Hiding in plain sight: Shellfish-killing phytoplankton in Washington State.

Summer bivalve shellfish mortalities have been observed in Puget Sound for nearly a century and attempts to understand and mitigate these losses have been only partially successful. Likewise, the understanding of the environmental conditions triggering shellfish mortalities and successful strategies for their mitigation are incomplete. In the literature, phytoplankton have played only a cursory role in summer shellfish mortalities in Washington State because spawning stress and bacteria were thought to be the primary causes. In recent years, the occurrence of Protoceratium reticulatum (Claparede & Lachmann) Buetschli and Akashiwo sanguinea (Hirasaka) Hansen & Moestrup, have been documented by the SoundToxins research and monitoring partnership in increasing numbers and duration and have been associated with declining shellfish health or mortality at various sites in Puget Sound. Blooms of these species occur primarily in summer months and have been shown to cause mass mortalities of shellfish in the U.S. and other parts of the world. In 2016-2017, yessotoxins (YTX) were measured in several species of Puget Sound bivalve shellfish, with a maximum concentration of 2.20 mg/kg in blue mussels, a value below the regulatory limit of 3.75 mg/kg established by the European Union for human health protection but documented to cause shellfish mortalities in other locations around the world. In July 2019, a bloom of P. reticulatum coincided with a summer shellfish mortality event, involving a dramatic surfacing of stressed, gaping Manila clams, suggesting that YTX could be the cause. YTX concentrations in their tissues were measured at a maximum of 0.28 mg/kg and histology of these clams demonstrated damage to digestive glands. A culture of P. reticulatum, isolated from North Bay during this massive bloom and shellfish mortality event, showed YTX reaching 26.6 pg/cell, the highest recorded toxin quota measured in the U.S. to date. Concentrations of YTX in phytoplankton samples reached a maximum of 920 ng/L during a P. reticulatum bloom in Mystery Bay on 13 August 2019 when cell abundance reached 1.82 million cells/L. The highest cellular YTX quota during that bloom that lasted into September was 10.8 pg/cell on 3 Sept 2019. Shellfish producers in Washington State have also noted shellfish larvae mortalities due to A. sanguinea passing through filtration intake systems into hatchery facilities. Early warning of shellfish-killing harmful algal bloom (HAB) presence in Puget Sound, through partnerships such as SoundToxins, provides options for shellfish growers to mitigate their effects through early harvest, movement of shellstock to upland facilities, or enhanced filtration at aquaculture facilities.

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.

Denman Island disease (causative agent Mikrocytos mackini) in a new host, Kumamoto oysters Crassostrea sikamea.

Mikrocytos mackini, causative agent of Denman Island disease in Pacific oysters Crassostrea gigas and other oyster species, was found in 2011 in a previously unreported host, the Kumamoto oyster C. sikamea, in Humboldt Bay, California, USA. The detection was also the first reported finding of M. mackini in California. Prevalence was estimated as high as approximately 27% from pooled samples analyzed by PCR. Higher prevalence appeared related to longer residence time in the bay and somewhat colder than typical winter seawater temperatures. No M. mackini was detected in Humboldt Bay juvenile Kumamoto oysters or Pacific oyster brood or seed stock in 2011 or 2012.

Effect of viral hemorrhagic septicemia virus on Pacific herring in Prince William Sound, Alaska, from 1989 to 2005.

We critically review the role of viral hemorrhagic septicemia virus (VHSV) in the 1992-1993 collapse of the Prince William Sound (PWS) herring fishery. VHSV was detected in samples of moribund Pacific herring from PWS in spring 1993 when about 63% of the expected fish failed to appear. A low prevalence and severity of VHSV were observed in adult pre-spawning PWS herring in most of the years from 1994 to 2002. The North American strain of VHSV became established about 500 yr ago in many northeast Pacific marine fish species, including herring. In Alaska, the typical annual prevalence of VHSV in pre-spawning herring ranges from 0 to 17%. New threshold analysis of a 9 yr study indicates that only about half of the virus-infected adult fish in PWS were clinically affected; ulcers formerly attributed to VHS have been overestimated by a factor of about 3. We conclude that VHSV was not a primary causative factor in the PWS herring population collapse or in its failure to recover. Because older age classes of herring were not disproportionately missing in 1993, the protozoan Ichthyophonus hoferi was also not a likely cause of losses. The 'Exxon Valdez' oil spill occurred in PWS, Alaska, U.S.A., in 1989. Evidence for interaction of oil and VHSV expression is also evaluated. A study exposing herring to varying concentrations of weathered crude oil showed increasing prevalences of VHSV correlated with oil concentration; however, repeated experiments with juvenile and adult fish failed to corroborate these results or link oil to VHSV infection in herring.

