Gill epithelial cell nuclear virus disease is prevalent and widespread in Mya arenaria clams of Chesapeake Bay, USA.

Several historic investigations have reported intranuclear virus infections of Mya arenaria soft-shell clams from the Atlantic coast of North America, but their descriptive details are limited. Among numerous multi-clam samples of Chesapeake Bay M. arenaria that were analyzed histopathologically during clam population surveys from 2000-2009, virus replication apparently caused extreme hypertrophy among the infected nuclei of gill epithelial cells. Infected cells were often abundant within the gill epithelia of affected clams, where their nuclear abnormalities suggested compromised genetic controls of critical cellular physiological functions. Infection prevalences were generally elevated, reaching 90% in 25% of samples. A grand mean prevalence of 67% resulted for all (69) M. arenaria samples of the decadal investigation, which included 1934 individual clams. Infected nuclei of gill epithelial cells were microscopically conspicuous by their extreme hypertrophic diameters of 10 µm or more and their prominent DNA-inclusion bodies. Cells with abnormal, hypertrophic nuclei were often abundant in the epithelia of M. arenaria gills. Transmission electron microscopy revealed abundant, replicating, icosahedral viral particles of 65-85 nm diameter within such hypertrophic nuclei. Viruses frequently occurred in paracrystalline nuclear arrays, showed granular internal contents, and had radial structures that suggested capsid surface ornamentation. Normal heterochromatin of infected nuclei appeared emarginated by dense central masses of replicating virions. Large, electron-dense DNA inclusion bodies routinely occurred at the internal margins of virus-infected nuclei. These may be virus replication centers based on their ultrastructural features and close proximity to replicated viral particles.

Immunoassays and diagnostic antibodies for Perkinsus spp. pathogens of marine molluscs.

Perkinsus sp. protozoans are parasites of a wide variety of molluscs around the world and are responsible for episodes of mass mortalities and large economic losses for aquaculture industries and fisheries. The first step towards the management of infectious episodes is the reliable detection of Perkinsus species. While historic methods for diagnosis of Perkinsus sp. infections in mollusc hosts include histological, in vitro, molecular-genetic, and immunoassays, antibody-based diagnostic assays may prove most practical with development of improved reagents and techniques. This paper reviews historic developments of antibodies against Perkinsus species, and of diagnostic immunoassays. Thirteen research papers reported the development of antibodies against Perkinsus sp. or their extracellular products, mainly P. olseni and P. marinus. Nine of those tested the cross-reactivity of their antibodies against different life stages or species than the one used as immunogen. While all antibodies raised against trophozoites labelled hypnospores, several antibodies raised against hypnospores did not label trophozoites, suggesting antigenic differences between those cell types. Antibody specificity studies showed that there is antigenic heterogeneity between Perkinsus species and Perkinsus-like organisms, and also that common epitopes occur among Perkinsus species, as well as some dinoflagellates. This review summarizes the current knowledge and aims at helping the future development of Perkinsus species-specific antibodies and immunoassays.

Parasites in two coexisting bivalves of the Patagonia coast, southwestern Atlantic Ocean: The Puelche oyster (Ostrea puelchana) and false oyster (Pododesmus rudis).

