Gary R. Meyer

Diseases of cultured Japanese scallops (Patinopecten yessoensis) in British Columbia, Canada

Abstract Preliminary grow-out studies with Patinopecten yessoensis in British Columbia, Canada, revealed the presence of at least three pathogens. At one location, the shell boring polychaete, Polydora websteri , was responsible for 84% mortality. Most surviving scallops were stunted and regrowth of the shell was often abnormal. At another locality, a protozoan of unknown taxonomic affinities (SPX) was observed. Following one year of excellent growth and survival (>90%), about 60% of the surviving scallops died within 3 months (June to August). Creamy-white pustules (up to 5 mm in diameter) were observed in 50% of the scallops. SPX was found in the connective tissues of 84% of the scallops with pustules and in 15% of those without pustules. The third pathogen was also an unidentified protozoan (SPG). However, unlike SPX, SPG was found in 5% to 10% of P. yessoensis from seven localities and infections were always light. In addition to the diseases with recognizable causes, focal lesions of unknown aetiology were associated with scallop mortalities (>65%) in six of seven growout localities during 1989–1990. Visible lesions consisting of pinkish-orange pustules (up to 15 mm in diameter) occurred in up to 30% of P. yessoensis . In addition, 30% to 55% of the scallops had microscopic lesions in the connective tissues of all organs.

Sequence homogeneity of internal transcribed spacer rDNA in Mikrocytos mackini and detection of Mikrocytos sp. in a new location.

Mikrocytos mackini is a microcell parasite of Pacific oysters only known to occur on the Pacific coast of North America. It is the only described species in the genus, although a genetically divergent Mikrocytos sp. organism has been reported once in both the Atlantic Ocean and China. We developed methods for sequencing the internal transcribed spacer (ITS) of rDNA for the purpose of characterizing extant diversity within M. mackini throughout its known geographic range, and surveying for other evidence of Mikrocytos sp. organisms. Our specific aims were to examine relatedness of M. mackini among sites to make inferences about its recent evolutionary history, and to provide baseline data for future development of a species-specific molecular detection method. We found a total lack of genetic variation within M. mackini across the complete ITS1-5.8S-ITS2 array in over 70 samples collected throughout its range. We hypothesize that this could be a result of a founder effect if the parasite had been introduced into its known range alongside its host, which was imported from Asia beginning around 1914 to about 1961. We detected a single divergent sequence at a short stretch of 18S that was identical to the Mikrocytos sp. detected elsewhere, which adds to the recent and growing body of evidence that Mikrocytos is much more broadly distributed than the limited range of M. mackini suggests. A 1903 bp section of rDNA from Mikrocytos sp. was generated that contained regions of high divergence from M. mackini (in ITS1 and ITS2) that could be exploited for molecular diagnostics.

Detection of a parasitic amoeba (Order Dactylopodida) in the female gonads of oysters in Brazil

The impacts of oocyte parasites on the reproductive success of molluscs are largely unknown. In this study, we evaluated the presence of gonad parasites in 6 species of marine bivalve molluscs native to southern Brazil. Cultured bivalves included the mangrove oyster Crassostrea gasar (sometimes called C. brasiliana), the brown mussel Perna perna, the lions paw scallop Nodipecten nodosus and the wing pearl oyster Pteria hirundo. Another species of mangrove oyster, C. rhizophorae, and the carib pointed venus clam Anomalocardia brasiliana (syn. A. flexuosa) were collected from the wild. Molluscs were collected in winter 2009 and summer 2010 for histopathological and molecular evaluation. An unknown ovarian parasite (UOP) was observed in histopathological sections of female gonads of C. gasar and C. rhizophorae. The UOP possessed features suggestive of amoebae, including an irregular outer membrane, frothy cytoplasm, a nucleus with a prominent central nucleolus and a closely associated basophilic parasome. PCR analysis was negative for Marteilioides chungmuensis, Perkinsus spp. and Paramoeba perurans. However, real-time PCR successfully amplified DNA from oyster gonads when using universal Paramoeba spp. primers. Also, conventional PCR amplified DNA using primers specific for Perkinsela amoebae-like organisms (syn. Perkinsiella), which are considered as endosymbionts of Parameoba spp., previously thought to be the parasome. Our results suggest that this UOP is a species of amoeba belonging to 1 of the 2 families of the order Dactylopodida, possibly related to Paramoeba spp. This study represents the first report of this type of organism in oysters. We found that C. gasar and C. rhizophorae were the most susceptible molluscs to these UOPs.

