Ultrastructural comparison of Bonamia spp. (Haplosporidia) infecting ostreid oysters.

The ultrastructure of Bonamia from Ostrea angasi from Australia, Crassostrea ariakensis from the USA, O. puelchana from Argentina and O. edulis from Spain was compared with described Bonamia spp. All appear conspecific with B. exitiosa. The Bonamia sp. from Chile had similarities to the type B. exitiosa from New Zealand (NZ), but less so than the other forms recognized as B. exitiosa. Two groups of ultrastructural features were identified; those associated with metabolism (mitochondrial profiles, lipid droplets and endoplasmic reticulum), and those associated with haplosporogenesis (Golgi, indentations in the nuclear surface, the putative trans-Golgi network, perinuclear granular material and haplosporosome-like bodies). Metabolic features were regarded as having little taxonomic value, and as the process of haplosporogenesis is not understood, only haplosporosome shape and size may be of taxonomic value. However, the uni-nucleate stages of spore-forming haplosporidians are poorly known and may be confused with Bonamia spp. uni-nucleate stages. The many forms of NZ B. exitiosa have not been observed in other hosts, which may indicate that it has a plastic life cycle. Although there are similarities between NZ B. exitiosa and Chilean Bonamia in the development of a larger uni-nucleate stage and the occurrence of cylindrical confronting cisternae, the clarification of the identity of Chilean Bonamia must await molecular studies.

Bonamia exitiosa transmission among, and incidence in, Asian oyster Crassostrea ariakensis under warm euhaline conditions.

Previously reported in Australia, New Zealand, and more recently in Europe, the protistan parasite Bonamia exitiosa was also reported in the mid-Atlantic region of the USA after causing serious mortalities there in the Asian oyster Crassostrea ariakensis. At the time, this oyster was being considered for introduction, and the potential consequences of introducing this species were being assessed using field and laboratory studies. B. exitiosa emerged as the most serious disease threat for this oyster species, especially under warm euhaline conditions and for oysters <50 mm in size. To better evaluate how quickly this parasite may be able to spread among C. ariakensis, we investigated B. exitiosa transmission and incidence in C. ariakensis. During a first trial, potential direct transmission of B. exitiosa was evaluated by cohabitating infected C. ariakensis with uninfected C. ariakensis under in vivo quarantine conditions. In a second experiment, B. exitiosa incidence was estimated in situ by determining its prevalence in C. ariakensis deployed in an enzootic area after 4, 7, 14, 21 and 28 d of exposure. Results suggest that under warm euhaline conditions B. exitiosa can be transmitted among C. ariakensis without requiring any other parasite source and that parasite incidence may be at least as high as 40% after only 4 d exposure to an enzootic area. These results underscored the severity of the bonamiasis disease threat to C. ariakensis and provided further evidence that efforts to build an aquaculture industry based on C. ariakensis in the eastern USA might have been thwarted by parasitic disease.

Haplosporidium raabei n. sp. (Haplosporidia): a parasite of zebra mussels, Dreissena polymorpha (Pallas, 1771).

Extensive connective tissue lysis is a common outcome of haplosporidian infection. Although such infections in marine invertebrates are well documented, they are relatively rarely observed in freshwater invertebrates. Herein, we report a field study using a comprehensive series of methodologies (histology, dissection, electron microscopy, gene sequence analysis, and molecular phylogenetics) to investigate the morphology, taxonomy, systematics, geographical distribution, pathogenicity, and seasonal and annual prevalence of a haplosporidian observed in zebra mussels, Dreissena polymorpha. Based on its genetic sequence, morphology, and host, we describe Haplosporidium raabei n. sp. from D. polymorpha - the first haplosporidian species from a freshwater bivalve. Haplosporidium raabei is rare as we observed it in histological sections in only 0·7% of the zebra mussels collected from 43 water bodies across 11 European countries and in none that were collected from 10 water bodies in the United States. In contrast to its low prevalences, disease intensities were quite high with 79·5% of infections advanced to sporogenesis.

Inter-relationships of haplosporidians deduced from ultrastructural studies.