The purpose of this study was to compare the parasites of two coexisting bivalves, the edible Puelche oyster (Ostrea puelchana) and the false oyster (Pododesmus rudis) that lives attached to O. puelchana shells, and to investigate their host specificity. Samples from wild populations, 465 O. puelchana and 131 P. rudis, were collected seasonally during two years in the San José Gulf (northern Patagonia, Argentina) and were processed using standard histological techniques. To increase the natural low prevalences of Bonamia spp. and Perkinsus spp. that are present in wild populations, an in situ experiment was performed by maintaining captive sentinel bivalves at high densities inside a plastic mesh bag to enhance parasite transmission. Polymerase chain reaction (PCR) assays were used to test for apparent Bonamia sp. infections among captive sentinel O. puelchana specimens (n = 80), and Ray's fluid thioglycollate medium (RFTM) assays and histological immunoassays tested for apparent Perkinsus sp. infections among captive sentinel P. rudis specimens (n = 100). Despite histological observations that revealed the presence of microcells resembling Bonamia sp. infecting hemocytes of some Puelche oysters, PCR assays did not confirm that parasite identification. Among captive sentinel P. rudis that showed histological evidence of Perkinsus sp. infections, neither RFTM nor immunoassays confirmed such parasites. Ostrea puelchana from wild populations were occasional hosts for both Rickettsia-like organism (RLOs) and Urastoma-like turbellarians. In contrast, six parasite taxa infected P. rudis from coexisting populations, including RLOs, Urastoma-like turbellarians, an intracellular gregarine species, Nematopsis-like oocysts, an unidentified coccidian and a Perkinsus qugwadi-like protozoan. These results demonstrated specific infection patterns of the identified parasites in relation to their hosts.

A novel monoclonal Perkinsus chesapeaki in vitro isolate from an Australian cockle, Anadara trapezia.

A monoclonal Perkinsus chesapeaki isolate was established from 1 of 10 infected Australian Anadara trapezia cockles. Morphological features were similar to those of described P. chesapeaki isolates, and also included a unique vermiform schizont cell-type. Perkinsus olseni-specific PCR primers amplified DNAs from all 10 cockles. Perkinsus chesapeaki-specific primers also amplified DNAs from 4/10 cockles, including DNA from the isolate source cockle. Three different sets of DNA sequences from the monoclonal isolate grouped with the homologous, previously deposited, P. chesapeaki sequences in phylogenetic analyses. In situ hybridization assays detected both P. chesapeaki and P. olseni cells in histological sections from the source cockle for monoclonal isolate ATCC PRA-425.

Development and applications of Ray's fluid thioglycollate media for detection and manipulation of Perkinsus spp. pathogens of marine molluscs.

During the early 1950s, Sammy M. Ray discovered that his high-salt modification of fluid thioglycollate sterility test medium caused dramatic in vitro enlargement of Perkinsus marinus (=Dermocystidium marinum) cells that coincidentally infected several experimentally cultured oyster gill tissue explants. Subsequent testing confirmed that the enlarged cells among some oyster tissues incubated in Ray's fluid thioglycollate medium (RFTM) were those of that newly described oyster pathogen. Non-proliferative in vitro enlargement, cell wall thickening, and subsequent blue-black iodine-staining of hypertrophied trophozoites (=hypnospores=prezoosporangia) following incubation in RFTM are unique characteristics of confirmed members of the protistan genus Perkinsus. A number of in vitro assays and manipulations with RFTM have been developed for selective detection and enumeration of Perkinsus sp. cells in tissues of infected molluscs, and in environmental samples. RFTM-enlarged Perkinsus sp. cells from tissues of infected molluscs also serve as useful inocula for initiating in vitro isolate cultures, and cells of several Perkinsus spp. from both in vitro cultures and infected mollusc tissues may be induced to zoosporulate by brief incubations in RFTM. DNAs from RFTM-enlarged Perkinsus sp. cells provide useful templates for PCR amplifications, and for sequencing and other assays to differentiate and identify the detected Perkinsus species. We review the history and components of fluid thioglycollate and RFTM media, and the characteristics of numerous RFTM-based diagnostic assays that have been developed and used worldwide since 1952 for detection and identification of Perkinsus spp. in host mollusc tissues and environmental samples. We also review applications of RFTM for in vitro manipulations and purifications of Perkinsus sp. pathogen cells.

Perkinsus sp. infections and in vitro isolates from Anadara trapezia (mud arks) of Queensland, Australia.

