The haplosporidian Bonamia exitiosa is present in Australia, but the identity of the parasite described as Bonamia (formerly Mikrocytos) roughleyi is uncertain.

Protistan oyster parasites in the genus Bonamia have been observed in recent years infecting new hosts on five continents, with most of these parasites genetically similar to austral species Bonamia exitiosa and Bonamia roughleyi. Identification of the newly observed parasites as one or another of these described species has been complicated by the fact that B. exitiosa and B. roughleyi are phylogenetically indistinguishable at the small-subunit ribosomal DNA (SSU rDNA) level, with samples of B. roughleyi type material no longer available for genetic re-analyses using more informative internal transcribed spacer (ITS) region DNA sequences. To resolve this issue, we evaluated B. roughleyi in field collections of hosts Saccostrea glomerata and Ostrea angasi (as well as Crassostrea gigas) in New South Wales, Australia in 2006 and 2007, and re-analyzed histological samples from the original description of this parasite species using in situ hybridization. Despite (1) reports of the oyster disease putatively caused by B. roughleyi during the time of collections, (2) the observation of gross lesions characteristic of the disease, and (3) the observation of B. roughleyi cells in association with the lesions, we detected a Bonamia sp. by PCR in just 1/42 O. angasi (2.4%), and 1/608 S. glomerata (0.2%), the latter oyster of which is the type host. SSU rDNA sequences of the amplicons were nearly identical to those of B. exitiosa and B. roughleyi, and phylogenetic analysis of ITS region sequences placed them on a B. exitiosa clade. A Haplosporidium sp. sequence similar to that of H. costale was PCR-amplified from nearly half the S. glomerata and O. angasi, but no Haplosporidium sp. was observed histologically. Our inability to identify a Bonamia sp. sequence in association with the B. roughleyi observed histologically suggests that this parasite is not a Bonamia sp. at all, and should be regarded as B. roughleyi nomen dubium. We conclude that the Bonamia sp. that we and other investigators detected in southeastern Australian S. glomerata and O. angasi was B. exitiosa.

Phylogenetics of Bonamia parasites based on small subunit and internal transcribed spacer region ribosomal DNA sequence data.

The genus Bonamia (Haplosporidia) includes economically significant oyster parasites. Described species were thought to have fairly circumscribed host and geographic ranges: B. ostreae infecting Ostrea edulis in Europe and North America, B. exitiosa infecting O. chilensis in New Zealand, and B. roughleyi infecting Saccostrea glomerata in Australia. The discovery of B. exitiosa-like parasites in new locations and the observation of a novel species, B. perspora, in non-commercial O. stentina altered this perception and prompted our wider evaluation of the global diversity of Bonamia parasites. Samples of 13 oyster species from 21 locations were screened for Bonamia spp. by PCR, and small subunit and internal transcribed spacer regions of Bonamia sp. ribosomal DNA were sequenced from PCR-positive individuals. Infections were confirmed histologically. Phylogenetic analyses using parsimony and Bayesian methods revealed one species, B. exitiosa, to be widely distributed, infecting 7 oyster species from Australia, New Zealand, Argentina, eastern and western USA, and Tunisia. More limited host and geographic distributions of B. ostreae and B. perspora were confirmed, but nothing genetically identifiable as B. roughleyi was found in Australia or elsewhere. Newly discovered diversity included a Bonamia sp. in Dendostrea sandvicensis from Hawaii, USA, that is basal to the other Bonamia species and a Bonamia sp. in O. edulis from Tomales Bay, California, USA, that is closely related to both B. exitiosa and the previously observed Bonamia sp. from O. chilensis in Chile.

PTEROBDELLA OCCIDENTALIS N. SP. (HIRUDINIDA: PISCICOLIDAE) FOR P. ABDITOVESICULATA (MOORE, 1952) FROM THE LONGJAW MUDSUCKER, GILLICHTHYS MIRABILIS, AND STAGHORN SCULPIN, LEPTOCOTTUS ARMATUS, AND OTHER FISHES IN THE EASTERN PACIFIC.

