Draft genomes of Perkinsus olseni and Perkinsus chesapeaki reveal polyploidy and regional differences in heterozygosity.

Perkinsus spp. parasites have significant impact on aquaculture and wild mollusc populations. We sequenced the genomes of five monoclonal isolates of Perkinsus olseni and one Perkinsus chesapeaki from international sources. Sequence analysis revealed similar levels of repetitive sequence within species, a polyploid genome structure, and substantially higher heterozygosity in Oceanian-sourced isolates. We also identified tandem replication of the rRNA transcriptional unit, with high strain variation. Characterized gene content was broadly similar amongst all Perkinsus spp. but P. olseni Oceanian isolates contained an elevated number of genes compared to other P. olseni isolates and cox3 could not be identified in any Perkinsus spp. sequence. Phylogenetics and average nucleotide identity scans were consistent with all P. olseni isolates being within one species. These are the first genome sequences generated for both P. olseni and P. chesapeaki and will allow future advances in diagnostic design and population genomics of these important aquatic parasites.

Characterisation of the Pacific Oyster Microbiome During a Summer Mortality Event.

The Pacific oyster, Crassostrea gigas, is a key commercial species that is cultivated globally. In recent years, disease outbreaks have heavily impacted C. gigas stocks worldwide, with many losses incurred during summer. A number of infectious agents have been associated with these summer mortality events, including viruses (particularly Ostreid herpesvirus 1, OsHV-1) and bacteria; however, cases where no known aetiological agent can be identified are common. In this study, we examined the microbiome of disease-affected and disease-unaffected C. gigas during a 2013-2014 summer mortality event in Port Stephens (Australia) where known oyster pathogens including OsHV-1 were not detected. The adductor muscle microbiomes of 70 C. gigas samples across 12 study sites in the Port Stephens estuary were characterised using 16S rRNA (V1-V3 region) amplicon sequencing, with the aim of comparing the influence of spatial location and disease state on the oyster microbiome. Spatial location was found to be a significant determinant of the disease-affected oyster microbiome. Furthermore, microbiome comparisons between disease states identified a significant increase in rare operational taxonomic units (OTUs) belonging to Vibrio harveyi and an unidentified member of the Vibrio genus in the disease-affected microbiome. This is indicative of a potential role of Vibrio species in oyster disease and supportive of previous culture-based examination of this mortality event.

Australian bass Macquaria novemaculeata susceptibility to experimental megalocytivirus infection and utility as a model disease vector.

Megalocytiviruses, particularly red seabream iridovirus, infect a broad range of fish including both freshwater and marine species. Although a limited number of infectious spleen and kidney necrosis virus (ISKNV) strains have been reported in association with mortality events in marine aquaculture species, the potential host range for ISKNV strains, particularly of those that have been detected in ornamental fish, has not been well characterised. There have also been few reports on the susceptibility of euryhaline fish species that could potentially transmit megalocytiviruses between freshwater and marine environments. We found that the euryhaline Australian native percichthyid fish, Australian bass Macquaria novemaculeata, is susceptible experimentally to ISKNV (strain DGIV-10), obtained from a freshwater ornamental fish, dwarf gourami Trichogaster lalius. Australian bass developed clinical disease following direct inoculation and also following cohabitation with infected fish, and were able to transmit DGIV-10 to naïve Murray cod Maccullochella peelii. This study demonstrated the potential for a euryhaline species to become infected with, and transmit, the megalocytivirus ISKNV between fish populations.

Experimental transmission of infectious spleen and kidney necrosis virus (ISKNV) from freshwater ornamental fish to silver sweep Scorpis lineolata, an Australian marine fish.

The Australian native marine fish species, silver sweep Scorpis lineolata, is susceptible to the megalocytivirus Infectious spleen and kidney necrosis virus (strain DGIV-10) obtained from a freshwater ornamental fish, dwarf gourami Trichogaster lalius. This was demonstrated by direct inoculation and through cohabitation. Transmission by cohabitation was also demonstrated from inoculated freshwater Murray cod Maccullochella peelii to euryhaline Australian bass Macquaria novemaculeata and to marine silver sweep. The virus was also transmitted from infected marine silver sweep to euryhaline Australian bass and then to freshwater Murray cod. This study is the first to demonstrate the virulence of a megalocytivirus derived from ornamental fish in an Australian marine species and the first to show a feasible pathway for the exchange of megalocytiviruses between freshwater and marine finfish hosts. These results demonstrate that megalocytiviruses from freshwater ornamental fish have the potential to spread to diverse aquatic environments.

Mass mortalities of unknown aetiology in Pacific oysters Crassostrea gigas in Port Stephens, New South Wales, Australia.

