Optimizing surveillance for early disease detection: Expert guidance for Ostreid herpesvirus surveillance design and system sensitivity calculation.

To keep pace with rising opportunities for disease emergence and spread, surveillance in aquaculture must enable the early detection of both known and new pathogens. Conventional surveillance systems (designed to provide proof of disease freedom) may not support detection outside of periodic sampling windows, leaving substantial blind spots to pathogens that emerge in other times and places. To address this problem, we organized an expert panel to envision optimal systems for early disease detection, focusing on Ostreid herpesvirus 1 (OsHV-1), a pathogen of panzootic consequence to oyster industries. The panel followed an integrative group process to identify and weight surveillance system traits perceived as critical to the early detection of OsHV-1. Results offer a road map with fourteen factors to consider when building surveillance systems geared to early detection; factor weights can be used by planners and analysts to compare the relative value of different designs or enhancements. The results were also used to build a simple, but replicable, model estimating the system sensitivity (SSe) of observational surveillance and, in turn, the confidence in disease freedom that negative reporting can provide. Findings suggest that optimally designed observational systems can contribute substantially to both early detection and disease freedom confidence. In contrast, active surveillance as a singular system is likely insufficient for early detection. The strongest systems combined active with observational surveillance and engaged joint industry and government involvement: results suggest that effective partnerships can generate highly sensitive systems, whereas ineffective partnerships may seriously erode early detection capability. Given the costs of routine testing, and the value (via averted losses) of early detection, we conclude that observational surveillance is an important and potentially very effective tool for health management and disease prevention on oyster farms, but one that demands careful planning and participation. This evaluation centered on OsHV-1 detection in farmed oyster populations. However, many of the features likely generalize to other pathogens and settings, with the important caveat that the pathogens need to manifest via morbidity or mortality events in the species, life stages and environments under observation.

The first detection of a novel OsHV-1 microvariant in San Diego, California, USA.

The spread, emergence, and adaptation of pathogens causing marine disease has been problematic to fisheries and aquaculture industries for the last several decades creating the need for strategic management and biosecurity practices. The Pacific oyster (Crassostrea gigas), a highly productive species globally, has been a target of disease and mortality caused by a viral pathogen, the Ostreid herpesvirus 1 (OsHV-1) and its microvariants (OsHV-1 µvars). During routine surveillance to establish health history at a shellfish aquaculture nursery system in San Diego, California, the presence of OsHV-1 in Pacific oyster juveniles was detected. Quantification of OsHV-1 in tissues of oysters revealed OsHV-1 viral loads > 106 copies/mg. We characterized and identified the OsHV-1 variant by sequencing of ORFs 4 (C2/C6) and 43 (IA1/IA2), which demonstrated that this variant is a novel OsHV-1 microvariant: OsHV-1 µvar SD. A pilot transmission study indicates that OsHV-1 µvar SD is infectious with high viral loads ~ 7.57 × 106 copies/mg detected in dead individuals. The detection of OsHV-1 µvar SD in a large port mirrors previous studies conducted in Australia where aquaculture farms and feral populations near port locations may be at a higher risk of OsHV-1 emergence. Further research is needed to understand the impacts of OsHV-1 µvar SD, such as transmission studies focusing on potential vectors and characterization of virulence as compared to other OsHV-1 µvars. To increase biosecurity of the global aquaculture industry, active and passive surveillance may be necessary to reduce spread of pathogens and make appropriate management decisions.

Differential Mortality and High Viral Load in Naive Pacific Oyster Families Exposed to OsHV-1 Suggests Tolerance Rather than Resistance to Infection.