Pterobdella occidentalis n. sp. (Hirudinida: Piscicolidae) is described from the longjaw mudsucker, Gillichthys mirabilis Cooper, 1864, and the staghorn sculpin, Leptocottus armatus Girard, 1854, in the eastern Pacific, and the diagnosis of Pterobdella abditovesiculata (Moore, 1952) from the 'o'opu 'akupa, Eleotris sandwicensis Vaillant and Sauvage, 1875, from Hawaii is amended. The morphology of both species conforms with the genus Pterobdella in possessing a spacious coelom, well-developed nephridial system, and 2 pairs of mycetomes. Originally described as Aestabdella abditovesiculata, P. occidentalis (present along the U.S. Pacific Coast), can be distinguished from most congeners by its metameric pigmentation pattern and diffuse pigmentation on the caudal sucker. Based on mitochondrial gene sequences, including cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit I (ND1), P. occidentalis forms a distinct polyphyletic clade with Pterobdella leiostomi from the western Atlantic. Based on COI, ND1, and the 18S rRNA genes, other leech species most closely related to P. occidentalis include Pterobdella arugamensis from Iran, Malaysia, and possibly Borneo, which likely represent distinct species, and Pterobdella abditovesiculata from Hawaii, one of only a few endemic fish parasites in Hawaii. Like P. abditovesiculata, P. arugamensis, and Petrobdella amara, P. occidentalis is often found in estuarine environments, frequently infecting hosts adapted to a wide range of salinity, temperature, and oxygen. The physiological plasticity of P. occidentalis and the longjaw mudsucker host, and the ease of raising P. occidentalis in the lab, make it an excellent candidate for the study of leech physiology, behavior, and possible bacterial symbionts.

A rapid phenotype change in the pathogen Perkinsus marinus was associated with a historically significant marine disease emergence in the eastern oyster.

The protozoan parasite Perkinsus marinus, which causes dermo disease in Crassostrea virginica, is one of the most ecologically important and economically destructive marine pathogens. The rapid and persistent intensification of dermo in the USA in the 1980s has long been enigmatic. Attributed originally to the effects of multi-year drought, climatic factors fail to fully explain the geographic extent of dermo's intensification or the persistence of its intensified activity. Here we show that emergence of a unique, hypervirulent P. marinus phenotype was associated with the increase in prevalence and intensity of this disease and associated mortality. Retrospective histopathology of 8355 archival oysters from 1960 to 2018 spanning Chesapeake Bay, South Carolina, and New Jersey revealed that a new parasite phenotype emerged between 1983 and 1990, concurrent with major historical dermo disease outbreaks. Phenotypic changes included a shortening of the parasite's life cycle and a tropism shift from deeper connective tissues to digestive epithelia. The changes are likely adaptive with regard to the reduced oyster abundance and longevity faced by P. marinus after rapid establishment of exotic pathogen Haplosporidium nelsoni in 1959. Our findings, we hypothesize, illustrate a novel ecosystem response to a marine parasite invasion: an increase in virulence in a native parasite.

Observation of a Bonamia sp. infecting the oyster Ostrea stentina in Tunisia, and a consideration of its phylogenetic affinities.

The small non-commercial oyster Ostrea stentina co-occurs with commercially important Ostrea edulis in the Mediterranean Sea, yet its disposition with respect to the destructive pathogens Bonamia ostreae and Marteilia refringens is unknown. We began an evaluation of the Bonamia spp. infection status of O. stentina from Hammamet, Tunisia, in June 2007 using polymerase chain reaction diagnostics followed by histology and in situ hybridization. Of 85 O. stentina sampled, nine were PCR-positive for a Bonamia sp. using a Bonamia genus-specific assay; of these nine, one displayed the uninucleate microcells associated with oyster hemocytes characteristic of Bonamia spp. There was no associated pathology. DNA sequencing of the parasite from this one infected individual revealed it to be of a member of the Bonamia exitiosa/Bonamia roughleyi clade, an identification supported by positive in situ hybridization results with probes specific for members of this clade, and by the morphology of the parasite cells: nuclei were central, as in B. exitiosa, not eccentric, as in B. ostreae. There is no basis for identifying the Tunisian parasite as either B. exitiosa or B. roughleyi, however, as these species are genetically indistinguishable. Likewise, there is no basis for identifying any of the other Bonamia spp. with affinities to the B. exitiosa/B. roughleyi clade, from Argentina, Australia, Spain, and the eastern USA, as one or the other of these named species. Though they are clearly distinct from Bonamia perspora and B. ostreae, justification for drawing species boundaries among the primarily austral microcells with affinities to B. exitiosa and B. roughleyi remains elusive.