From January to June 2013 and November to January 2014, mass mortalities were reported in Pacific oysters Crassostrea gigas cultivated in Port Stephens estuary, New South Wales, Australia. In some cases, 100% mortality was reported in both triploid and diploid C. gigas, although native species of oyster cultivated in the same areas remained unaffected. Histological examination of oysters sampled from the time of mortality events revealed consistent but non-specific pathology, involving a diffuse haemocytic infiltrate in the connective tissue surrounding the digestive gland, extending into the mantle in some instances, but no other signs of any infectious aetiological agent. We conducted a structured survey in early January 2014 to compare samples of C. gigas from affected and unaffected areas by bacteriology and histopathology. Quantitative PCR excluded involvement of ostreid herpesvirus-1 (OsHV-1) in these mortality events. To determine whether a directly transmissible aetiological agent was responsible for the mortalities, naïve C. gigas sourced from an estuary where no evidence of mortality was reported were challenged with material derived from affected oysters. Significant mortality was only observed in naïve C. gigas directly inoculated with purified cultures of Vibrio spp. isolated from affected oysters, but this could not be replicated by cohabitation with naïve C. gigas. Analysis of environmental data indicated that mortality events generally coincided with periods of low salinity and high temperature. The results from this study suggest that the cause of the mortality events was multifactorial in nature and not due to any single directly transmissible aetiological agent.

Longitudinal study of winter mortality disease in Sydney rock oysters Saccostrea glomerata.

Winter mortality (WM) is a poorly studied disease affecting Sydney rock oysters Saccostrea glomerata in estuaries in New South Wales, Australia, where it can cause significant losses. WM is more severe in oysters cultured deeper in the water column and appears linked to higher salinities. Current dogma is that WM is caused by the microcell parasite Bonamia roughleyi, but evidence linking clinical signs and histopathology to molecular data identifying bonamiasis is lacking. We conducted a longitudinal study between February and November 2010 in 2 estuaries where WM has occurred (Georges and Shoalhaven Rivers). Results from molecular testing of experimental oysters for Bonamia spp. were compared to clinical disease signs and histopathology. Available environmental data from the study sites were also collated and compared. Oyster condition declined over the study period, coinciding with decreasing water temperatures, and was inversely correlated with the presence of histological lesions. While mortalities occurred in both estuaries, only oysters from the Georges River study site showed gross clinical signs and histological changes characteristic of WM (lesions were prevalent and intralesional microcell-like structures were sometimes noted). PCR testing for Bonamia spp. revealed the presence of an organism belonging to the B. exitiosa-B. roughleyi clade in some samples; however, the very low prevalence of this organism relative to histological changes and the lack of reactivity of affected oysters in subsequent in situ hybridisation experiments led us to conclude that this Bonamia sp. is not responsible for WM. Another aetiological agent and a confluence of environmental factors are a more likely explanation for the disease.

Scoliosis and kyphosis in blue-spotted and marbled flathead fish associated with a Myxobolus acanthogobii-like parasite.

Spinal deformities in finfish have the potential to impact aquaculture industries and wild populations by increasing morbidity, mortality, and reducing growth rates. Myxobolus acanthogobii has been implicated in causing scoliosis and lordosis in various aquatic species in Japan. We investigated 4 cases of spinal deformity in 2 flathead (Platycephalus) species that were submitted to the Elizabeth Macarthur Agricultural Institute (EMAI) in New South Wales (NSW), Australia, between 2015 and 2021. Flathead are commercially significant species that are popular among Australian consumers, and are also sought-after species targeted by recreational fishers. Gross deformities are concerning to the community and may impact the quality and quantity of specimens available for consumption. Three blue-spotted flathead (P. caeruleopunctatus) and one marbled flathead (P. marmoratus) were submitted, all with marked scoliosis and kyphosis; 1-2-mm cysts were present on the dorsum of the brain, most often over the optic lobe or cerebellum. Cytology and differential interference microscopy of cyst material revealed numerous oval spores, x̄ 14 ± SD 0.75 µm × x̄ 11.5 ± SD 0.70 µm, with 2 pyriform polar capsules, the morphology of which is consistent with a Myxobolus sp. PCR assay and 18S rDNA sequencing of the cyst material identified a Myxobolus sp. with 96% identity to M. acanthogobii. The identification of this Myxobolus sp. confirms the presence of parasites with the potential to cause spinal deformity in significant aquatic species in NSW waterways.

Identification and characterisation of an ostreid herpesvirus-1 microvariant (OsHV-1 µ-var) in Crassostrea gigas (Pacific oysters) in Australia.

