A rapid phenotype change in the pathogen Perkinsus marinus was associated with a historically significant marine disease emergence in the eastern oyster

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.

CRISPR/Cas9 Ribonucleoprotein-Based Genome Editing Methodology in the Marine Protozoan Parasite Perkinsus marinus

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.

Environmental factors drive 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 parasites from host to water column. In order to elucidate how environmental factors can influence the abundance of this parasite in the water column, we conducted a series of experiments testing the effects of time of day, temperature, and salinity on release of P. marinus cells from infected oysters. We found that P. marinus cells were released on a diurnal cycle, with most cells released during the hottest and brightest period of the day (12:00-18:00). Temperature also had a strong and immediate effect on number of cells released, but salinity did not, only influencing the intensity of infection over the course of several months. Taken together, our results demonstrate that 1) the number of parasites in the water column fluctuates according to a diurnal cycle, 2) temperature and salinity act on different timescales to influence parasite abundance, and 3) live infected oysters may substantially contribute to the abundance of transmissive parasites in the water column under particular environmental conditions.

Accommodation of the sex-ratio in eastern oysters Crassostrea virginica to variation in growth and mortality across the estuarine salinity gradient

Protandric oysters generate a relatively uniform reproductive potential over a wide range of environmental conditions that impose variations in growth rate and life span. Sex-at-length keys applied to survey data show that the female fraction routinely fell between 0.4 and 0.5, regardless of location in the salinity gradient. About 70% of population biomass is female over the same salinity range. Due to the necessary local modulation of the rate of male-to-female conversion to limit the influence of varying growth and life span over the salinity gradient, the number of males always exceeds by a small amount the number of females; thus improving the likelihood of a female having neighbouring males, a necessity for an immobile broadcast spawner. However, oysters at the extremes of the estuarine gradient all yielded populations with divergent sex-ratios. One consequence of reduced generation time brought about by increased mortality from disease should be selection favouring the switch from male to female at smaller size, if disease mortality is strongly female-biased. The site with the longest record of high mortality manifests such an increase. Sites up coastal rivers, putative refuges from disease, harbour animals with the slowest male-to-female conversion rates. Arguably these animals are most similar to the ancestral oyster pre-disease. Marketed animals range from 62% to 69% female. The principal influence of the fishery, and of oyster disease, would seem to be a reduction in lifetime egg production. Dermo disease may have reduced lifetime fecundity of females by nearly a factor of four.