Minchinia mercenariae n. sp. (Haplosporidia) in the hard clam Mercenaria mercenaria: implications of a rare parasite in a commercially important host.

During routine histopathology of 180 juvenile hard clams, Mercenaria mercenaria, from a site in Virginia, USA, in 2007, we discovered a single individual heavily infected with a parasite resembling a haplosporidian, some members of which cause lethal bivalve diseases. Scanning electron microscopy of spores and sequencing of small subunit ribosomal DNA confirmed a new species: Minchinia mercenariae n. sp. Further sampling of clams at the site found prevalences up to 38% using polymerase chain reaction (PCR). No parasites were found in routine histological screening of the same individuals, but re-examination of clams judged positive by in situ hybridization (ISH) revealed very faintly staining plasmodia. No unusual mortalities have occurred among the sampled groups. Analysis of clams from Massachusetts to Florida by PCR failed to detect the parasite, but a haplosporidian found in a clam from New Jersey in 2001 was subsequently identified by ISH as M. mercenariae. No other haplosporidians have been reported in thousands of hard clams from the US east coast examined histologically since the mid-1980s. The discovery underscores critical questions about how to assess the risks associated with parasites in groups known to be lethal, but that themselves are not considered a problem.

Interacting effects of temperature and salinity on Bonamia sp. parasitism in the Asian oyster Crassostrea ariakensis.

The proposition to introduce the Asian oyster Crassostrea ariakensis to the mid-Atlantic region of the USA is being considered with caution, particularly after the discovery of a novel microcell haplosporidian parasite, Bonamia sp., in North Carolina. Although this parasite was found to be pathogenic in C. ariakensis under warm euhaline conditions, its persistence in C. ariakensis exposed to various temperature and salinity combinations remained unresolved. In this laboratory experiment, we tested the influence of temperature in combination with a wide range of salinities (10, 20 and 30 psu) on Bonamia sp. Temperature was either changed from warm (>20 degrees C) to cold (6 degrees C for 6 weeks) and back to warm or maintained constant and warm. Warm temperature was associated with higher host mortality than cold temperature, suggesting that temperature influenced Bonamia sp. pathogenicity. The effect of salinity was revealed under warm temperature with highest mortality levels observed in infected C. ariakensis exposed to 30 psu. When temperature was increased following low-temperature exposure, Bonamia sp. was not detected; however sub-optimal experimental conditions may have contributed to this result, making it difficult to draw conclusions regarding the reemergence of the parasite after low-temperature exposure. Although the overwintering of Bonamia sp. in C. ariakensis will need to be further investigated, the results presented here suggest that Bonamia sp. may be able to persist in C. ariakensis under a combination of low temperature and meso- to euhaline salinities.

Strong seasonality of Bonamia sp. infection and induced Crassostrea ariakensis mortality in Bogue and Masonboro Sounds, North Carolina, USA.

