ABSTRACT FROM AUTHOR]

Perkinsus species are notorious unicellular marine parasites that infect commercially important mollusk species including clams and oysters. Recent accumulation of molecular information will greatly facilitate the understanding of Perkinsus biology and development of strategies to control infection. However, the limited availability of methods for genetic manipulation has hindered molecular-based studies of the parasites. In particular, the lack of a drug selection system requires manual isolation of fluorescent cells under a microscope to establish transfected cell lines. Here, we introduce a drug selection system using a glycopeptide antibiotic, bleomycin, and a vector containing the resistance gene Sh-ble . Perkinsus marinus is sensitive to bleomycin, and 100 μg/ml of this drug completely blocks its proliferation. Concomitant expression of Sh-ble enables us to specifically select transfected cells in the presence of the drug. We believe that this system provides new opportunities for functional analyses of this parasite. [ABSTRACT FROM AUTHOR]

The protozoan parasite Perkinsus marinus is the etiological agent of “dermo disease”. This pathogen is considered by the World Organization for Animal Health (OIE) as reportable due to the high mortalities that it produces in the eastern oyster Crassostrea virginica in the US. In 2006, this parasite was detected in the pleasure oyster Crassostrea corteziensis in Nayarit on the Pacific coast of Mexico, indicating a new host and an extension of its known distribution. Epizootiological data of P. marinus in the pleasure oyster are unknown. With the objective of determining the prevalence and intensity in relation with temperature and salinity throughout time, as well as for studying interactions of host size and sex with the parasite, a monthly sampling was carried out in two aquaculture sites of Nayarit from 2007 to 2014. A total of 7700 oysters were analyzed. In both localities, prevalence was low in winter (<6%) when temperature and salinity fluctuated around 24 °C and 33, respectively; and the highest prevalence values occurred during summer (37%) when temperature and salinity were around 30 °C and 20, respectively. Infection intensity increased in summer, but severe cases remained on average <10%. Larger oysters showed the highest prevalence and intensity, and higher prevalence were generally observed in females. No unusual mortalities directly related with P. marinus were observed. [ABSTRACT FROM AUTHOR]

Parasites can exert strong effects on population to ecosystem level processes, but data on parasites are limited for many global regions, especially tropical marine systems. Characterizing parasite diversity and distributions are the first steps towards understanding the potential impacts of parasites. The Panama Canal serves as an interesting location to examine tropical parasite diversity and distribution, as it is a conduit between two oceans and a hub for international trade. We examined metazoan and protistan parasites associated with ten oyster species collected from both Panamanian coasts, including the Panama Canal and Bocas del Toro. We found multiple metazoan taxa (pea crabs, Stylochus spp., Urastoma cyrinae). Our molecular screening for protistan parasites detected four species of Perkinsus (Perkinsus marinus, Perkinsus chesapeaki, Perkinsus olseni, Perkinsus beihaiensis) and several haplosporidians, including two genera (Minchinia, Haplosporidium). Species richness was higher for the protistan parasites than for the metazoans, with haplosporidian richness being higher than Perkinsus richness. Perkinsus species were the most frequently detected and most geographically widespread among parasite groups. Parasite richness and overlap differed between regions, locations and oyster hosts. These results have important implications for tropical parasite richness and the dispersal of parasites due to shipping associated with the Panama Canal. [ABSTRACT FROM AUTHOR]

Thirty-six Brook Silversides, Labidesthes sicculus, and 15 Golden Silversides, Labidesthes vanhyningi, collected from the Ouachita and Red River drainage basins of Arkansas and Oklahoma, U.S.A., were examined for parasites. Six (17%) of the L. sicculus and 3 (20%) of the L. vanhyningi were infected with Dermocystidium sp., 1 (7%) L. vanhyningi harbored an immature tapeworm, Proteocephalus sp., and I (7%) L. vanhyningi was infected with the copepod Ergasilus fimduli. We document new host records for all 3 parasites as well as 2 new state records for Dermocystidium sp. and E. funduli. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

Durante este estudio se determinó la presencia del protozoario Perkinsus marinus en el ostión Crassostrea virginica en las lagunas de Mecoacán, Carmén y Machona en el estado de Tabasco al Susreste de México. Para obtener el articulo completo pueden contactar al Editor de la Revista Ecosistemas y Recursos Agropecuarios en el siguiente correo electrónico era@ujat.mx y se les enviará sin algún costo.

During the early 1950s, Sammy M. Ray discovered that his high-salt modification of fluid thioglycollate sterility test medium caused dramatic in vitro enlargement of Perkinsus marinus (= Dermocystidium marinum ) cells that coincidentally infected several experimentally cultured oyster gill tissue explants. Subsequent testing confirmed that the enlarged cells among some oyster tissues incubated in Ray’s fluid thioglycollate medium (RFTM) were those of that newly described oyster pathogen. Non-proliferative in vitro enlargement, cell wall thickening, and subsequent blue–black iodine-staining of hypertrophied trophozoites (=hypnospores = prezoosporangia) following incubation in RFTM are unique characteristics of confirmed members of the protistan genus Perkinsus . A number of in vitro assays and manipulations with RFTM have been developed for selective detection and enumeration of Perkinsus sp. cells in tissues of infected molluscs, and in environmental samples. RFTM-enlarged Perkinsus sp. cells from tissues of infected molluscs also serve as useful inocula for initiating in vitro isolate cultures, and cells of several Perkinsus spp. from both in vitro cultures and infected mollusc tissues may be induced to zoosporulate by brief incubations in RFTM. DNAs from RFTM-enlarged Perkinsus sp. cells provide useful templates for PCR amplifications, and for sequencing and other assays to differentiate and identify the detected Perkinsus species. We review the history and components of fluid thioglycollate and RFTM media, and the characteristics of numerous RFTM-based diagnostic assays that have been developed and used worldwide since 1952 for detection and identification of Perkinsus spp. in host mollusc tissues and environmental samples. We also review applications of RFTM for in vitro manipulations and purifications of Perkinsus sp. pathogen cells. [ABSTRACT FROM AUTHOR]

Prevalence of the protozoan Perkinsus spp. in the gills of the pleasure oyster Crassostrea corteziensis from two estuaries in Nayarit, Mexico, was measured. The protozoan was identified by PCR amplification of the internal transcribed spacer (ITS) region of the rDNA of Perkinsus spp. The pathogen was found in 92% of oysters from Boca de Camichín and 77% of oysters from Pozo Chino. ITS sequences characterized from C. corteziensis showed 96-100% similarity to Perkinsus marinus. The most frequent ITS sequence (GenBank ) had 100% identity with the ITS locus of P. marinus from New Jersey, Maryland, South Carolina and Texas, and the second most frequent observed sequence (GenBank ) was 100% identical to ITS sequences of P. marinus from New Jersey, South Carolina, Louisiana, and Bahía Kino, Sonora, Mexico. The 14 sequences from the non-transcribed spacer (NTS) showed 98% similarity to P. marinus from Texas. The most frequent polymorphism identified was at nucleotide 446 of the ITS region; however, the NTS showed the highest nucleotide diversity, thereby suggesting that this region is suitable for genotype identification. Moreover, the most conserved ITS marker is better for species-specific diagnosis. Both the ITS and NTS sequences of P. marinus obtained from C. corteziensis were grouped in two clades, identifying two allelic variants of P. marinus. [ABSTRACT FROM AUTHOR]