Pacific oysters, Crassostrea gigas, are one of the most productive aquaculture species in the world. However, they are threatened by the spread of Ostreid herpesvirus-1 (OsHV-1) and its microvariants (collectively "µvars"), which cause mass mortalities in all life stages of Pacific oysters globally. Breeding programs have been successful in reducing mortality due to OsHV-1 variants following viral outbreaks; however, an OsHV-1-resistant oyster line does not yet exist in the United States (US), and it is unknown how OsHV-1 µvars will affect US oyster populations compared to the current variant, which is similar to the OsHV-1 reference, found in Tomales Bay, CA. The goals of this study were to investigate the resistance of C. gigas juveniles produced by the Molluscan Broodstock Program (MBP) to three variants of OsHV-1: a California reference OsHV-1, an Australian µvar, and a French µvar. This is the first study to directly compare OsHV-1 µvars to a non-µvar. The survival probability of oysters exposed to the French (FRA) or Australian (AUS) µvar was significantly lower (43% and 71%, respectively) than to the reference variant and controls (96%). No oyster family demonstrated resistance to all three OsHV-1 variants, and many surviving oysters contained high copy numbers of viral DNA (mean ~3.53 × 108). These results indicate that the introduction of OsHV-1 µvars could have substantial effects on US Pacific oyster aquaculture if truly resistant lines are not achieved, and highlight the need to consider resistance to infection in addition to survival as traits in breeding programs to reduce the risk of the spread of OsHV-1 variants.

Unraveling concordant and varying responses of oyster species to Ostreid Herpesvirus 1 variants.

The Ostreid herpesvirus 1 (OsHV-1) and variants, particularly the microvariants (µVars), are virulent and economically devastating viruses impacting oysters. Since 2008 OsHV-1 µVars have emerged rapidly having particularly damaging effects on aquaculture industries in Europe, Australia and New Zealand. We conducted field trials in Tomales Bay (TB), California where a non-µVar strain of OsHV-1 is established and demonstrated differential mortality of naturally exposed seed of three stocks of Pacific oyster, Crassostrea gigas, and one stock of Kumamoto oyster, C. sikamea. Oysters exposed in the field experienced differential mortality that ranged from 64 to 99% in Pacific oysters (Tasmania>Midori = Willapa stocks), which was much higher than that of Kumamoto oysters (25%). Injection trials were done using French (FRA) and Australian (AUS) µVars with the same oyster stocks as planted in the field and, in addition, two stocks of the Eastern oyster, C. virginica. No mortality was observed in control oysters. One C. virginica stock suffered ~10% mortality when challenged with both µVars tested. Two Pacific oyster stocks suffered 75 to 90% mortality, while one C. gigas stock had relatively low mortality when challenged with the AUS µVar (~22%) and higher mortality when challenged with the French µVar (~72%). Conversely, C. sikamea suffered lower mortality when challenged with the French µVar (~22%) and higher mortality with the AUS µVar (~44%). All dead oysters had higher viral loads (~1000×) as measured by quantitative PCR relative to those that survived. However, some survivors had high levels of virus, including those from species with lower mortality. Field mortality in TB correlated with laboratory mortality of the FRA µVar (69% correlation) but not with that of the AUS µVar, which also lacked correlation with the FRA µVar. The variation in response to OsHV-1 variant challenges by oyster species and stocks demonstrates the need for empirical assessment of multiple OsHV-1 variants.

First comparison of French and Australian OsHV-1 µvars by bath exposure.

Economically devastating mortality events of farmed and wild shellfish due to infectious disease have been reported globally. Currently, one of the most significant disease threats to Pacific oyster Crassostrea gigas culture is the ostreid herpesvirus 1 (OsHV-1), in particular the emerging OsHV-1 microvariant genotypes. OsHV-1 microvariants (OsHV-1 µvars) are spreading globally, and concern is high among growers in areas unaffected by OsHV-1. No study to date has compared the relative virulence among variants. We provide the first challenge study comparing survival of naïve juvenile Pacific oysters exposed to OsHV-1 µvars from Australia (AUS µvar) and France (FRA µvar). Oysters challenged with OsHV-1 µvars had low survival (2.5% exposed to AUS µvar and 10% to FRA µvar), and high viral copy number as compared to control oysters (100% survival and no virus detected). As our study was conducted in a quarantine facility located ~320 km from the ocean, we also compared the virulence of OsHV-1 µvars using artificial seawater made from either facility tap water (3782 µmol kg-1 seawater total alkalinity) or purchased distilled water (2003 µmol kg-1). Although no differences in survival or viral copy number were detected in oysters exposed to seawater made using tap or distilled water, more OsHV-1 was detected in tanks containing the lower-alkalinity seawater, indicating that water quality may be important for virus transmission, as it may influence the duration of viral viability outside of the host.