Asian oyster Crassostrea ariakensis is being considered for introduction to Atlantic coastal waters of the USA. Successful aquaculture of this species will depend partly on mitigating impacts by Bonamia sp., a parasite that has caused high C. ariakensis mortality south of Virginia. To better understand the biology of this parasite and identify strategies for management, we evaluated its seasonal pattern of infection in C. ariakensis at two North Carolina, USA, locations in 2005. Small (<50 mm) triploid C. ariakensis were deployed to upwellers on Bogue Sound in late spring (May), summer (July), early fall (September), late fall (November), and early winter (December) 2005; and two field sites on Masonboro Sound in September 2005. Oyster growth and mortality were evaluated biweekly at Bogue Sound, and weekly at Masonboro, with Bonamia sp. prevalence evaluated using parasite-specific PCR. We used histology to confirm infections in PCR-positive oysters. Bonamia sp. appeared in the late spring Bogue Sound deployment when temperatures approached 25 degrees C, six weeks post-deployment. Summer- and early fall-deployed oysters displayed Bonamia sp. infections after 3-4 weeks. Bonamia sp. prevalences were 75% in Bogue Sound, and 60% in Masonboro. While oyster mortality reached 100% in late spring and summer deployments, early fall deployments showed reduced (17-82%) mortality. Late fall and early winter deployments, made at temperatures <20 degrees C, developed no Bonamia sp. infections at all. Seasonal Bonamia sp. cycling, therefore, is influenced greatly by temperature. Avoiding peak seasonal Bonamia sp. activity will be essential for culturing C. ariakensis in Bonamia sp.-enzootic waters.

Misuse of PCR assay for diagnosis of mollusc protistan infections.

Polymerase chain reaction (PCR) assays are useful tools for pathogen surveillance, but they are only proxy indications of pathogen presence in that they detect a DNA sequence. To be useful for detection of actual infections, PCR assays must be thoroughly tested for sensitivity and specificity, and ultimately validated against a technique, typically histology, which allows visualization of the parasite in host tissues. There is growing use of PCR assays for pathogen surveillance, but too often the assumption is made that a positive PCR result verifies an infection in a tested host. This assumption is valid only if the assay has been properly validated for the geographic area and for the hosts examined. Researchers should interpret unvalidated PCR assay results with caution, and editors and reviewers should insist that robust validations support all assertions that PCR results confirm infections.

Molecular epizootiology of Perkinsus marinus and P. chesapeaki infections among wild oysters and clams in Chesapeake Bay, USA.

Perkinsus marinus and P. chesapeaki host ranges among wild Chesapeake Bay, USA, region bivalves were examined by surveying Crassostrea virginica oysters and members of several sympatric clam species from 11 locations. Perkinsus genus- and species-specific PCR assays were performed on DNA samples from 731 molluscs, and species-specific in situ hybridization assays were performed on a selected subset of histological samples whose PCR results indicated dual or atypical Perkinsus sp. infections. PCR assays detected P. marinus in 92% of oysters, but the P. chesapeaki PCR assay was positive for only 6% of oysters, and P. marinus was detected by PCR in only one clam. The very low prevalence of P. marinus infections in clams is noteworthy because all surveyed clams were sympatric with oyster populations showing high P. marinus infection prevalences. P. chesapeaki commonly infected Mya arenaria, Macoma balthica, and Tagelus plebeius clams, as well as the previously unreported P. chesapeaki host clams Mulinia lateralis, Rangia cuneata, and Cyrtopleura costata. Among 30 in vitro isolates propagated from surveyed hosts, 8 P. marinus isolates were exclusively from Crassostrea virginica oysters, and all 22 P. chesapeaki isolates were from clam hosts of 5 different species. Although both P. marinus and P. chesapeaki were previously both shown to be experimentally infective for oyster and clam hosts, this survey of wild bivalves in the Chesapeake Bay region reveals that P. marinus infections occur almost exclusively in oysters, and P. chesapeaki infections predominate among members of at least 6 clam species.

Experimental cross-infections by Perkinsus marinus and P. chesapeaki in three sympatric species of Chesapeake Bay oysters and clams.

