Jessica Moss

Description of Perkinsus beihaiensis n. sp., a new Perkinsus sp. Parasite in Oysters of Southern China

ABSTRACT. Oysters were collected from coastal locations in China from 1999–2006 for parasite analyses by molecular, culture, and histological techniques. Polymerase chain reaction‐based assays targeting the internal transcribed spacer (ITS) region of the ribosomal RNA gene complex were performed to detect the presence of Perkinsus species. Sequencing and phylogenetic analysis of amplified Perkinsus sp. DNAs indicated that a novel Perkinsus sp. infects Crassostrea hongkongensis, Crassostrea ariakensis, and other bivalve hosts from Fujian to Guangxi provinces in southern China. Prevalence of this Perkinsus sp. reaches as high as 60% in affected oyster populations. Analyses of nucleotide sequences of the rRNA ITS region and of large subunit rRNA and actin genes, consistently confirmed the genus affiliation of this Perkinsus sp., but distinguished it from currently accepted Perkinsus species. Parasite cell types, such as signet ring trophozoites of 2–8 μm diameter, were observed by histology, and application of both genus Perkinsus and Perkinsus species‐specific in situ hybridization probes consistently labelled the same Perkinsus sp. cells in histological sections from infected oyster tissues. Combined phylogenetic and histological results support the identity of a new parasite species, Perkinsus beihaiensis n. sp.

Review of Panulirus argus virus 1--a decade after its discovery.

In 2000, a pathogenic virus was discovered in juvenile Caribbean spiny lobsters Panulirus argus from the Florida Keys, U.S.A. Panulirus argus virus 1 (PaV1) is the first naturally occurring pathogenic virus reported from lobsters, and it profoundly affects their ecology and physiology. PaV1 is widespread in the Caribbean with infections reported in Florida (U.S.A.), St. Croix, St. Kitts, Yucatan (Mexico), Belize, and Cuba. It is most prevalent and nearly always lethal in the smallest juvenile lobsters, but this declines with increasing lobster size; adults harbor the virus, but do not present the characteristic signs of the disease. No other PaV1 hosts are known. The prevalence of PaV1 in juvenile lobsters from the Florida Keys has been stable since 1999, but has risen to nearly 11% in the eastern Yucatan since 2001. Heavily infected lobsters become sedentary, cease feeding, and die of metabolic exhaustion. Experimental routes of viral transmission include ingestion, contact, and for newly settled juveniles, free virus particles in seawater. Prior to infectiousness, healthy lobsters tend to avoid diseased lobsters and so infected juvenile lobsters mostly dwell alone, which appears to reduce disease transmission. However, avoidance of diseased individuals may result in increased shelter competition between healthy and diseased lobsters, and greater predation on infected lobsters. Little is known about PaV1 outside of Mexico and the USA, but it poses a potential threat to P. argus fisheries throughout the Caribbean.

ADVANCED PERKINSUS MARINUS INFECTIONS IN CRASSOSTREA ARIAKENSIS MAINTAINED UNDER LABORATORY CONDITIONS

Abstract The Suminoe oyster, Crassostrea ariakensis, has been under investigation since the early 1990s for potential use in restoring the commercial harvest or for aquaculture of oysters in the Chesapeake Bay, USA. Initial studies focusing on C. ariakensis documented a significant level of tolerance to the protozoan parasite Perkinsus marinus, a pathogen found in almost all reaches of the Bay and widely acknowledged as one of the main reasons for the decline in the eastern oyster, Crassostrea virginica, harvest since the late 1980s. Crassostrea ariakensis was demonstrated to acquire P. marinus, however infection intensities, as measured using Rays thioglycollate medium assay indices, generally were found to be light. As part of a series of experiments to study potential impacts on the Chesapeake Bay region of pathogens found in C. ariakensis in Asia, a challenge experiment was conducted to study the pathogenicity of Perkinsus olseni to C. ariakensis. During this study, we observed the acquisition of moderate and heavy infection intensities of P. marinus in triploid C. ariakensis oysters being maintained in the laboratory. Results suggest that there may be some risk of mortality from P. marinus if C. ariakensis is held under stressful conditions at least in hatchery or laboratory settings.

Myospora metanephrops (n. g., n. sp.) from marine lobsters and a proposal for erection of a new order and family (Crustaceacida; Myosporidae) in the Class Marinosporidia (Phylum Microsporidia).