Ecological Factors Mediate Immunity and Parasitic Co-Infection in Sea Fan Octocorals.

The interplay among environment, demography, and host-parasite interactions is a challenging frontier. In the ocean, fundamental changes are occurring due to anthropogenic pressures, including increased disease outbreaks on coral reefs. These outbreaks include multiple parasites, calling into question how host immunity functions in this complex milieu. Our work investigates the interplay of factors influencing co-infection in the Caribbean sea fan octocoral, Gorgonia ventalina, using metrics of the innate immune response: cellular immunity and expression of candidate immune genes. We used existing copepod infections and live pathogen inoculation with the Aspergillus sydowii fungus, detecting increased expression of the immune recognition gene Tachylectin 5A (T5A) in response to both parasites. Cellular immunity increased by 8.16% in copepod infections compared to controls and single Aspergillus infections. We also detected activation of cellular immunity in reef populations, with a 13.6% increase during copepod infections. Cellular immunity was similar in the field and in the lab, increasing with copepod infections and not the fungus. Amoebocyte density and the expression of T5A and a matrix metalloproteinase (MMP) gene were also positively correlated across all treatments and colonies, irrespective of parasitic infection. We then assessed the scaling of immune metrics to population-level disease patterns and found random co-occurrence of copepods and fungus across 15 reefs in Puerto Rico. The results suggest immune activation by parasites may not alter parasite co-occurrence if factors other than immunity prevail in structuring parasite infection. We assessed non-immune factors in the field and found that sea fan colony size predicted infection by the copepod parasite. Moreover, the effect of infection on immunity was small relative to that of site differences and live coral cover, and similar to the effect of reproductive status. While additional immune data would shed light on the extent of this pattern, ecological factors may play a larger role than immunity in controlling parasite patterns in the wild. Parsing the effects of immunity and ecological factors in octocoral co-infection shows how disease depends on more than one host and one parasite and explores the application of co-infection research to a colonial marine organism.

First evaluation of resistance to both a California OsHV-1 variant and a French OsHV-1 microvariant in Pacific oysters.

BACKGROUND: Variants of the Ostreid herpesvirus 1 (OsHV-1) cause high losses of Pacific oysters globally, including in Tomales Bay, California, USA. A suite of new variants, the OsHV-1 microvariants (µvars), cause very high mortalities of Pacific oysters in major oyster-growing regions outside of the United States. There are currently no known Pacific oysters in the United States that are resistant to OsHV-1 as resistance has yet to be evaluated in these oysters. As part of an effort to begin genetic selection for resistance to OsHV-1, 71 families from the Molluscan Broodstock Program, a US West Coast Pacific oyster breeding program, were screened for survival after exposure to OsHV-1 in Tomales Bay. They were also tested in a quarantine laboratory in France where they were exposed to a French OsHV-1 microvariant using a plate assay, with survival recorded from three to seven days post-infection. RESULTS: Significant heritability for survival were found for all time points in the plate assay and in the survival phenotype from a single mortality count in Tomales Bay. Genetic correlations between survival against the French OsHV-1 µvar in the plate assay and the Tomales Bay variant in the field trait were weak or non-significant. CONCLUSIONS: Future breeding efforts will seek to validate the potential of genetic improvement for survival to OsHV-1 through selection using the Molluscan Broodstock Program oysters. The lack of a strong correlation in survival between OsHV-1 variants under this study's exposure conditions may require independent selection pressure for survival to each variant in order to make simultaneous genetic gains in resistance.

Oysters and eelgrass: potential partners in a high pCO2 ocean.