In controlled laboratory transmission experiments, uniform doses of axenic in vitro isolate cultures of Perkinsus marinus from a Crassostrea virginica oyster, and of independent P. chesapeaki isolates from Chesapeake Bay Mya arenaria and Macoma balthica clams, were used to reciprocally challenge Perkinsus sp.-free C. virginica, M. arenaria, and M. balthica experimental hosts. Following mantle cavity inoculations, all 3 experimental hosts acquired high incidences (30 to 100%) of infections by each of the 3 Perkinsus sp. isolates, based on PCR assays of DNAs from experimental host tissues that were collected through 60 d post-inoculation. Lesions containing proliferating pathogen cells were documented histologically in tissues of all experimental host species challenged with all isolates of both Perkinsus species. Experimental Perkinsus sp. challenge isolates were re-isolated and propagated in vitro from infected tissues of host molluscs from most (5 of 9) experimental treatment groups. Koch's postulates were generally satisfied to confirm experimental infections in all bivalve molluscs that were challenged with 3 isolates of 2 Perkinsus spp. These results suggest potential broad and overlapping host specificities for the 2 current Chesapeake Bay-endemic Perkinsus species: P. marinus and P. chesapeaki.

Hemoflagellates of Oregon marine fishes with the description of new species of Trypanosoma and Trypanoplasma.

Of 2,122 marine fishes representing 36 species collected in the northeastern Pacific Ocean in the vicinity of Newport, Oregon from 1971 to 1973, 541 individuals (25.5%) representing 8 species (22.2%) were infected with hemoflagellates. Four morphologically distinct trypanosomes and 3 distinct trypanoplasms were found in fishes collected offshore, but no hemoflagellates were observed in fishes from Yaquina Bay estuary. Trypanosoma pacifica was found in English sole Parophrys vetulus, Pacific sanddab Citharichthys sordidus, and slender sole Lyopsetta exilis, and survived in 5 other species after intraperitoneal injection. Trypanosoma gargantua was found in big skate Raja binoculata, and the leech Orientobdella confluens was able to transmit the trypanosome in experimental conditions. Trypanosoma khani n. sp. occurred in P. vetulus, petrale sole Eopsetta jordani, and Dover sole Microstomus pacificus. Trypanosoma murmanense was found in L. exilis collected from 200 m, but not in L. exilis collected from 80 m. Trypanoplasma beckeri parasitized the cabezon Scorpaenichthys marmoratus. Trypanoplasma bobolsoni n. sp. was found in E. jordani, L. exilis, and P. vetulus, and survived in 2 other species after intraperitoneal injection. A distinct, but unnamed trypanoplasm, was found in P. vetulus.

Multiplication of Trypanosoma pacifica (Euglenozoa: Kinetoplastea) in English sole, Parophrys vetulus, from Oregon coastal waters.

Multiplication of Trypanosoma pacifica was common in the fish host from observations of live flagellates and Giemsa-stained blood smears. Multiplication began with the elongation of the kinetoplast, thickening of the posterior portion of the body, and appearance of a new flagellum near the kinetoplast. The new flagellum was very rigid when less than 3 microm in length, but it became flexible as it elongated. When the new flagellum was approximately 12 microm in length, cell division began and the kinetoplast also began to divide. The timing of nuclear division was variable. Generally, it did not occur until division of the kinetoplast had begun, but occasionally binucleate individuals were observed before cell or kinetoplast division was apparent. As division continued, 1 nucleus migrated past the dividing kinetoplast into the future daughter trypanosome. Finally, the kinetoplast completed division and the trypanosomes separated. Cell division was unequal, with the daughter trypanosome being smaller than the parent and with a more weakly developed undulating membrane.

Two new species of Austrobdella (Hirudinida: Piscicolidae) from Chile.