In this study we describe, the first microsporidian parasite from nephropid lobsters. Metanephrops challengeri were captured from an important marine fishery situated off the south coast of New Zealand. Infected lobsters displayed an unusual external appearance and were lethargic. Histology was used to demonstrate replacement of skeletal and other muscles by merogonic and sporogonic stages of the parasite, while transmission electron microscopy revealed the presence of diplokaryotic meronts, sporonts, sporoblasts and spore stages, all in direct contact with the host sarcoplasm. Analysis of the ssrDNA gene sequence from the lobster microsporidian suggested a close affinity with Thelohania butleri, a morphologically dissimilar microsporidian from marine shrimps. Whilst morphological features of the lobster parasite are consistent with members of the family Nosematidae, molecular data place the parasite closer to members of the family Thelohanidae. Due to the contradiction between morphological and molecular taxonomic data, we propose the erection of a new genus in which the lobster parasite is the type species (Myospora metanephrops). Furthermore, we recommend the erection of a new family (Myosporidae) and a new order (Crustaceacida) to contain this genus. The taxonomic framework presented could be further applied to the re-classification of existing members of the Phylum Microsporidia.

A molecular phylogeny of Bopyroidea and Cryptoniscoidea (Crustacea: Isopoda)

Epicaridean isopods are parasitic on other crustaceans. They represent a diverse group of highly derived taxa in two superfamilies and 10 families. Little work has been done on the phylogeny of these parasites because of the difficulty in defining homologous characters for adults above the genus level. The females exhibit morphological reduction of characters and the males have few distinguishing characters. Moreover, epicarideans have only rarely been included in past studies of isopod phylogeny. Our objective was to derive a phylogeny of epicaridean taxa based on 18S rDNA, then use that phylogeny to examine the relationships of the bopyrid subfamilies, bopyroid families and epicarideans to cymothoid isopods. We tested the monophyly of the Epicaridea, evaluated hypotheses on relationships among epicaridean families and subfamilies, examined the evolution of the abdominal mode of infestation on caridean, gebiidean, axiidean and anomuran hosts and examined coevolution between epicarideans and their crustacean hosts. The molecular phylogeny indicated that Epicaridea were monophyletic with respect to Cymothooidea. Bopyroidea formed a monophyletic group without Dajidae and Entophilinae (now as Entophilidae). Both latter taxa grouped with Cryptoniscoidea, and this group was the sister taxon to the redefined Bopyroidea in all trees. The bopyrid subfamily Ioninae is the sister taxon to the other bopyrid subfamilies (except Entophilidae). Ioninae was elevated to family status but found not to be monophyletic; a new subfamily, Keponinae, was erected for all genera formerly placed in Ioninae except the type genus. The abdominal mode of parasitism appears to have evolved independently among the subfamilies. Coevolution between host and parasite phylogenies showed extensive incongruence, indicating frequent host-switching as a general pattern in Epicaridea. http://www.zoobank.org/urn:lsid:zoobank.org:pub:30ECFB13-2795-494E-AABE-6B5F84A57A67

Perkinsus olseni in vitro Isolates from the New Zealand Clam Austrovenus stutchburyi

ABSTRACT. Perkinsus olseni infections are reported at 10%–84% prevalences among Austrovenus stutchburyi clams (cockles) in northern New Zealand coastal waters. However, P. olseni has not yet been propagated in vitro from New Zealand clams. In our sample of A. stutchburyi clams from Mangemangaroa Stream, New Zealand, 24% (8/34) showed low‐intensity Perkinsus sp. infections among mantle and gill tissues incubated in alternative Rays fluid thioglycollate medium (ARFTM), and 5% (4/79) showed Perkinsus sp. lesions by histological analyses. Among clams that were screened using a polymerase chain reaction (PCR) assay, 16% (3/19) were positive for Perkinsus sp. DNA. Alternative Rays fluid thioglycollate medium‐enlarged hypnospores from tissues of five infected clams yielded three in vitro Perkinsus sp. isolate cultures that were cloned before sequencing internal transcribed spacer (ITS) regions of their rRNA gene complex. For one isolate, ATCC PRA‐205, large subunit (LSU) rRNA and actin genes were also sequenced. All nucleotide sequences from all isolates consistently identified them as P. olseni, as did their in vitro cell cycles and zoosporulation characteristics. All in vitro isolate cultures and their respective monoclonal derivative strains were cryopreserved and deposited for archiving and distribution by the American Type Culture Collection (http://www.atcc.org).

Continuous Culture of Perkinsus mediterraneus, a Parasite of the European Flat Oyster Ostrea edulis, and Characterization of Its Morphology, Propagation, and Extracellular Proteins in Vitro

ABSTRACT. Continuous in vitro cultures of Perkinsus mediterraneus were established from tissues of infected European flat oysters, Ostrea edulis. The parasite proliferated in protein‐free medium and divided by schizogony in vitro. Cell morphology was similar to that observed for P. mediterraneus in tissues of naturally infected O. edulis and for other Perkinsus spp. cultured in vitro. Parasite cells enlarged approximately 8‐fold when placed in alternative Rays fluid thioglycollate medium, and stained black with Lugols iodine solution, a response characteristic of Perkinsus spp. DNA sequences matched those determined previously for P. mediterraneus, and phylogenetic analyses on three different data sets indicated that this was a Perkinsus species with a close relationship to another recently described species, Perkinsus honshuensis. Parasite viability was high (>90%) in vitro, but the proliferation rate was low, with densities generally increasing 2‐to‐6‐fold between subcultures at 6‐wk intervals. Enzyme analysis of cell‐free culture supernatants revealed protease‐, esterase‐, glycosidase‐, lipase‐, and phosphatase‐like activities. Incubation with class‐specific protease inhibitors showed that P. mediterraneus produced serine proteases, and eight proteolytic bands with molecular weights ranging from 34 to 79 kDa were detected in the supernatants by gelatin sodium dodecylsulfate‐polyacrylamide gel electrophoresis.