We reviewed papers reporting haplosporidian ultrastructure to compare inter-relationships based on ultrastructure with those based on molecular data, to identify features that may be important in haplosporidian taxonomy, and to consider parasite taxonomy in relation to host taxonomy. There were links between the following: (1) the plasmodia of an abalone parasite, Haplosporidium nelsoni and Urosporidium crescens in the release of haplosporosomes; (2) H. costale and H. armoricanum in haplosporosome shape and presence and shape of Golgi in spores; (3) basal asporous crustacean haplosporidians which form haplosporosomes from formative bodies (FBs) in vegetative stages--H. nelsoni, which forms haplosporosomes from FBs in plasmodial cytoplasm, and H. louisiana, Minchinia spp. and Bonamia perspora, which form haplosporosomes from FBs in spores; (4) crustacean haplosporidians, Bonamia spp. and M. occulta in the predominance of uni- and binucleate stages; and (5) lipid-like vesicles in sporoplasms of H. costale, H. armoricanum, H. lusitanicum, H. pickfordi, H. montforti, and B. perspora. In general, these relationships reflect phylogenies based on molecular studies. As well as spore form and ornamentation, haplosporogenesis in spores appears to be taxonomically important. Parasite and host taxonomy were linked in the infection of lower invertebrates by Urosporidium spp., the infection of oysters by Bonamia spp., and of molluscs by Minchinia spp. Haplosporidium spp. are patently an artificial, paraphyletic group probably comprising many taxa. Consequently, the taxonomy of haplosporidians needs a thorough revision.

Leeches on mudskippers: host-parasite interaction at the water's edge.

A host-parasite relationship was observed, for the first time, between a piscicolid leech and a species of amphibious goby (Scartelaos tenuis) from an intertidal mud flat in southern Iran. Morphological and molecular investigations assign the leech to Zeylanicobdella arugamensis. Of the 3 endemic and sympatric mudskipper species living in the Persian Gulf (S. tenuis, Boleophthalmus dussumieri, and Periophthalmus waltoni), leeches were only found on S. tenuis (prevalence and mean intensity = 71.4% and 2.3 +/- 2.5, respectively), which is also the most-aquatic mudskipper species. Scartelaos tenuis is not the largest species, but more leeches (> or =4 leeches/host) were found on larger specimens (>12 cm standard length [SL]). Nonetheless, in aquaria, leeches also attached on P. waltoni. This suggests either an ecological partitioning of host-parasite complexes, determined by host habitat selection, or leech limited-resistance to air exposure, or both.

Shellfish tissues evaluated for Perkinsus spp. using the Ray's fluid thioglycollate medium culture assay can be used for downstream molecular assays.

Ray's fluid thioglycollate medium (RFTM) culture assay is the standard, recommended method for surveillance of Perkinsus spp. infections in marine molluscs. In this assay, shellfish tissues are incubated in RFTM, stained with Lugol's iodine solution to render Perkinsus spp. cells blue-black, and evaluated microscopically to rate infection intensities. A limitation of this assay, however, is the lack of pathogen species specificity. Generally, identification of Perkinsus spp. requires DNA sequence analysis of parallel or additional samples since the exposure to iodine is believed to hamper DNA amplification from samples processed by the RFTM assay. However, we show that P. marinus DNA can be successfully amplified by PCR from Crassostrea virginica tissues cultured in RFTM and stained with Lugol's iodine. The beneficial consequence is that, where necessary, DNA sequence data may be obtained from RFTM-cultured tissues, allowing the identification of the Perkinsus sp. responsible for an observed infection. This would obviate further sampling, representing gain of time and reduction in cost, where a Perkinsus sp. is unexpectedly observed in new host(s) or location(s) but where parallel samples are not available for molecular diagnostics. Laboratories without molecular diagnostic tools for Perkinsus spp. may fix presumptive Perkinsus sp.-positive culture material in 95% ethanol for transport to, and subsequent analysis by, a laboratory that does have this capacity.

Spore ornamentation of Haplosporidium hinei n. sp. (Haplosporidia) in pearl oysters Pinctada maxima (Jameson, 1901).

An infection of pearl oysters, Pinctada maxima, attributed to a Haplosporidium sp. by Hine and Thorne (1998) has been detected on 3 occasions and is considered to represent a serious concern to the pearling industry in Australia. The spore ornamentation of the parasite was determined by scanning electron microscopy and transmission electron microscopy. Spores of the parasite were pleomorphic, or elongated 3.5-4 microm x 2.5-3.0 microm in size. Two filaments were wound around the spore and originated from 2 'knob-like' posterior thickenings. Both filaments passed up one side of the spore together until just below the operculum whereupon each split and passed obliquely under the lip of the opercula lid. Each filament wrapped around the spore 4 times. The posterior thickenings seem to appear late in the development of the spore and were composed of spore wall material. A second set of branching tubular filaments composed of a different material was observed on the spore body although not on mature spores possessing a 'knob-like' posterior thickening. The ornamentation on the spores of the pearl oyster parasite was unique amongst described haplosporidian species where spore ornamentation is known. The parasite is named in this manuscript as Haplosporidium hinei n. sp.