Austrobdella coliumicus n. sp. is described from Coliumo Bay, Chile. It is characterized by a continuous, external coelomic canal (= marginal lacuna), 5 pairs of testisacs, accessory gland cells, a body not distinctly divided into trachelosome and urosome, 2 pairs of dorsal ocelli on the trachelosome, dorsal and ventral segmental ocelli present on the urosome, green overall pigmentation with transverse brown bands, and the absence of conducting tissue. Austrobdella coliumicus is distinguished from other species of Austrobdella by the presence of 2 pairs of ocelli on the trachelosome and a more cylindrical body. It is unusual that this leech was collected from inside the mantle cavity of the razor clam, Ensis macha. Austrobdella losmoliniensis n. sp. is described from the electric ray, Discopyge tschudii, collected at Los Molinos, Chile. It is characterized by a continuous, external coelomic canal; 5 pairs of testisacs; accessory gland cells; a body distinctly divided into trachelosome and urosome; 1 pair of eyes on the oral sucker; overall black pigmentation with unpigmented areas; and the absence of conducting tissue. Austrobdella losmoliniensis can be distinguished from other species of Austrobdella by the combination of 1 pair of eyes on the oral sucker and black pigmentation.

A collection of fish leeches (Hirudinida: Piscicolidae) from Japan and surrounding waters, including redescriptions of three species.

Leeches were observed incidentally on fishes in collections made from 1975 to 2006 in Japan and surrounding waters, or from mariculture facilities or public aquaria in Japan. Seven species of leeches in 7 genera were collected--Crangonobdella maculosa, Johanssonia arctica, Limnotrachelobdella okae, Platybdella olriki, Stibarobdella bimaculata, Taimenobdella amurensis, and Trachelobdella livanovi. The transfer of Calliobdella livanovi to Trachelobdella is supported, and Trachelobdella livanovi and Taimenobdella amurensis are redescribed based on new specimens. Stibarobdella bimaculata is synonymized with Stibarobdella macrothela based on eyes, tubercle patterns, and sucker size ratios. Taimenobdella amurensis, C. maculosa, J. arctica, S. macrothela, and P. olriki are reported for the first time from Japan. New hosts are reported for L. okae, T. livanovi, S. macrothela, C. maculosa, J. arctica, and P. olriki. Stibarobdella moorei was not collected during this study, but a well-preserved specimen collected in Japan was discovered in the Muséum National d'Histoire Naturelle, Paris, France, and it allowed a redescription of this species. Stibarobdella loricata is synonymized with S. moorei based on tubercle patterns and the presence of papillae and a marginal fringe on the oral sucker.

A redescription of the fish leech Pterobdella amara (= Rhopalobdella japonica) (Hirudinida: Piscicolidae) based on specimens from the type locality in India and from Australia.

Pterobdella amara is redescribed from specimens from the type locality in India, and from the type host in Queensland, Australia. A neotype is designated from the type locality. The presence of conspicuous finlike bodies along the lateral margins of P. amara was greatly exaggerated in the original description. The trachelosome and anterior portion of the urosome are wide and flat, but there are no finlike bodies present. The urosome tapers abruptly posterior to the flattened portion, becomes cylindrical, and remains of constant width to the caudal sucker. The oral sucker is small and the caudal sucker is terminal. The body is smooth and lacks pigmentation, except for 1 pair of eyes on the oral sucker. Important internal characters are 2 pairs of mycetomes, 5 pairs of testisacs, confluent gonopores, extensive accessory gland cells on the male atrium and ejaculatory ducts, a spacious coelom consisting of dorsal and ventral sinuses with expansive connections between each at urosome ganglia, and interganglionic expansions of the dorsal sinus. Rhopalobdella japonica from stingrays in Japan is synonymized with P. amara and becomes a subjective junior synonym. This leech is a parasite in the oral cavity of the stingrays Pastinachus sephen and Himantura uarnak in India and Australia, and Dasyatis akajei in Japan.

A new species of Malmiana (Oligochaeta: Hirudinida: Piscicolidae) from tidepool fishes in northern California.