Molecular taxonomy of Mikrocytos boweri sp. nov. from Olympia oysters Ostrea lurida in British Columbia, Canada.

Mikrocytos mackini is a microcell parasite that usually infects Crassostrea gigas distributed along the Pacific Northwest coast of North America. For many years, M. mackini was the only known species in the genus, but there have been multiple recent findings of genetically divergent forms of Mikrocytos in different hosts and in distantly located geographic locations. This note describes M. boweri sp. nov. found in Olympia oysters Ostrea lurida collected from and native to British Columbia, Canada, primarily using a molecular taxonomic approach.

Review of Mikrocytos microcell parasites at the dawn of a new age of scientific discovery

The genus Mikrocytos is traditionally known for Mikrocytos mackini, the microcell parasite that typically infects Pacific oysters along the west coast of North America. Multiple factors have conspired to create difficulty for scientific research on Mikrocytos parasites. These include their tiny cell size, infections that are often of light intensity, lack of suitable cell lines and techniques for in vitro culture, and the seasonal nature of infections. The extreme rate of molecular evolution in Mikrocytos stymied new species discovery and confounded attempts to resolve its phylogenetic position for many years. Fortunately, 2 recent landmark studies have paved the way forward for future research by drastically changing our understanding of the evolution and diversity of these parasites. No longer an orphan eukaryotic lineage, the phylogenetic placement of Mikrocytos has been confidently resolved within Rhizaria and as sister taxon to Haplosporidia. The genus has also found a taxonomic home within the newly-discovered order, Mikrocytida - a globally distributed lineage of parasites infecting a wide range of invertebrate hosts. Here we review available scientific information on Mikrocytos parasites including their evolution and diversity, host and geographic ranges, epizootiology, and detection of the regulated pathogen, M. mackini. We also make recommendations towards a consistent taxonomic framework for this genus by minimally suggesting the use of 18S rDNA sequence, host species information, and histopathological presentation in new species descriptions. This is timely given that we are likely embarking on a new era of scientific advancements, including species discovery, in this genus and its relatives.

Rediscovery of the Yesso scallop pathogen Perkinsus qugwadi in Canada, and development of PCR tests.

Perkinsus qugwadi, a pathogenic protozoan parasite of Yesso scallops Patinopecten yessoensis, is found only in cultured populations in British Columbia, Canada. This pathogen was first identified in 1988 and caused significant mortalities at some locations during the early 1990s. Prevalence of infection decreased dramatically following 1995, and the disease was last reported in 1997, leading to speculation that the Yesso scallop stocks in Canada had developed resistance to the disease, or that P. qugwadi had disappeared. However, the present study revealed that infection with P. qugwadi and associated mortality is still occurring in scallops from at least one location in British Columbia. One of the PCR tests developed for P. qugwadi detected the parasite in a 105-fold dilution of DNA extracted from a heavily infected sample and detected 52% more positive scallops than histology; however, the assay also cross-reacted with P. honshuensis and P. olseni. The other PCR test was less sensitive and detected 34% more positives, but did not react to any of the other Perkinsus species tested, suggesting that these PCR tests are powerful tools for screening for the presence of P. qugwadi. Phylogenetic analysis of 1796 bp of SSU rRNA gene sequence clearly indicated that P. qugwadi is positioned basally to other Perkinsus species.

