Susan E. Ford

Climate Change Influences on Marine Infectious Diseases: Implications for Management and Society

Infectious diseases are common in marine environments, but the effects of a changing climate on marine pathogens are not well understood. Here we review current knowledge about how the climate drives host-pathogen interactions and infectious disease outbreaks. Climate-related impacts on marine diseases are being documented in corals, shellfish, finfish, and humans; these impacts are less clearly linked for other organisms. Oceans and people are inextricably linked, and marine diseases can both directly and indirectly affect human health, livelihoods, and well-being. We recommend an adaptive management approach to better increase the resilience of ocean systems vulnerable to marine diseases in a changing climate. Land-based management methods of quarantining, culling, and vaccinating are not successful in the ocean; therefore, forecasting conditions that lead to outbreaks and designing tools/approaches to influence these conditions may be the best way to manage marine disease.

Evaluation of methods using ray's fluid thioglycollate medium for diagnosis of Perkinsus marinus infection in the eastern oyster, Crassostrea virginica

Abstract Accurate detection and quantification of parasite body burden are critical for understanding many aspects of host-parasite interactions. The standard assay for diagnosing Perkinsus marinus infections in Crassostrea virginica involves incubation of oyster tissue in Rays fluid thioglycollate medium (RFTM), followed by iodine staining and microscopic examination for parasites. The original RFTM tissue assay is destructive and provides only a ranked level of infection intensity. A recent modification provides a technique to enumerate P. marinus after incubation in RFTM and determines total body burden. Application of this technique to hemolymph samples has provided a nondestructive assay. We provide a critical evaluation of these three assays. Data from previous studies were not adequate to critically evaluate performance of the tissue and hemolymph assays. Sensitivity and accuracy of these assays were therefore compared against weight-standardized body burden in Delaware Bay oysters over the course of a year. Determination of total body burden was significantly more sensitive than the other assays. Neither tissue nor hemolymph assays provided accurate estimates of individual infection intensities and were insensitive at low infection levels. The tissue assay was easier to use and slightly more accurate than the hemolymph assay, and both provided reasonable estimates of average infection level in the population. In summary, total body burden assessment is recommended when highly accurate measures of infection intensity or prevalence are necessary, the tissue assay is recommended for monitoring epizootics because of its simplicity and accuracy at the population level, and the hemolymph assay is only recommended when oysters must be kept alive.

History and epizootiology of Haplosporidium nelsoni (MSX), an oyster pathogen in Delaware Bay, 1957–1980

Abstract Between 1957 and 1959, a previously unknown sporozoan parasite, now designated as Haplosporidium nelsoni (formerly Minchinia nelsoni ), or MSX, killed 90–95% of the oysters in lower Delaware Bay. Native oysters have been studied for more than 20 years since then to determine long-term disease and mortality patterns resulting from this host-parasite association. Development of resistance to MSX-kill in native oysters has reduced disease mortality to about half the original level, even though the pathogen continues to be very active in the bay. Since the initial epizootic, MSX levels have fluctuated in a cyclic pattern with peaks every 6 to 8 years. Periods of low disease pressure follow very cold winters, while average or above average winter temperatures correlate with high MSX activity. During peak years, every oyster in the lower bay may become infected. Although the parasite is salinity limited, salinities in the lower bay, the area from which oysters are marketed, are nearly always favorable for MSX, and fluctuations in river flow have almost no effect on MSX in this region. Infection periods recur each summer. Some oysters die soon after becoming infected; others survive through winter, but die in spring as the pathogen compounds normal overwinter stresses. Many survivors are able to suppress or rid themselves of infections when temperatures approach 20°C in late spring. Resistance to MSX-kill in native oysters is not correlated with an ability to prevent infection, but with restriction of parasites to localized, nonlethal lesions. The persistence of “hot spots” for infection in areas where oysters are sparse, the lack of spores in infected oysters, and failure to transmit the disease experimentally lead to the hypothesis that an alternate or reservoir host produces infective stages of MSX.