Climate change is affecting the health and physiology of marine organisms and altering species interactions. Ocean acidification (OA) threatens calcifying organisms such as the Pacific oyster, Crassostrea gigas. In contrast, seagrasses, such as the eelgrass Zostera marina, can benefit from the increase in available carbon for photosynthesis found at a lower seawater pH. Seagrasses can remove dissolved inorganic carbon from OA environments, creating local daytime pH refugia. Pacific oysters may improve the health of eelgrass by filtering out pathogens such as Labyrinthula zosterae (LZ), which causes eelgrass wasting disease (EWD). We examined how co-culture of eelgrass ramets and juvenile oysters affected the health and growth of eelgrass and the mass of oysters under different pCO2 exposures. In Phase I, each species was cultured alone or in co-culture at 12°C across ambient, medium, and high pCO2 conditions, (656, 1,158 and 1,606 µatm pCO2 , respectively). Under high pCO2 , eelgrass grew faster and had less severe EWD (contracted in the field prior to the experiment). Co-culture with oysters also reduced the severity of EWD. While the presence of eelgrass decreased daytime pCO2 , this reduction was not substantial enough to ameliorate the negative impact of high pCO2 on oyster mass. In Phase II, eelgrass alone or oysters and eelgrass in co-culture were held at 15°C under ambient and high pCO2 conditions, (488 and 2,013 µatm pCO2 , respectively). Half of the replicates were challenged with cultured LZ. Concentrations of defensive compounds in eelgrass (total phenolics and tannins), were altered by LZ exposure and pCO2 treatments. Greater pathogen loads and increased EWD severity were detected in LZ exposed eelgrass ramets; EWD severity was reduced at high relative to low pCO2 . Oyster presence did not influence pathogen load or EWD severity; high LZ concentrations in experimental treatments may have masked the effect of this treatment. Collectively, these results indicate that, when exposed to natural concentrations of LZ under high pCO2 conditions, eelgrass can benefit from co-culture with oysters. Further experimentation is necessary to quantify how oysters may benefit from co-culture with eelgrass, examine these interactions in the field and quantify context-dependency.

The Use of Filter-feeders to Manage Disease in a Changing World.

Rapid environmental change is linked to increases in aquatic disease heightening the need to develop strategies to manage disease. Filter-feeding species are effective biofilters and can naturally mitigate disease risk to humans and wildlife. We review the role of filter-feeders, with an emphasis on bivalves, in altering disease outcomes via augmentation and reduction. Filtration can reduce transmission by removing pathogens from the water column via degradation and release of pathogens in pseudofeces. In other cases, filtration can increase pathogen transmission and disease risk. The effect of filtration on pathogen transmission depends on the selectivity of the filter-feeder, the degree of infectivity by the pathogen, the mechanism(s) of pathogen transmission and the ability of the pathogen to resist degradation. For example, some bacteria and viruses can resist degradation and accumulate within a filter-feeder leading to disease transmission to humans and other wildlife upon ingestion. Since bivalves can concentrate microorganisms, they are also useful as sentinels for the presence of pathogenic microorganisms. While somewhat less studied, other invertebrates, including ascidians and sponges may also provide ecosystem services by altering pathogen transmission. In all scenarios, climate change may affect the potential for filter-feeders to mitigate disease risk. We conclude that an assessment including empirical data and modeling of system-wide impacts should be conducted before selection of filter-feeders to mitigate disease. Such studies should consider physiology of the host and microbe and risk factors for negative impacts including augmentation of other pathogens.

Plant characteristics associated with widespread variation in eelgrass wasting disease.

Seagrasses are ecosystem engineers of essential marine habitat. Their populations are rapidly declining worldwide. One potential cause of seagrass population declines is wasting disease, which is caused by opportunistic pathogens in the genus Labyrinthula. While infection with these pathogens is common in seagrasses, theory suggests that disease only occurs when environmental stressors cause immunosuppression of the host. Recent evidence suggests that host factors may also contribute to disease caused by opportunistic pathogens. In order to quantify patterns of disease, identify risk factors, and investigate responses to infection, we surveyed shoot density, shoot length, epiphyte load, production of plant defenses (phenols), and wasting disease prevalence in eelgrass Zostera marina across 11 sites in the central Salish Sea (Washington state, USA), a region where both wasting disease and eelgrass declines have been documented. Wasting disease was diagnosed by the presence of necrotic lesions, and Labyrinthula cells were identified with histology. Disease prevalence among sites varied from 6 to 79%. The probability of a shoot being diseased was higher in longer shoots, in patches of higher shoot density, and in shoots with higher levels of biofouling from epiphytes. Phenolic concentration was higher in diseased leaves. We hypothesize that this results from the induction of phenols during infection. Additional research is needed to evaluate whether phenols are an adaptive defense against Labyrinthula infection. The high site-level variation in disease prevalence emphasizes the potential for wasting disease to be causing some of the observed decline in eelgrass beds.