Disease and mortality among Yesso scallops Patinopecten yessoensis putatively caused by infection with Francisella halioticida

During the fall of 2015, up to 40% mortality occurred in juvenile Yesso scallops Patinopecten yessoensis at an aquaculture site in Baynes Sound, British Columbia, Canada. Macroscopic lesions were present in 11% of the scallops, and histopathology consisting of multifocal and diffuse haemocyte infiltration was observed in 44% of the specimens examined. Histologically, small Gram-negative intracellular bacteria-like particles were observed within necrotic haemocytes of the lesions, suggesting a bacterial aetiology. DNA was extracted from adductor muscle lesions of diseased scallops, and the 16s rDNA gene as well as the DNA-directed RNA polymerase beta subunit (rpoB) were amplified by PCR. Sequence analyses of the resulting 413 and 925 bp fragments were a 100% match to the reference sequence for Francisella halioticida, originally described as the cause of mortality in abalone from Japan. Isolation and culture of the bacteria was not possible at the time, as no further diseased specimens were available. Results from in situ hybridization assays as well as examination by transmission electron microscopy provide further evidence supporting the hypothesis that F. halioticida was the most probable causative agent of the lesions and mortality.

Molecular detection of Mikrocytos mackini in Pacific oysters using quantitative PCR

Mikrocytos mackini is an internationally regulated pathogen and causative agent of Denman Island disease in Pacific oysters Crassostrea gigas. Recent phylogenetic breakthroughs have placed this parasite within a highly divergent and globally distributed eukaryotic lineage that has been designated a new taxonomic order, Mikrocytida. The discovery of this new radiation of parasites is accompanied by a heightened awareness of the many knowledge gaps that exist with respect to the general biology, epizootiology, and potential impact of mikrocytid parasites on hosts, ecosystems, and commercial fisheries. It has also highlighted current shortcomings regarding our ability to detect these organisms. In this study, we developed a species-specific, sensitive, and quantitative method for detecting M. mackini DNA from host tissues using probe-based real-time qPCR technology. A limit of sensitivity between 2 and 5 genome copy equivalents was achieved in a reaction matrix containing ≥ 40 ng/μL host gDNA without inhibition. This detection proved superior to existing methods based on conventional PCR, histology or gross pathology and is the first species-specific diagnostic test for M. mackini. Quantitative assessment of parasite DNA using this assay remained accurate to between 10 and 50 copies identifying that during infection, M. mackini DNA was significantly more prevalent in hemolymph, labial palp, and mid-body cross-sections compared to mantle or adductor muscle. DNA extracted from a mid-body cross-section also provided the highest likelihood for detection during diagnostic screening of infected oysters. Taken together, these findings provide strong analytical evidence for the adoption of qPCR as the new reference standard for detecting M. mackini and give preliminary insight into the distribution of the parasite within host tissues. Standardised operating methodologies for sample collection and qPCR testing are provided to aid in the international regulatory diagnosis of M. mackini and serve as a useful platform for the future development of multiplexed or alternate mikrocytid species detection.

Denman Island disease in Washington State, USA: distribution and prevalence in Pacific and Olympia oysters

We sampled over 2400 wild, feral, and cultured Pacific oysters Crassostrea gigas and Olympia oysters Ostrea lurida in Washington State, USA, from 2002 to 2006 to estimate the prevalence of infection with Mikrocytos mackini, the causative agent of Denman Island disease. Both histology and qualitative PCR methods were used. Estimates of true prevalence of M. mackini infection in C. gigas, after accounting for imperfect test sensitivity, ranged from mean values of 0 to 10.0% by histology and 0 to 8.4% based on pooled PCR samples. M. mackini was not detected in any of the O. lurida samples. Results suggest a lower prevalence of the pathogen and severity of this oyster disease in Washington than that indicated in previous reports from British Columbia, Canada, potentially attributable to higher seawater temperatures in the Washington sample locations.

Rare occurrence of heart lesions in Pacific oysters Crassostrea gigas caused by an unknown bacterial infection

On rare occasions, small cream-coloured cysts have been observed in the heart and pericardial cavity of Pacific oysters Crassostrea gigas from British Columbia, Canada. Histopathology revealed the presence of large colonies of bacteria (up to 800 µm in diameter) causing significant host response and hypertrophy of the heart epithelium. The causative bacteria were characterized as follows: Gram-negative, coccoid to small rod-shaped, typically <1.5 µm in size, cell walls highly endowed with surface fimbriae and division via binary fission. Although these bacteria shared some morphological characteristics with the order Rickettsiales, they did not require an intracellular existence for multiplication. Unfortunately, a cultured isolate was not available, and a retrospective attempt to further characterize the bacteria using DNA sequence analysis of a fragment from the 16S rDNA region proved to be uninformative.