Haemocyte parameters associated with resistance to brown ring disease in Ruditapes spp. clams

Brown ring disease (BRD) is a shell disease caused by Vibrio tapetis. This pathogen disturbs the periostracal lamina causing the appearance of a brown conchiolin deposit on the inner face of the shell, within the extrapallial space. Although differences in resistance to BRD have been documented, their relationship to possible defense functions has never been investigated. In this study, flow cytometry was used to analyze cellular parameters in asymptomatic and experimentally infected Ruditapes philippinarum from France and the west coast of the USA. Parallel analyses were made on Ruditapes decussatus, the native European clam, which is highly resistant to BRD. In the haemolymph and extrapallial fluid of animals without BRD, total haemocyte counts, the percentage of granulocytes, and the phagocytic activity against latex beads or V. tapetis by the haemocytes were significantly higher in American R. philippinarum than in French R. philippinarum. In most cases, levels in R. decussatus were the highest of all three groups. Four weeks following challenge with V. tapetis, BRD prevalence reached 52 in American clams and 100% in French specimens, but only 37% in R. decussatus. In symptomatic animals, phagocytosis of V. tapetis increased significantly in the resistant species of clam, R. decussatus, was unchanged in US clams, and decreased significantly in FR specimens when compared to asymptomatic individuals from each population. Ingestion of V. tapetis by haemocytes in the extrapallial fluid, which is in contact with the periostracal lamina, could be the main defense mechanism used to counter the pathogen. Our results suggest that resistance to BRD may well be related to the concentration of granular haemocytes and the phagocytic activity of haemocytes.

INFECTION AND MORTALITY PATTERNS IN STRAINS OF OYSTERS CRASSOSTREA VIRGINICA SELECTED FOR RESISTANCE TO THE PARASITE HAPLOSPORIDIUM NELSONI (MSX)

Strains of oysters Crassostrea virginica resistant to mortality caused by the parasite Haplosporidium nelsoni (MSX) were developed and tested through 6 generations. In addition, strains in each generation were followed for up to 6 yr of continuous exposure to the parasite in nature. Selected strains responded to challenge by the parasite with gradually improved survival in successive generations. They were slower to develop patent infections than were unselected groups and were able to delay mortality after infections did develop, but under repeated exposure most oysters eventually died with H. nelsoni parasitism. Many selected strains, however, reached market size before significant mortalities occurred. The data suggest that resistance to H. nelsoni mortality is under the influence of many genes. No clear defense mechanism has been described and we hypothesize that resistance to H. nelsoni may, in part, involve a physiological state in which selected oysters temporarily fail to provide a suitable habitat for the parasite. Temporary insusceptibility would, in this view, be followed by an increased ability to tolerate the parasite when conditions for its development are present. Selection would then favor individuals that are able to prolong periods of insusceptibility and/or to carry out basic life processes while parasitized.

Haplosporidium nelsoni (MSX) on delaware bay seed oyster beds: A host-parasite relationship along a salinity gradient

Abstract Epizootic mortalities of oysters in Delaware and Chesapeake Bays during the late 1950s and early 1960s diminished in an upbay direction, indicating that the causative agent, the sporozoan parasite, Haplosporidium nelsoni (formerly Minchinia nelsoni), or MSX, was salinity limited. Since 1959, native Delawere Bay oysters have been sampled for presence of MSX and for mortality along a salinity gradient that ranges at mid-tide and mean river flow from 20 to 23 ppt on the lower bay planted grounds, and from 9 to 18 ppt on the upper bay seed beds. This study has clarified the relationship of salinity to the distribution and effect of the parasite in native oysters in the estuary. The sampling period included a drought in the mid-1960s when very low Delaware River flows produced high salinities in the upper bay. It also included a period of very high flows, and low salinities, in the early 1970s. On Arnolds and Cohansey, two of the uppermost seed beds, with mean salinities of 9 and 12 ppt, respectively, MSX activity was significant only during the drought. Farther downbay, at New Beds and Bennies Bed (15–16 ppt), the disease was present in all years, but with clearly elevated activity during the drought. On the planted grounds, however, where the effects of MSX are felt most strongly by the oyster industry, there was no correlation of disease levels with river flow. In fact, MSX prevalence was higher there in the early 1970s when river flows were high, than it was during the drought. When disease and mortality statistics for each location along the salinity gradient were pooled for the entire sampling period, two distinct patterns emerged: (1) Prevalence (proportion of oysters diagnosed as having MSX) showed a regular decrease from high to low salinity that paralleled the salt gradient. (2) However, infection intensity, a better measure of disease stress than prevalence, showed a sharp drop between the planting grounds and the seed beds, then no further change with lower salinity. MSX-related mortality followed a pattern similar to infection intensity: on a long-term average, 30% of all oysters have died with MSX infections during their first year after planting on the leased grounds. On the seed beds, regardless of location, annual disease-related kill was only 4 to 9%. Correlation of mortality rates with infection intensity rather than with prevalence is explained by the fact that infections must reach a certain intensity before they become lethal. The sharp drop in infection intensity and MSX-related mortality occurs between the upbay edge of the leased grounds and the lowermost seed bed, locations that are separated by less than 2 miles and an average of only 2 ppt salinity. This suggests a salinity threshold that has little effect on the distribution of infective stages of MSX or on their ability to infect, but that severely limits the parasites capacity to develop once it has entered the oyster.