Perkinsus sp. protists were found infecting Anadara trapezia mud ark cockles at 6 sites in Moreton Bay, Queensland, Australia, at prevalences of 4 to 100% during 2011 as determined by surveys using Ray's fluid thioglycollate medium. Perkinsus sp. lesions were found among gill and visceral connective tissues in histological samples from several cockles, where basophilic, eccentrically vacuolated Perkinsus sp. signet ring trophozoites and proliferating, Perkinsus sp. schizont cells were documented. Two Perkinsus sp. isolates were propagated in vitro during August 2013 from gill tissues of a single infected A. trapezia cockle from Wynnum in Moreton Bay. DNA from those isolate cells amplified universally by a Perkinsus genus-specific PCR assay, and rDNA-internal transcribed spacer sequences respectively grouped them with P. olseni and P. chesapeaki in phylogenetic analyses. This is the first report of P. chesapeaki in Australia, and the first report of a P. chesapeaki in vitro isolate from an Australian mollusc host. Although P. olseni was originally described in 1981 as a pathogen of abalone in South Australia, and has subsequently been identified as a prevalent pathogen of numerous other molluscs worldwide, this is also the first report of a P. olseni-like in vitro isolate from an Australian mollusc host.

Two Perkinsus spp. infect Crassostrea gasar oysters from cultured and wild populations of the Rio São Francisco estuary, Sergipe, northeastern Brazil.

Brazilian production of bivalve molluscs is small but expanding, especially in the northeastern region where the native oysters Crassostrea rhizophorae and C. gasar are abundant, and tropical weather promotes their rapid growth. Studies on bivalve pathology are scarce in Brazil, with only a few employing techniques for detecting protozoan pathogens listed by the World Organisation for Animal Health (OIE). In 2008, a Perkinsus sp. was reported for the first time in Brazil, infecting C. rhizophorae oysters from a wild population in Ceará state, NE Brazil. Recently P. marinus was detected in the same oyster species in nearby Paraíba state. These findings highlighted the need to expand knowledge on the presence and impacts of Perkinsus spp. on Brazilian oyster populations. The current investigation evaluated Perkinsus sp. infections among wild and cultured C. gasar mangrove oysters from the estuary of the Rio São Francisco, Sergipe state, NE Brazil. Our results show that Perkinsus sp. infections occurred commonly in oysters of both groups, at prevalences that were frequently higher among cultured oysters. Prevalences varied seasonally, with maximum values during summer (January) of 57% and 80% for wild and cultured oysters respectively, and minimum values during winter (July). Results of DNA sequencing, in situ hybridization assays, and phylogenetic analyses showed dual- and single-pathogen infections by P. marinus and/or P. olseni in the tested oysters.

Diseases of oysters Crassostrea ariakensis and C. virginica reared in ambient waters from the Choptank River, Maryland and the Indian River Lagoon, Florida.

To assess potential benefits and liabilities from a proposed introduction of Asian Suminoe oysters, susceptibilities of exotic Crassostrea ariakensis and native C. virginica oysters were compared during exposures to pathogens endemic in temperate, mesohaline waters of Chesapeake Bay and sub-tropical, polyhaline Atlantic waters of southern Florida, USA. Cohorts of diploid, sibling oysters of both species were periodically tested for diseases while reared in mesocosms receiving ambient waters from the Choptank River, Maryland (>3 yr) or the Indian River Lagoon, Florida (10 to 11 mo). Haplosporidium sp. infections (e.g. MSX disease) were not detected in oysters from either site. Perkinsus sp. infections (dermo disease) occurred among members of both oyster species at both sites, but infections were generally of low or moderate intensities. A Bonamia sp. was detected by PCR of DNAs from tissues of both oyster species following exposure to Florida waters, with maximum PCR prevalences of 44 and 15% among C. ariakensis and C. virginica oysters respectively during June 2007. Among C. ariakensis oysters sampled during April to July 2007, a Bonamia sp. was detected in 31% of oysters by PCR (range 11 to 35%) and confirmed histologically in 10% (range 0 to 15%). Among simultaneously sampled C. virginica oysters, a Bonamia sp. was detected in 7% by PCR (range 0 to 15%), but histological lesions were absent. Although this is the first report of a Bonamia sp. from Florida waters, sequences of small subunit (SSU) rDNA and in situ hybridization (ISH) assays both identified the Florida pathogen as Bonamia exitiosa, which also infects oysters in the proximate waters of North Carolina, USA.