Complementary approaches to diagnosing marine diseases: a union of the modern and the classic.

Linking marine epizootics to a specific aetiology is notoriously difficult. Recent diagnostic successes show that marine disease diagnosis requires both modern, cutting-edge technology (e.g. metagenomics, quantitative real-time PCR) and more classic methods (e.g. transect surveys, histopathology and cell culture). Here, we discuss how this combination of traditional and modern approaches is necessary for rapid and accurate identification of marine diseases, and emphasize how sole reliance on any one technology or technique may lead disease investigations astray. We present diagnostic approaches at different scales, from the macro (environment, community, population and organismal scales) to the micro (tissue, organ, cell and genomic scales). We use disease case studies from a broad range of taxa to illustrate diagnostic successes from combining traditional and modern diagnostic methods. Finally, we recognize the need for increased capacity of centralized databases, networks, data repositories and contingency plans for diagnosis and management of marine disease.

Up in Arms: Immune and Nervous System Response to Sea Star Wasting Disease.

Echinoderms, positioned taxonomically at the base of deuterostomes, provide an important system for the study of the evolution of the immune system. However, there is little known about the cellular components and genes associated with echinoderm immunity. The 2013-2014 sea star wasting disease outbreak is an emergent, rapidly spreading disease, which has led to large population declines of asteroids in the North American Pacific. While evidence suggests that the signs of this disease, twisting arms and lesions, may be attributed to a viral infection, the host response to infection is still poorly understood. In order to examine transcriptional responses of the sea star Pycnopodia helianthoides to sea star wasting disease, we injected a viral sized fraction (0.2 µm) homogenate prepared from symptomatic P. helianthoides into apparently healthy stars. Nine days following injection, when all stars were displaying signs of the disease, specimens were sacrificed and coelomocytes were extracted for RNA-seq analyses. A number of immune genes, including those involved in Toll signaling pathways, complement cascade, melanization response, and arachidonic acid metabolism, were differentially expressed. Furthermore, genes involved in nervous system processes and tissue remodeling were also differentially expressed, pointing to transcriptional changes underlying the signs of sea star wasting disease. The genomic resources presented here not only increase understanding of host response to sea star wasting disease, but also provide greater insight into the mechanisms underlying immune function in echinoderms.

Whole transcriptome analysis reveals changes in expression of immune-related genes during and after bleaching in a reef-building coral.

Climate change is negatively affecting the stability of natural ecosystems, especially coral reefs. The dissociation of the symbiosis between reef-building corals and their algal symbiont, or coral bleaching, has been linked to increased sea surface temperatures. Coral bleaching has significant impacts on corals, including an increase in disease outbreaks that can permanently change the entire reef ecosystem. Yet, little is known about the impacts of coral bleaching on the coral immune system. In this study, whole transcriptome analysis of the coral holobiont and each of the associate components (i.e. coral host, algal symbiont and other associated microorganisms) was used to determine changes in gene expression in corals affected by a natural bleaching event as well as during the recovery phase. The main findings include evidence that the coral holobiont and the coral host have different responses to bleaching, and the host immune system appears suppressed even a year after a bleaching event. These results support the hypothesis that coral bleaching changes the expression of innate immune genes of corals, and these effects can last even after recovery of symbiont populations. Research on the role of immunity on coral's resistance to stressors can help make informed predictions on the future of corals and coral reefs.

Densovirus associated with sea-star wasting disease and mass mortality.

Populations of at least 20 asteroid species on the Northeast Pacific Coast have recently experienced an extensive outbreak of sea-star (asteroid) wasting disease (SSWD). The disease leads to behavioral changes, lesions, loss of turgor, limb autotomy, and death characterized by rapid degradation ("melting"). Here, we present evidence from experimental challenge studies and field observations that link the mass mortalities to a densovirus (Parvoviridae). Virus-sized material (i.e., <0.2 µm) from symptomatic tissues that was inoculated into asymptomatic asteroids consistently resulted in SSWD signs whereas animals receiving heat-killed (i.e., control) virus-sized inoculum remained asymptomatic. Viral metagenomic investigations revealed the sea star-associated densovirus (SSaDV) as the most likely candidate virus associated with tissues from symptomatic asteroids. Quantification of SSaDV during transmission trials indicated that progression of SSWD paralleled increased SSaDV load. In field surveys, SSaDV loads were more abundant in symptomatic than in asymptomatic asteroids. SSaDV could be detected in plankton, sediments and in nonasteroid echinoderms, providing a possible mechanism for viral spread. SSaDV was detected in museum specimens of asteroids from 1942, suggesting that it has been present on the North American Pacific Coast for at least 72 y. SSaDV is therefore the most promising candidate disease agent responsible for asteroid mass mortality.