VARIABILITY IN MOLLUSCAN HEMOCYTES : A FLOW CYTOMETRIC STUDY

Reported variability in numbers and relative proportions of hemocytes in marine bivalves may be related to environmental conditions and laboratory method differences. An automated identification assay, flow cytometry, removes much laboratory bias, but its usefulness is limited because the putative cell types in delineated subpopulations have never been confirmed. The present study was designed to: (1) confirm the identity of oyster hemocyte subpopulations described by flow cytometry, and (2) use flow cytometry in an experimental analysis of potential causes of variation. Light-scatter flow cytometry consistently differentiated three subpopulations in oysters from two mid-Atlantic (USA) sites. Cell sorting and microscopy identified them as granular, small granular, and agranular (hyalinocytes and apparently degranulated) hemocytes. Subpopulation proportions estimated by microscopy and by flow cytometry were significantly correlated (r(2) = 0.27 to 0.50). In a 4-week laboratory experiment, neither temperature (12 vs. 22 degrees C) nor food (fed vs. not fed) had a statistically significant effect on total or differential counts, or on hemocyte viability. Most of the variability was attributable to individual differences and was very similar to that reported for vertebrates. We hypothesize that variability in molluscan hemocytes may be more immediately linked to individual metabolic condition than to ambient changes.

Long-term Trends in Oyster Population Dynamics in Delaware Bay: Regime Shifts and Response to Disease

Abstract We evaluate a 54-y survey time series for the Delaware Bay oyster beds in New Jersey waters to identify the characteristics of regime shifts in oyster populations and the influence of MSX and Dermo diseases on population stability. Oyster abundance was high during the 1970s through 1985. Oyster abundance was low at the inception of the time series in 1953, remained low through 1969, and has been low since 1985 and very low since 2000. Natural mortality was low in most years prior to the appearance of MSX in 1957. From 1957 through 1966, natural mortality generally remained above 10% annually and twice exceeded 20%. Natural mortality remained well below 15% during the 1970s and into the early 1980s when oyster abundance was continuously high. The largest mortality event in the time series, an MSX epizootic that resulted in the death of 47% of the stock, occurred in 1985. Mortality rose again with the incursion of Dermo in 1990 and has remained above 15% for most years since that time and frequently has exceeded 20%. The primary impact of MSX and Dermo diseases has been to raise natural mortality and ultimately to cause a dispersed stock to retreat into its habitat of refuge in the moderately low salinity reach of the bay. The time series of oyster abundance on the New Jersey oyster beds of Delaware Bay is dominated by two regime shifts, the 1970 abundance increase that was maintained for about 15 y thereafter, and the 1985 abundance decrease that continues through today. These two regime shifts ushered in long-term periods of apparent constancy in population dynamics. The 1985 regime shift was induced by the largest MSX epizootic on record that produced high mortalities throughout a population distributed broadly throughout its habitat range after 15 y of high abundance. A putative new regime commenced circa 2000 as a consequence of a series of Dermo epizootics. Mortalities routinely exceeded 20% of the population annually during this period, with the consequence of a greater degree of stock consolidation than any previous time in the 54-y record. Extreme consolidation of the stock would appear to be a characteristic of the populations response to Dermo disease. The 1970–1984 and post-1985 regimes each were ushered in by a confluence of events unique in the 54-y time series. Each was characterized by a period of relative stability in population abundance. However, the stability in total population abundance belies a more dynamic process of stock redistribution during both time intervals, demonstrating that the appearance of constancy in stock abundance is not necessarily a result of invariant stock dynamics. Rather, the Delaware Bay oyster time series suggests that regime shifts delimit periods during which differential, often offsetting, local trends impart similar abundance levels, and thus constancy at the level of the stock masks substantive changes in local population dynamics potentially fostering future catastrophic changes in population-level attributes. Understanding such regime shifts will likely determine the success of decadal management goals more so than measures designed to influence population abundance.