A Paraffin Microtomy Method for Improved and Efficient Production of Standardized Plastic Microfibers.

Microfibers are one of the most abundant microplastic particle types found in the environment, where they cause negative impacts on organisms and possibly on human health. Microfibers should be included in a wide range of laboratory studies; however, microfibers for scientific studies are not commercially available. Current methods to make microfibers generally create particles with large size ranges and poor precision, and efficient production of particles ≤100 µm is difficult. Laboratory studies of the biological and toxicological effects and chemical interactions of microfibers require uniform, small microfibers in sufficient numbers for environmentally relevant experiments. We developed a novel fiber embedding technique and modified a seminal cryomicrotomy method to produce precise microfibers in quantities suitable for environmentally relevant concentrations. Polyethylene terephthalate (PET) and nylon fibers were strategically wound onto a spindle, embedded in paraffin wax, and sectioned using a standard paraffin microtome. After processing with a suitable organic solvent to remove the wax, microfiber size distributions were assessed. The small microfibers (10-42 µm) were accurate to the target lengths with excellent precision and a production rate ≥13.5 times higher than previous methods. As a proof of application, three lengths of manufactured PET fibers were stained with Nile red and exposed to eastern oyster larvae (Crassostrea virginica) for 24 h. Larvae ingested the smaller fiber lengths (14 and 28 µm), and the Nile red-stained fibers were visible and distinguishable in the guts of the larvae. This experiment was the first to demonstrate ingestion of plastic particles other than microspheres by oyster larvae. The present method facilitates the use of small microfibers in laboratory experiments, allowing for a more complete understanding of microplastic effects in the environment. Environ Toxicol Chem 2022;41:944-953. © 2021 SETAC.

Exotic Perkinsus sp. protozoa in an imported Vietnamese ornamental clam (Tridacna crocea) maintained in a home aquarium.

An adult, hermaphroditic Tridacna crocea ornamental clam imported from Vietnam into the USA became terminally moribund with sloughed byssal tissue and incomplete extension of the poorly responsive mantle and was necropsied. Necropsy findings included emaciation, visceral mass edema, and rare multifocal, 1-mm diameter, off-white to light tan gill nodules. Histopathology revealed marked inflammation and necrosis within the visceral mass and gills, with interstitial edema and atrophy of glandular, gonadal, and muscular tissues. Inflamed tissues contained large numbers of 10-15 microm extracellular, spherical organisms with a signet-ring morphology consistent with Perkinsus spp. trophozoites. The organisms often formed clusters of two to four cells and were surrounded by a host reaction consisting of a 1-4 microm rim of amorphous eosinophilic material and two to four host hemocytes. Incubation of infected host tissues in alternative Ray's fluid thioglycollate medium (ARFTM) confirmed the presence of Perkinsus sp. hypnospores that stained blue-black with Lugol's iodine. Polymerase chain reaction assays with sequencing of products revealed a high level of nucleotide similarity, but no exact match, to known P. olseni isolates. Perkinsus sp. organisms, including P. olseni and P. marinus, which are internationally reportable, are highly pathogenic destructive protozoa capable of disrupting ecosystems populated by naïve mollusks within the USA and negatively affecting both domestic and international shellfish industries. This is the first report of an exotic Perkinsus sp. pathogen in an imported ornamental clam maintained long term in a home aquarium. However, ongoing research indicates that T. crocea from Vietnam are commonly infected by such organisms. Veterinarians, aquarium facility mangers, and veterinary clients with hobby aquariums should use appropriate caution and responsible disposal practices for clam carcasses and for water in which imported ornamental clams have been housed. Such practices will reduce the possibility of dispersing viable, exotic Perkinsus sp. organisms into domestic waters.