Between November 2010 and January 2011, triploid Crassostrea gigas (Pacific oysters) cultivated in the Georges River, New South Wales, experienced >95% mortality. Mortalities also occurred in wild diploid C. gigas in the Georges River and shortly thereafter in the adjacent Parramatta River estuary upstream from Sydney Harbour. Neighbouring Saccostrea glomerata (Sydney rock oysters) did not experience mortalities in either estuary. Surviving oysters were collected to investigate the cause of mortalities. Histologically all oysters displayed significant pathology, and molecular testing revealed a high prevalence of ostreid herpesvirus-1 (OsHV-1). Quantitative PCR indicated that many C. gigas were carrying a high viral load at the time of sampling, while the load in S. glomerata was significantly lower (p < 0.001). Subsequent in situ hybridisation experiments confirmed the presence of a herpesvirus in C. gigas but not S. glomerata tissues, suggesting that S. glomerata is not susceptible to infection with OsHV-1. Naïve sentinel triploid C. gigas placed in the Georges River estuary in January 2011 quickly became infected and experienced nearly 100% mortality within 2 wk of exposure, indicating the persistence of the virus in the environment. Phylogenetic analysis of sequences derived from the C2/C6 region of the virus revealed that the Australian strain of OsHV-1 belongs to the microvariant (µ-var) cluster, which has been associated with severe mortalities in C. gigas in other countries since 2008. Environmental data revealed that the Woolooware Bay outbreaks occurred during a time of considerable environmental disturbance, with increased water temperatures, heavy rainfall, a toxic phytoplankton bloom and the presence of a pathogenic Vibrio sp. all potentially contributing to oyster stress. This is the first confirmed report of OsHV-1 µ-var related C. gigas mortalities in Australia.

Phylogenomic diversity of Vibrio species and other Gammaproteobacteria isolated from Pacific oysters (Crassostrea gigas) during a summer mortality outbreak.

The Pacific oyster (PO), Crassostrea gigas, is an important commercial marine species but periodically experiences large stock losses due to disease events known as summer mortality. Summer mortality has been linked to environmental perturbations and numerous viral and bacterial agents, indicating this disease is multifactorial in nature. In 2013 and 2014, several summer mortality events occurred within the Port Stephens estuary (NSW, Australia). Extensive culture and molecular-based investigations were undertaken and several potentially pathogenic Vibrio species were identified. To improve species identification and genomically characterise isolates obtained from this outbreak, whole-genome sequencing (WGS) and subsequent genomic analyses were performed on 48 bacterial isolates, as well as a further nine isolates from other summer mortality studies using the same batch of juveniles. Average nucleotide identity (ANI) identified most isolates to the species level and included members of the Photobacterium, Pseudoalteromonas, Shewanella and Vibrio genera, with Vibrio species making up more than two-thirds of all species identified. Construction of a phylogenomic tree, ANI analysis, and pan-genome analysis of the 57 isolates represents the most comprehensive culture-based phylogenomic survey of Vibrios during a PO summer mortality event in Australian waters and revealed large genomic diversity in many of the identified species. Our analysis revealed limited and inconsistent associations between isolate species and their geographical origins, or host health status. Together with ANI and pan-genome results, these inconsistencies suggest that to determine the role that microbes may have in Pacific oyster summer mortality events, isolate identification must be at the taxonomic level of strain. Our WGS data (specifically, the accessory genomes) differentiated bacterial strains, and coupled with associated metadata, highlight the possibility of predicting a strain's environmental niche and level of pathogenicity.

The molecular epidemiology of iridovirus in Murray cod (Maccullochella peelii peelii) and dwarf gourami (Colisa lalia) from distant biogeographical regions suggests a link between trade in ornamental fish and emerging iridoviral diseases.

Iridoviruses have emerged over 20 years to cause epizootics in finfish and amphibians in many countries. They may have originated in tropical Asia and spread through trade in farmed food fish or ornamental fish, but this has been difficult to prove. Consequently, MCP, ATPase and other viral genes were sequenced from archival formalin-fixed, paraffin-embedded tissues from farmed Murray cod (Maccullochella peelii peelii) that died during an epizootic in 2003 and from diseased gouramis that had been imported from Asia. There was almost complete homology (99.95%) over 4,527 bp between Murray cod iridovirus (MCIV) and an iridovirus (DGIV) present in dwarf gourami (Colisa lalia) that had died in aquarium shops in Australia in 2004, and very high homology with infectious spleen and kidney necrosis virus (ISKNV) (99.9%). These viruses are most likely to be a single species within the genus Megalocytivirus and probably have a common geographic origin. Primers for genus-specific PCR and for rapid discrimination of MCIV/DGIV/ISKNV and red sea bream iridovirus (RSIV), a notifiable pathogen, were developed. These were used in a survey to determine that the prevalence of DGIV infection in diseased gourami in retail aquarium shops in Sydney was 22% (95% confidence limits 15-31%). The global trade in ornamental fish may facilitate the spread of Megalocytivirus and enable emergence of disease in new host species in distant biogeographic regions.