Pathogens in Crassostrea ariakensis and other Asian oyster species: implications for non-native oyster introduction to Chesapeake Bay.

With the drastic decline of eastern oyster Crassostrea virginica populations in the Chesapeake Bay due to over-fishing, diseases and habitat destruction, there is interest in Maryland and Virginia in utilizing the non-native oyster species Crassostrea ariakensis for aquaculture, fishery resource enhancement, and ecological restoration. The International Council for the Exploration of the Sea (ICES) recommends that non-native species be examined for ecological, genetic and disease relationships in the native range prior to a deliberate introduction to a new region. Therefore, a pathogen survey of C. ariakensis and other sympatric oyster species was conducted on samples collected in the PR China, Japan and Korea using molecular diagnostics and histopathology. Molecular assays focused on 2 types of pathogens: protistan parasites in the genus Perkinsus and herpesviruses, both with known impacts on commercially important molluscan species around the world, including Asia. PCR amplification and DNA sequence data from the internal transcribed spacer region of the rRNA gene complex revealed the presence of 2 Perkinsus species not currently found in USA waters: P. olseni and an undescribed species. In addition, 3 genetic strains of molluscan herpesviruses were detected in oysters from several potential C. ariakensis broodstock acquisition sites in Asia. Viral gametocytic hypertrophy, Chlamydia-like organisms, a Steinhausia-like microsporidian, Perkinsus sp., Nematopsis sp., ciliates, and cestodes were also detected by histopathology.

Real-time PCR investigation of parasite ecology: in situ determination of oyster parasite Perkinsus marinus transmission dynamics in lower Chesapeake Bay.

Perkinsus marinus is a severe pathogen of the oyster Crassostrea virginica on the East Coast of the United States. Transmission dynamics of this parasite were investigated in situ for 2 consecutive years (May through October) at 2 lower Chesapeake Bay sites. Compared to previous studies where seasonal infection patterns in oysters were measured, this study also provided parasite water column abundance data measured using real-time PCR. As previously observed, salinity and temperature modulated parasite transmission dynamics. Using regression analysis, parasite prevalence, oyster mortalities and parasite water column abundance were significantly positively related to salinity. Perkinsus marinus weighted prevalence in wild oysters and parasite water column abundance both were significantly related to temperature, but the responses lagged 1 month behind temperature. Parasite water column abundance was the highest during August (up to 1,200 cells/l) and was significantly related to P. marinus weighted prevalence in wild oysters, and to wild oyster mortality suggesting that parasites are released in the environment via both moribund and live hosts (i.e. through feces). Incidence was not significantly related to parasite water column abundance, which seems to indicate the absence of a linear relationship or that infection acquisition is controlled by a more complex set of parameters.

Molecular diagnostics, field validation, and phylogenetic analysis of Quahog Parasite Unknown (QPX), a pathogen of the hard clam Mercenaria mercenaria.

Quahog Parasite Unknown (QPX) is a protistan parasite that causes disease and mortality in the hard clam Mercenaria mercenaria. PCR primers and DNA oligonucleotide probes were designed and evaluated for sensitivity and specificity for the QPX organism specifically and for the phylum Labyrinthulomycota in general. The best performing QPX-specific primer pair amplified a 665 bp region of the QPX small-subunit ribosomal DNA (SSU rDNA) and detected as little as 1 fg cloned QPX SSU rDNA and 20 fg QPX genomic DNA. The primers did not amplify DNA of uninfected hard clams M. mercenaria or of the thraustochytrids Schizochytrium aggregatum, Thraustochytrium aureum, and T. striatum. The general labyrinthulomycete primers, which were designed to offer broader specificity than the QPX primers, amplified a 435 bp region of SSU rDNA from QPX, and a 436 to 437 bp region of SSU rDNA from S. aggregatum, T. aureum, and T. striatum, but did not amplify that of the clam M. mercenaria. Field validation of the QPX-specific primer pair, through comparative sampling of 224 clams collected over a 16 mo period from a QPX endemic site in Virginia, USA, indicated that the PCR assay is equivalent to histological diagnosis if initially negative PCR products are reamplified. Oligonucleotide DNA probes specific for QPX and the phylum Labyrinthulomycota were evaluated for in situ hybridization assays of cell smears or paraffin-embedded tissues. Two DNA probes for QPX offered limited sensitivity when used independently; however, when used together as a probe cocktail, sensitivity was greatly enhanced. The probe cocktail hybridized to putative QPX organisms in tissues of hard clams collected from Virginia, New Jersey, Massachusetts and Canada, suggesting that the QPX organisms in these areas are either very closely related or the same species. The QPX probe cocktail did not hybridize with clam tissue or with the thraustochytrids S. aggregatum, T. aureum, and T. striatum. The labyrinthulomycete DNA probe hybridized with QPX and the 3 thraustochytrids, with no background hybridization to clam tissue. SSU rDNA sequences were obtained for the putative QPX organisms from geographically distinct sites. Phylogenetic analyses based on the QPX and Labyrinthulomycota sequences confirmed earlier reports that QPX is a member of this phylum, but could not definitively demonstrate that all of the QPX organisms were the same species.