Host demography influences the prevalence and severity of eelgrass wasting disease.

Many marine pathogens are opportunists, present in the environment, but causing disease only under certain conditions such as immunosuppression due to environmental stress or host factors such as age. In the temperate eelgrass Zostera marina, the opportunistic labyrinthulomycete pathogen Labyrinthula zosterae is present in many populations and occasionally causes severe epidemics of wasting disease; however, risk factors associated with these epidemics are unknown. We conducted both field surveys and experimental manipulations to examine the effect of leaf age (inferred from leaf size) on wasting disease prevalence and severity in Z. marina across sites in the San Juan Archipelago, Washington, USA. We confirmed that lesions observed in the field were caused by active Labyrinthula infections both by identifying the etiologic agent through histology and by performing inoculations with cultures of Labyrinthula spp. isolated from observed lesions. We found that disease prevalence increased at shallower depths and with greater leaf size at all sites, and this effect was more pronounced at declining sites. Experimental inoculations with 2 strains of L. zosterae confirmed an increased susceptibility of older leaves to infection. Overall, this pattern suggests that mature beds and shallow beds of eelgrass may be especially susceptible to outbreaks of wasting disease. The study highlights the importance of considering host and environmental factors when evaluating risk of disease from opportunistic pathogens.

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.

Immune response of the Caribbean sea fan, Gorgonia ventalina, exposed to an Aplanochytrium parasite as revealed by transcriptome sequencing.

Coral reef communities are undergoing marked declines due to a variety of stressors including disease. The sea fan coral, Gorgonia ventalina, is a tractable study system to investigate mechanisms of immunity to a naturally occurring pathogen. Functional studies in Gorgonia ventalina immunity indicate that several key pathways and cellular components are involved in response to natural microbial invaders, although to date the functional and regulatory pathways remain largely un-described. This study used short-read sequencing (Illumina GAIIx) to identify genes involved in the response of G. ventalina to a naturally occurring Aplanochytrium spp. parasite. De novo assembly of the G. ventalina transcriptome yielded 90,230 contigs of which 40,142 were annotated. RNA-Seq analysis revealed 210 differentially expressed genes in sea fans exposed to the Aplanochytrium parasite. Differentially expressed genes involved in immunity include pattern recognition molecules, anti-microbial peptides, and genes involved in wound repair and reactive oxygen species formation. Gene enrichment analysis indicated eight biological processes were enriched representing 36 genes, largely involved with protein translation and energy production. This is the first report using high-throughput sequencing to characterize the host response of a coral to a natural pathogen. Furthermore, we have generated the first transcriptome for a soft (octocoral or non-scleractinian) coral species. Expression analysis revealed genes important in invertebrate innate immune pathways, as well as those whose role is previously un-described in cnidarians. This resource will be valuable in characterizing G. ventalina immune response to infection and co-infection of pathogens in the context of environmental change.

Special issue Oceans and Humans Health: the ecology of marine opportunists.

Opportunistic marine pathogens, like opportunistic terrestrial pathogens, are ubiquitous in the environment (waters, sediments, and organisms) and only cause disease in immune-compromised or stressed hosts. In this review, we discuss four host-pathogen interactions within the marine environment that are typically considered opportunistic: sea fan coral-fungus, eelgrass-Labyrinthula zosterae, sea fan-Labyrinthulomycetes, and hard clam-Quahog Parasite Unknown with particular focus on disease ecology, parasite pathology, host response, and known associated environmental conditions. Disease is a natural part of all ecosystems; however, in some cases, a shift in the balance between the host, pathogen, and the environment may lead to epizootics in natural or cultured populations. In marine systems, host-microbe interactions are less understood than their terrestrial counterparts. The biological and physical changes to the world's oceans, coupled with other anthropogenic influences, will likely lead to more opportunistic diseases in the marine environment.