Histological Assessment of the Lobster (Homarus americanus) in the “100 Lobsters” Project

ABSTRACT The emergence of epizootic shell disease in the American lobster (Homarus americanus) has been devastating to the industry in the coastal waters of southern New England. A comprehensive assessment of the disease syndrome, known as the “100 Lobsters” Project, was initiated to examine health and physiological parameters among laboratories involved in the research on lobster shell disease. A histological study of the 100 lobsters was undertaken as part of that assessment. Tissues from 90 lobsters from Rhode Island and 19 lobsters from Maine were examined as a general health assessment of the 100 lobsters. Approximately half the lobsters from Rhode Island were selected because they had frank epizootic shell disease, whereas none of the lobsters from Maine exhibited the syndrome. In addition to epizootic shell disease, the histological findings revealed 3 other idiopathic syndromes—necrotizing hepatopancreatitis, idiopathic blindness, and nonspecific granulomas—in higher prevalences in lobsters from Rhode Island compared with those from Maine. Necrotizing hepatopancreatitis, a newly described disease syndrome in lobsters, was observed in 15% of the lobsters from Rhode Island. Idiopathic blindness was present in 54% of the lobsters from Rhode Island, and 16% of the animals from Maine. This is the first report of the syndrome in lobsters from Maine. None of the idiopathic syndromes was associated with epizootic shell disease. The detection of multiple disease syndromes such as epizootic shell disease, necrotizing hepatopancreatitis, and idiopathic blindness may be indicative of exposure to environmental stressors in Narragansett Bay, RI.

Distribution, Prevalence, and Genetic Analysis of Panulirus Argus Virus 1 (Pav1) from the Caribbean Sea

The pathogenic virus Panulirus argus virus 1 (PaV1) was first discovered in Caribbean spiny lobsters Panulirus argus from the Florida Keys (USA) in 1999 and has since been reported in Belize, Mexico, and Cuba; its distribution in the wider Caribbean is unknown. We collected tissue samples from adult spiny lobsters from 30 locations in 14 countries bordering the Caribbean Sea and used molecular diagnostics to assay for the presence of PaV1. PaV1 occurred primarily in the northern areas of the Caribbean, where its prevalence was highest. The virus was not found in lobsters from the southeastern Caribbean, and its prevalence was lowest in the southwestern Caribbean. DNA sequence analysis was performed on a fragment of the viral DNA to examine the genetic diversity of PaV1 on a Caribbean-wide scale. Sequence variation in the viral DNA fragment was high, with 61 unique alleles identified from 9 areas. The sharing of viral alleles in lobsters from distant locations supports the hypothesis of a strong genetic connectivity among lobsters within the Caribbean, and further supports the hypothesis that postlarvae infected with PaV1 may serve to disperse the virus over long distances.

Genetic Diversity in U.S. Hatchery Stocks of Crassostrea ariakensis (Fujita, 1913) and Comparison with Natural Populations in Asia

ABSTRACT Although several different U.S. hatchery stocks of the Asian Suminoe oyster Crassostrea ariakensis were used in laboratory and field trials assessing performance, and in comparative studies with the native oyster Crassostrea virginica, the genetic composition of these hatchery stocks has not yet been examined comprehensively. Using eight microsatellite markers we investigated the genetic variability among five hatchery stocks and compared the genetic makeup of these stocks with 8 wild populations from Asia. Results showed significant genetic differentiation among the 5 hatchery stocks that was 6-fold larger than that observed among wild populations. A significant reduction in genetic diversity was observed in hatchery stocks compared with wild source populations, indicating a genetic bottleneck. Two long-established stocks showed significant decreases in both allelic diversity and heterozygosity compared with the wild Japanese source population, whereas three recently established stocks showed less severe reductions in allelic diversity and a nonsignificant change in levels of heterozygosity compared with their source Chinese populations. These microsatellite markers also proved useful for assignment of hatchery individuals back to their source stocks with a high degree of confidence. Although assignment of wild individuals back to their population of origin proved less reliable, approximately 70% of wild individuals could be assigned either to their source population or to geographically proximal populations. Our results suggest that results obtained from experiments that used hatchery animals of a single C. ariakensis stock for biological and ecological studies should be interpreted cautiously, because they may not always accurately reflect the behavior of wild populations or of other hatchery stocks.