Validating Livanow: molecular data agree that leeches, Branchiobdellidans, and Acanthobdella peledina form a monophyletic group of oligochaetes.

To investigate the phylogenetic relationships of leeches, branchiobdellidans, and acanthobdellidans, whole nuclear 18S rDNA and over 650 bp of mitochondrial cytochrome c oxidase subunit I were acquired from 101 annelids, including 36 leeches, 18 branchiobdellidans, Acanthobdella peledina, as well as 28 oligochaetes and combined with homologous data for 17 polychaete outgroup taxa. Parsimony analysis of the combined aligned dataset supported monophyly of leeches, branchiobdellidans, and acanthobdellidans in 100% of jackknife replicates. Monophyly of the oligochaete order Lumbriculida with Acanthobdellida, Branchiobdellida, and Hirudinea was supported in 84% of jackknife replicates. These results provide support for the hypotheses that leeches and branchiobdellidans are sister groups, that acanthobdellidans are sister to them, and that together with the family Lumbriculidae they all constitute a clade within Oligochaeta. Results support synonymy of the classes Clitellata and the more commonly used Oligochaeta. Leeches branchiobdellidans, and acanthobdellidans should be regarded as orders equal to their closest relatives, the order Lumbriculida.

Spore ornamentation of Haplosporidium pickfordi Barrow, 1961 (Haplosporidia), a parasite of freshwater snails in Michigan, USA.

Spore ornamentation is increasingly recognized as a key character for species differentiation and genus assignment in the phylum Haplosporidia. Unfortunately, spore ornamentation is known for only a small number of described species so it is difficult to assign most species to genera with any confidence. Scanning and transmission electron microscopy were used to determine the presence and morphology of spore ornamentation of Haplosporidium pickfordi collected from the digestive gland of the snail Physella parkeri in Douglas Lake, Michigan. Spores possess filaments that are derived from the spore wall and originate from two separate areas at the posterior end of the spore. When spores are first isolated from host tissue, filaments are fused into a sheet that wraps around the spore, passing under the opercular lid. These filaments gradually unravel when spores are held in water and after about 14 d most filaments project freely from the posterior end of the spore. The number of filaments could not be determined with certainty, but appears to be approximately nine. Filaments are 100 nm in diam. and up to 50 microm in length. The presence of spore wall-derived filaments confirms the placement of the parasite in the genus Haplosporidium.

Haplosporidiosis in the Pacific oyster Crassostrea gigas from the French Atlantic coast.

Two cases of haplosporidian infection occurred during 1993 in Pacific oysters Crassostrea gigas from the French Atlantic coast. The localization and ultrastructure of the plasmodia are described. In situ hybridization of infected tissue sections was conducted with DNA probes for oyster-infecting haplosporidians. The Haplosporidium nelsoni-specific DNA probe MSX1347 hybridized with the C. gigas parasite, and the H. costale-specific probe SSO1318 did not hybridize. Total genomic DNA was extracted from the infected tissue sections for polymerase chain reaction (PCR) amplification of the haplosporidian. PCR amplifications with H. nelsoni-specific primers and with 'universal' actin primers did not yield the expected products of 573 and 700 bp, respectively. A series of primers was designed to amplify short regions of small subunit ribosomal DNA (SSU rDNA) from most haplosporidians. The primers encompass a highly variable region of the SSU rDNA and did not amplify oyster DNA. PCR amplification of the infected C. gigas genomic DNA with these primers yielded the expected-sized product from the primer pair targeting the shortest region (94 bp). This PCR product was sequenced and it was identical to the corresponding SSU rDNA region of H. nelsoni.

Rhopalobdella japonica n. gen., n. sp. (Hirudinea, Piscicolidae) from Dasyatis akajei (Chondrichthyes: Dasyatididae) in the northwestern Pacific.

