Understanding Crassostrea virginica tolerance of Perkinsus marinus through global gene expression analysis.

Disease tolerance, a host's ability to limit damage from a given parasite burden, is quantified by the relationship between pathogen load and host survival or reproduction. Dermo disease, caused by the protozoan parasite P. marinus, negatively impacts survival in both wild and cultured eastern oyster (C. virginica) populations. Resistance to P. marinus has been the focus of previous studies, but tolerance also has important consequences for disease management in cultured and wild populations. In this study we measured dermo tolerance and evaluated global expression patterns of two sensitive and two tolerant eastern oyster families experimentally challenged with distinct doses of P. marinus (0, 106, 107, and 108 parasite spores per gram wet weight, n = 3-5 individuals per family per dose). Weighted Gene Correlation Network Analysis (WGCNA) identified several modules correlated with increasing parasite dose/infection intensity, as well as phenotype. Modules positively correlated with dose included transcripts and enriched GO terms related to hemocyte activation and cell cycle activity. Additionally, these modules included G-protein coupled receptor, toll-like receptor, and tumor necrosis factor pathways, which are important for immune effector molecule and apoptosis activation. Increased metabolic activity was also positively correlated with treatment. The module negatively correlated with infection intensity was enriched with GO terms associated with normal cellular activity and growth, indicating a trade-off with increased immune response. The module positively correlated with the tolerant phenotype was enriched for transcripts associated with "programmed cell death" and contained a large number of tripartite motif-containing proteins. Differential expression analysis was also performed on the 108 dosed group using the most sensitive family as the comparison reference. Results were consistent with the network analysis, but signals for "programmed cell death" and serine protease inhibitors were stronger in one tolerant family than the other, suggesting that there are multiple avenues for disease tolerance. These results provide new insight for defining dermo response traits and have important implications for applying selective breeding for disease management.

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.

Perkinsus marinus suppresses in vitro eastern oyster apoptosis via IAP-dependent and caspase-independent pathways involving TNFR, NF-kB, and oxidative pathway crosstalk.

The protozoan parasite Perkinsus marinus causes Dermo disease in eastern oysters, Crassostrea virginica, and can suppress apoptosis of infected hemocytes using incompletely understood mechanisms. This study challenged hemocytes in vitro with P. marinus for 1 h in the presence or absence of caspase inhibitor Z-VAD-FMK or Inhibitor of Apoptosis protein (IAP) inhibitor GDC-0152. Hemocytes exposure to P. marinus significantly reduced granulocyte apoptosis, and pre-incubation with Z-VAD-FMK did not affect P. marinus-induced apoptosis suppression. Hemocyte pre-incubation with GDC-0152 prior to P. marinus challenge further reduced apoptosis of granulocytes with engulfed parasite, but not mitochondrial permeabilization. This suggests P. marinus-induced apoptosis suppression may be caspase-independent, affect an IAP-involved pathway, and occur downstream of mitochondrial permeabilization. P. marinus challenge stimulated hemocyte differential expression of oxidation-reduction, TNFR, and NF-kB pathways. WGCNA analysis of P. marinus expression in response to hemocyte exposure revealed correlated protease, kinase, and hydrolase expression that could contribute to P. marinus-induced apoptosis suppression.