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.

Development of a real time quantitative PCR assay for the hard clam pathogen Quahog Parasite Unknown (QPX).

Quahog Parasite Unknown (QPX) is a thraustochytrid pathogen responsible for catastrophic mortalities of the northern quahog (hard clam) Mercenaria mercenaria. A real-time quantitative polymerase chain reaction (qPCR) assay was developed to assist research efforts on QPX ecology and pathology. Sensitivity of the assay was evaluated with serial dilutions of QPX-cultured cells to determine the lowest concentration of DNA that remained detectable in both the presence and absence of extraneous environmental substances. QPX cells were quantified before DNA extraction to calibrate standard curves to cell counts. Based on our results, the qPCR assay is able to quantify QPX within the range of 1 to several thousand organisms per reaction. Specificity of the assay was assessed by testing 29 thraustochytrid-like protists isolated from suspension-feeding bivalves from China, Oregon, Maryland, and Virginia. Application of the assay was demonstrated with positive qPCR results from naturally contaminated environmental samples including marine aggregates (i.e. marine snow), clam pseudofeces, and inflammatory nodules from infected clams. This quantitative assay for QPX will provide a valuable tool for characterizing QPX parasite abundances in coastal environments and for improving clam disease diagnostics.

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.

Two epizootic diseases in Chesapeake Bay commercial clams, Mya arenaria and Tagelus plebeius.

Declining Chesapeake Bay harvests of softshell clams, together with historical and emerging reports of epizootic diseases in Mya arenaria, prompted a survey in summer 2000 of the health status of selected commercial clam populations. All sampled populations (8 M arenaria softshell clam, 2 Tagelus plebeius razor clam) were infected by Perkinsus sp. protozoans at prevalences ranging from 30 to 100% of sampled clams. Nucleotide sequences for the internal transcribed spacer (ITS) region of the rRNA gene complex were determined for clonal in vitro Perkinsus sp. isolates propagated from both M. arenaria and T plebeius. Multiple polymorphic sequences were amplified from each isolate, but phylogenetic analysis placed all sequences into 2 clades of a monophyletic group, which included both recently described clam parasites P. chesapeaki and P. andrewsi. Sequences amplified from each clonal isolate were found in both sister clades, one containing P. andrewsi and the other P. chesapeaki. Most (7 of 8) M. arenaria samples were also affected with disseminated neoplasia (DN), at prevalences of 3 to 37%, but neither T. plebeius sample showed DN disease. Disease mortalities projected for sampled clam populations, especially those affected by both diseases, may further deplete subtidal commercial clam populations in mesohaline portions of Chesapeake Bay.

Signal recognition particle RNA in dinoflagellates and the Perkinsid Perkinsus marinus.

In dinoflagellates and perkinsids, the molecular structure of the protein translocating machinery is unclear. Here, we identified several types of full-length signal recognition particle (SRP) RNA genes from Karenia brevis (dinoflagellate) and Perkinsus marinus (perkinsid). We also identified the four SRP S-domain proteins, but not the two Alu domain proteins, from P. marinus and several dinoflagellates. We mapped both ends of SRP RNA transcripts from K. brevis and P. marinus, and obtained the 3' end from four other dinoflagellates. The lengths of SRP RNA are predicted to be ∼260-300 nt in dinoflagellates and 280-285 nt in P. marinus. Although these SRP RNA sequences are substantially variable, the predicted structures are similar. The genomic organization of the SRP RNA gene differs among species. In K. brevis, this gene is located downstream of the spliced leader (SL) RNA, either as SL RNA-SRP RNA-tRNA gene tandem repeats, or within a SL RNA-SRP RNA-tRNA-U6-5S rRNA gene cluster. In other dinoflagellates, SRP RNA does not cluster with SL RNA or 5S rRNA genes. The majority of P. marinus SRP RNA genes array as tandem repeats without the above-mentioned small RNA genes. Our results capture a snapshot of a potentially complex evolutionary history of SRP RNA in alveolates.