Microarray analysis of gene expression in eastern oyster (Crassostrea virginica) reveals a novel combination of antimicrobial and oxidative stress host responses after dermo (Perkinsus marinus) challenge

Dermo disease, caused by Perkinsus marinus, is one of the most severe diseases of eastern oysters, Crassostrea virginica. It causes serious mortalities in both wild and aquacultured oysters. Using existing expressed sequence tag (EST) resources, we developed a 12K in situ oligonucleotide microarray and used it for the analysis of gene expression profiles of oysters during the interactions between P. marinus and its oyster host. Significant gene expression regulation was found at day 30 post-challenge in the eastern oyster. Putative identities of the differentially expressed genes revealed a set of genes involved in several processes including putative antimicrobial defenses, pathogen recognition and uptake, anti-oxidation and apoptosis. Consistent with results obtained from previous, smaller-scale experiments, expression profiles revealed a large set of genes likely involved in an active mitigating response to oxidative stress and apoptosis induced by P. marinus. Additionally, a unique galectin from C. virginica, CvGal, which serves as a preferential receptor for P. marinus trophozoites, was found to be significantly down-regulated in gill tissue of oysters with both light and heavy infection, suggesting an attempt to control parasite uptake and proliferation in the later stages of infection. Potential histone-derived antimicrobial responses to P. marinus were also revealed in the gene expression profiles.

Transcriptome analysis reveals strong and complex antiviral response in a mollusc.

Viruses are highly abundant in the oceans, and how filter-feeding molluscs without adaptive immunity defend themselves against viruses is not well understood. We studied the response of a mollusc Crassostrea gigas to Ostreid herpesvirus 1 µVar (OsHV-1μVar) infections using transcriptome sequencing. OsHV-1μVar can replicate extremely rapidly after challenge of C. gigas as evidenced by explosive viral transcription and DNA synthesis, which peaked at 24 and 48 h post-inoculation, respectively, accompanied by heavy oyster mortalities. At 120 h post-injection, however, viral gene transcription and DNA load, and oyster mortality, were greatly reduced indicating an end of active infections and effective control of viral replication in surviving oysters. Transcriptome analysis of the host revealed strong and complex responses involving the activation of all major innate immune pathways that are equipped with expanded and often novel receptors and adaptors. Novel Toll-like receptor (TLR) and MyD88-like genes lacking essential domains were highly up-regulated in the oyster, possibly interfering with TLR signal transduction. RIG-1/MDA5 receptors for viral RNA, interferon-regulatory factors, tissue necrosis factors and interleukin-17 were highly activated and likely central to the oysters antiviral response. Genes related to anti-apoptosis, oxidation, RNA and protein destruction were also highly up-regulated, while genes related to anti-oxidation were down-regulated. The oxidative burst induced by the up-regulation of oxidases and severe down-regulation of anti-oxidant genes may be important for the destruction of viral components, but may also exacerbate oyster mortality. This study provides unprecedented insights into antiviral response in a mollusc. The mobilization and complex regulation of expanded innate immune-gene families highlights the oyster genomes adaptation to a virus-rich marine environment.