Malmiana buthi n. sp. is described from the body and fins of the fluffy sculpin, Oligocottus snyderi, the tidepool sculpin, Oligocottus maculosus, and the woolly sculpin, Clinocottus analis, collected from tidepools at Horseshoe Cove on the Bodega Marine Reserve in Sonoma County, California. Prevalence of the leech was 32.6% on live-caught O. snyderi; mean intensity on O. snyderi was 3.3 leeches per fish, with a range from 1 to 7. The leech is not known to exceed 8 mm total length. The body is smooth, lacking papillae, tubercles, or pulsatile vesicles. Two pairs of crescentiform eyes are present on the oral sucker, and 1 pair of punctiform eyes occurs on the second annulus of the trachelosome. The caudal sucker has 14 small punctiform ocelli spaced evenly around the margin. The last 9 segments of the urosome have pairs of large punctiform ocelli both dorsally and ventrally. Body and caudal sucker pigmentation is uniformly reddish brown dorsally and ventrally with segental, lateral, unpigmented areas on both the urosome and trachelosome; pigmentation on the oral sucker is in the form of a cross. There are 5 pairs of testisacs; accessory gland cells on the atrial cornua and vector tissue are absent.

Spore ornamentation of Haplosporidium nelsoni and Haplosporidium costale (Haplosporidia), and incongruence of molecular phylogeny and spore ornamentation in the Haplosporidia.

Spore ornamentation of Haplosporidium nelsoni and Haplosporidium costale was determined by scanning electron microscopy. For H. nelsoni, the spore surface was covered with individual ribbons that were tightly bound together and occurred as a single sheet. In some spores, this layer was overlaid with a network of branching fibers, about 0.05 microm in diameter, which often was dislodged from the spore at the aboral pole. For H. costale, ornamentation consisted of a sparse network of branching fibers on the spore surface. Molecular phylogenetic analysis of the phylum Haplosporidia revealed that Urosporidium, Bonamia, and Minchinia were monophyletic but that Haplosporidium was paraphyletic. All species of Minchinia have ornamentation composed of epispore cytoplasm, supporting the monophyly of this genus. The presence of spores with a hinged operculum and spore wall-derived ornamentation in Bonamia perspora confounds the distinction between Bonamia and Haplosporidium. Species with ornamentation composed of outer spore wall material and attached to the spore wall do not form a monophyletic group in the molecular phylogenetic analysis. These results suggest that the widely accepted practice of assigning all species with spore wall-derived ornamentation to Haplospordium cannot be supported and that additional genera are needed in which to place some species presently assigned to Haplosporidium.

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.

Systematic evaluation of factors controlling Perkinsus marinus transmission dynamics in lower Chesapeake Bay.

The transmission of Perkinsus marinus in eastern oysters Crassostrea virginica in relation to water temperature, host oyster mortality, and water-column abundance of anti-P. marinus antibody-labeled cells was systematically examined for 20 mo at a site in the lower York River, Virginia, USA. Uninfected sentinel oysters were naturally exposed to the parasite at 2 wk intervals throughout the course of the study to determine the periodicity and rates of parasite transmission. The timing and magnitude of disease-associated oyster mortalities in a local P. marinus-infected oyster population were estimated by monitoring a captive subset of the local oyster population. Flow cytometric immunodetection methods were employed to estimate the abundance of P. marinus cells in water samples collected 3 times each week. The acquisition of P. marinus infections by naïve sentinel oysters occurred sporadically at all times of the year; however, the highest incidence of infection occurred during the months of August and September. This window of maximum parasite transmission coincided with the death of infected hosts within the captive local oyster population. Counts of antibody-labeled cells ranged from 10 to 11900 cells l(-1), with the highest abundances in July and August coincident with maximum summer temperatures. A statistically significant relationship between water-column parasite abundance and infection-acquisition rate was not observed; however, highest parasite-transmission rates in both years occurred during periods of elevated water-column abundance of parasite cells. These results support the prevailing model of P. marinus transmission dynamics by which maximum transmission rates are observed during periods of maximum P. marinus-associated host mortality. However, our results also indicate that transmission can occur when host mortality is low or absent, so alternative mortality-independent dissemination mechanisms are likely. The results also suggest that atypically early-summer oyster mortality from Haplosporidium nelsoni infection, at a time when infections of P. marinus are light, has a significant indirect influence on P. marinus transmission dynamics. Elimination of these hosts prior to late-summer P. marinus infection-intensification effectively reduces the overall number of P. marinus cells disseminated.