Introns, alternative splicing, spliced leader trans-splicing and differential expression of pcna and cyclin in Perkinsus marinus.

To gain understanding on the structure and regulation of growth-related genes of the parasitic alveolatePerkinsus marinus, we analyzed genes encoding proliferating cell nuclear antigen (pcna) and cyclins (cyclin). Comparison of the full-length cDNAs with the corresponding genomic sequences revealedtrans-splicing of the mRNAs of these genes with a conserved 21-22 nt spliced leader. Over 10 copies ofpcnawere detected, with identical gene structures and similar nucleotide (nt) sequences (88-99%), encoding largely identical amino acid sequences (aa). Two distinct types ofcyclin(Pmacyclin1 andPmacyclin2) were identified, with 66-69% nt and 81-85% aa similarities.Pmacyclin2 was organized in tandem repeats, and was alternatively spliced, giving rise to five subtypes of transcripts. For bothpcnaandcyclingenes, 6-10 introns were found. Quantitative RT-PCR assays showed thatpcnaandPmacyclin2 expression levels were low with small variations during a 28-h time course, whereasPmacyclin1 transcript abundance was 10-100 times higher, and increased markedly during active cell division, suggesting that it is a mitoticcyclinand can be a useful growth marker for this species. The gene structure and expression features along with phylogenetic results position this organism between dinoflagellates and apicomplexans, but its definitive affiliation among alveolates requires further studies.

Spliced leader RNAs, mitochondrial gene frameshifts and multi-protein phylogeny expand support for the genus Perkinsus as a unique group of alveolates.

The genus Perkinsus occupies a precarious phylogenetic position. To gain a better understanding of the relationship between perkinsids, dinoflagellates and other alveolates, we analyzed the nuclear-encoded spliced-leader (SL) RNA and mitochondrial genes, intron prevalence, and multi-protein phylogenies. In contrast to the canonical 22-nt SL found in dinoflagellates (DinoSL), P. marinus has a shorter (21-nt) and a longer (22-nt) SL with slightly different sequences than DinoSL. The major SL RNA transcripts range in size between 80-83 nt in P. marinus, and ∼ 83 nt in P. chesapeaki, significantly larger than the typical ≤ 56-nt dinoflagellate SL RNA. In most of the phylogenetic trees based on 41 predicted protein sequences, P. marinus branched at the base of the dinoflagellate clade that included the ancient taxa Oxyrrhis and Amoebophrya, sister to the clade of apicomplexans, and in some cases clustered with apicomplexans as a sister to the dinoflagellate clade. Of 104 Perkinsus spp. genes examined 69.2% had introns, a higher intron prevalence than in dinoflagellates. Examination of Perkinsus spp. mitochondrial cytochrome B and cytochrome C oxidase subunit I genes and their cDNAs revealed no mRNA editing, but these transcripts can only be translated when frameshifts are introduced at every AGG and CCC codon as if AGGY codes for glycine and CCCCU for proline. These results, along with the presence of the numerous uncharacterized 'marine alveolate group I' and Perkinsus-like lineages separating perkinsids from core dinoflagellates, expand support for the affiliation of the genus Perkinsus with an independent lineage (Perkinsozoa) positioned between the phyla of Apicomplexa and Dinoflagellata.

Description of Perkinsus beihaiensis n. sp., a new Perkinsus sp. parasite in oysters of Southern China.

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 microm 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.

Perkinsus olseni in vitro isolates from the New Zealand clam Austrovenus stutchburyi.

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 Ray's 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 Ray's 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).