Friend or foe: the association of Labyrinthulomycetes with the Caribbean sea fan Gorgonia ventalina.

A new syndrome in sea fans Gorgonia ventalina consisting of multifocal purple spots (MFPS) has been observed in the Caribbean Sea. Surveys of MFPS on sea fans were conducted from 2006 to 2010 at a shallow and deep site in La Parguera, Puerto Rico (PR). At the shallow site, MFPS increased between 2006 and 2010 (site average ranged from 8 to 23%), with differences found at depths over time using an analysis of covariance (ANCOVA, p < 0.0001). As a potential causative agent we examined a Labyrinthulomycota-like ovoid parasite that was observed to be abundant in MFPS lesions in light micrographs. Labyrinhylomycetes were successfully isolated, cultured and characterized in sea fans from Florida and PR. Sequence information obtained from the small subunit (SSU) rRNA gene indicated that Labyrinthulomycetes in most sea fans (healthy and MFPS sea fans from Florida; MFPS from PR) and the cultured microorganism are in the genus Aplanochytrium, although some healthy sea fans from PR contained members of the genus Thraustochytrium. Both genera fall within the family Thraustochytriidae. Histology confirmed observations of thraustochytrids within apparently healthy and MFPS sea fans from PR, and specific staining indicated a host melanization response only in colonies containing Labyrinthulomycetes or fungal infections. Growth trials indicate that the temperature-growth optima for the cultured microorganism is ~30°C. In inoculation experiments, the cultured Aplanochytrium did not induce purple spots, and histology revealed that many of the apparently healthy recipients contained Labyrinthulomycetes prior to inoculation. Taken together, these results indicate that the Labyrinthulomycetes associated with sea fans is likely an opportunistic pathogen. Further studies are needed to understand the pathogenesis of this microorganism in sea fans and its relationship with MFPS.

Quantifying Ostreid herpesvirus (OsHV-1) genome copies and expression during transmission.

Understanding the pathogenic potential of a new pathogen strain or a known pathogen in a new locale is crucial for management of disease in both wild and farmed animals. The Ostreid herpesvirus-1 (OsHV-1), a known pathogen of early-life-stage Pacific oysters, Crassostrea gigas, has been associated with mortalities of juvenile oysters in many locations around the world including Tomales Bay, California. In two trials, the California OsHV-1 strain was transmitted from infected juvenile C. gigas to naïve C. gigas larvae. Survival of control larvae was high throughout both trials (97-100%) and low among those exposed to OsHV-1. No OsHV-1-exposed larvae survived to day 9 in trial 1, while trial 2 was terminated at day 7 when survival was 36.90 ± 8.66%. To assess the amount of OsHV-1 DNA present, we employed quantitative polymerase chain reaction (qPCR) assays based on the A fragment and OsHV-1 catalytic subunit of a DNA polymerase δ (DNA pol) gene. Viral genome copy numbers based on qPCR assays peaked between 3 and 5 days. To measure the presence of viable and actively transcribing virus, the DNA pol gene qPCR assay was optimized for RNA analysis after being reverse transcribed (RT-qPCR). A decline in virus gene expression was measured using RT-qPCR: relative to earlier experimental time points copy numbers were significantly lower on day 9, trial 1 (p < 0.05) and day 7, trial 2 (p < 0.05). Peaks in copies of active virus per genome occurred during two periods in trial 1 (days 1 and 5/7, p < 0.05) and one period in trial 2 (day 1, p < 0.05). Transmission electron microscopy confirmed OsHV-1 infection; herpesvirus-like nucleocapsids, capsids, and extracellular particles were visualized. We demonstrated the ability to transmit OsHV-1 from infected juvenile oysters to naïve larvae, which indicates the spread of OsHV-1 between infected hosts in the field and between commercial farms is possible. We also developed an important tool (OsHV-1-specific RT-qPCR for an active virus gene) for use in monitoring for active virus in the field and in laboratory based transmission experiments.