Re-emergence of Vibrio tubiashii in bivalve shellfish aquaculture: severity, environmental drivers, geographic extent and management.

During 2006 and 2007, we documented the re-emergence of severe episodes of vibriosis caused by Vibrio tubiashii in shellfish hatcheries on the west coast of North America. Lost larval and juvenile production included 3 previously undescribed hosts, Pacific (Crassostrea gigas) and Kumamoto (C. sikamea) oysters and geoduck clams Panope abrupta, with a 2007 decline in larval oyster production of approximately 59% in one hatchery. Losses of larval and juvenile bivalves were linked to V. tubiashii blooms in the coastal environment, which were associated with the apparent mixing of unusually warm surface seawater and intermittently upwelled cooler, nutrient- and Vibrio spp.- enriched seawater. The ocean temperature elevation anomaly in 2007 was not clearly linked to an El Niño event, as was a similar episode in 1998. Concentrations of the dominant shellfish-pathogenic vibrios were as high as 1.6 x 10(5) cfu ml(-1) in the cold, upwelled water. The bacteria possessed the genes coding for a protease and hemolysin described for V. tubiashii, and pathogenic isolates secreted these peptides. Lesions resulting from a classic invasive disease and a toxigenic noninvasive disease occurred in oyster and geoduck clam larvae. Management and prevention require reduction of incoming concentrations of the bacteria, reduction of contamination in water and air supplies and in stock chemical solutions, removal of bacterial toxins, and interruption of the cycle of bacterial amplification in the hatchery and in microalgal food supplies.

Herpes virus in juvenile Pacific oysters Crassostrea gigas from Tomales Bay, California, coincides with summer mortality episodes.

Pacific Crassostrea gigas and eastern C. virginica oysters were examined between June 2002 and April 2003 from 8 locations along the east, west and south USA coasts for oyster herpes virus (OsHV) infections using the A primer set in a previously developed PCR test. Only surviving Pacific oysters from a mortality event in Tomales Bay, California, USA, where annual losses of oysters have occurred each summer since 1993, were infected with a herpes-like virus in 2002. PCR examination using template amounts of both 50 and 500 ng were essential for OsHV detection. Sequence analysis indicated that the Tomales Bay OsHV was similar to that identified in France with the exception of a single base pair substitution in a 917 bp fragment of the viral genome. However, unlike the French OsHV-1, the Tomales Bay OsHV did not amplify with the primer pair of a second OsHV-1 PCR assay, suggesting that further characterization of these viruses is warranted. No evidence of Cowdry type A viral infections characteristic of herpes virus infections or other pathogens were observed in OsHV-infected oysters. Hemocytosis, diapedesis and hemocyte degeneration characterized by nuclear pycnosis and fragmentation were observed in infected oysters, which is consistent with previous observations of OsHV infections in France. Together these data suggest that OsHV may be associated with the annual summer Pacific oyster seed mortality observed in Tomales Bay, but establishment of a causal relationship warrants further investigation.

Pathogenicity testing of shellfish hatchery bacterial isolates on Pacific oyster Crassostrea gigas larvae.

Bacterial diseases are a major cause of larval mortality in shellfish hatcheries. Even with proper sanitation measures, bacterial pathogens cannot be eliminated in all cases. The pathogenicity of bacteria isolated from Pacific Northwest shellfish hatcheries to Pacific oyster Crassostrea gigas larvae was investigated. We found 3 highly pathogenic strains and 1 mildly pathogenic strain among 33 isolates tested. These strains appear to be members of the genus Vibrio. Although there have been many studies of bivalve bacterial pathogens, a standard method to assess bacterial pathogenicity in bivalve larvae is needed. Thus, we developed 2 methods using either 15 ml conical tubes or tissue culture plates that were employed for rapidly screening bacterial strains for pathogenicity to Pacific oyster larvae. The tissue culture plates worked well for screening both mildly pathogenic strains and LD50 (lethal dose) assays. This method allowed for non-intrusive and non-destructive observation of the oyster larvae with a dissecting microscope. The LD50 for the 3 highly pathogenic strains ranged between 1.6 and 3.6 x 10(4) colony forming units (CFU) ml(-1) after 24 h and between 3.2 x 102 and 1.9 x 10(3) CFU ml(-1) after 48 h.