A new genus and species of piscicolid leech in the Platybdellinae inhabits the oral cavity of Dasyatis akajei in the northwestern Pacific Ocean near Tanabe, Japan. The genus Rhopalobdella n. gen. is characterized externally by very small oral and caudal suckers and a smooth body that is widest just posterior to the clitellum. Eyespots and ocelli are lacking. The coelom is spacious with large segmental connecting sinuses between dorsal and ventral sinuses. There are 5 pairs of testisacs, an unusually extensive epididymis, and a very large bursa. Conducting tissue is absent. There are 2 pairs of esophageal diverticula and very well developed nephridia. Rhopalobdella japonica n. gen. n. sp. is characterized by a urosome that tapers strongly to the caudal sucker and by a single gonopore; the common oviduct opens into the posterior portion of the bursa. The coelomic and excretory systems resemble Aestabdella, but in other respects the genera are quite different. This is the first marine leech reported from rays in the northwestern Pacific.

A quantitative competitive polymerase chain reaction assay for the oyster pathogen Perkinsus marinus.

A quantitative competitive polymerase chain reaction (QCPCR) assay was developed for the oyster parasite Perkinsus marinus. PCR primers for the rRNA gene region of P. marinus amplified DNA isolated from P. marinus but not from Perkinsus atlanticus, Crassostrea virginica, or the dinoflagellates Peridinium sp., Gymnodinium sp., or Amphidinium sp. A mutagenic primer was used to create a competitor plasmid molecule identical to the P. marinus target DNA sequence except for a 13-bp deletion. Both P. marinus and competitor DNA amplified with equivalent efficiencies. Each of 25 oysters was processed by 5 P. marinus diagnostic methods--Ray's fluid thioglycollate medium (FTM) tissue assay, FTM hemolymph assay, whole oyster body burden assay, QCPCR of combined gill and mantle (gill/mantle) tissue, and QCPCR of hemolymph. The QCPCR assay enabled detection of 0.01 fg of P. marinus DNA in 1.0 microg of oyster tissue. QCPCR of gill/mantle tissue or hemolymph as well as the body burden assay detected infections in 24 of 25 oysters. Ray's FTM tissue assay detected only 19 infections. The FTM hemolymph assay detected only 22 infections. Regression analysis of QCPCR results and FTM results indicated that the QCPCR assays were effective in quantitating P. marinus infections in oyster tissues.

Increased Virulence in an Introduced Pathogen: Haplosporidium nelsoni (MSX) in the Eastern Oyster Crassostrea virginica.

The protistan parasite Haplosporidium nelsoni has caused extensive mortality in the eastern oyster Crassostrea virginica along the mid-Atlantic coast of the United States since 1957. The origin of H. nelsoni has remained unresolved. Molecular diagnostic tools were used to examine the hypothesis that a haplosporidian parasite in the Pacific oyster C. gigas is H. nelsoni. A DNA probe specific for H. nelsoni reacted positively in in situ hybridizations with haplosporidian plasmodia from C. gigas collected in Korea, Japan, and California. Primers that specifically amplify H. nelsoni DNA in the polymerase chain reaction amplified product from Californian C. gigas infected with the haplosporidian parasite. The DNA sequence of the 565-base pair amplified product was identical to the H. nelsoni sequence except for a single nucleotide transition, a similarity of 99.8%. These results are conclusive evidence that the parasite in C. gigas is H. nelsoni and strongly support previous speculation that the parasite was introduced into Californian populations of C. gigas from Japan. Results also support previous speculation that H. nelsoni was introduced from the Pacific Ocean to C. virginica on the East Coast of the United States, likely with known importations of C. gigas. These results document greatly increased virulence in a naive host-parasite association and reinforce potential dangers of intentional, but improper, introductions of exotic marine organisms for aquaculture or resource restoration.

Higher level relationships of leeches (Annelida: Clitellata: Euhirudinea) based on morphology and gene sequences.

The evolutionary patterns of divergence of seven euhirudinean families were investigated by cladistic analysis of 33 euhirudinean species. Oligochaetes, Acanthobdella peledina, and branchiobdellidans were included as outgroup taxa. Cladistic analysis employed 1.8 kb of nuclear 18S ribosomal DNA and 651 bp of mitochondrial cytochrome c oxidase subunit I in addition to morphological data. The use of two molecular data sets, one nuclear gene and one mitochondrial gene, as well as morphological data combined historical information evolving under a variety of different constraints and therefore was less susceptible to the biases that could confound the use of only one type of data. Results suggest that the nuclear 18S rDNA gene yields a meaningful historical signal for determining higher level relationships. The more rapidly evolving CO-I gene was informative for recent or local areas of the evolutionary hypothesis, such as within-family relationships. Analyses combining all data from the three character sets yielded one most-parsimonious tree. Most of the higher taxa in recent leech systematics were well corroborated in the resulting topology. However, these results suggested paraphyly of the order Rhynchobdellida, which contradicts the presence of a proboscis as a synapomorphy. The medicinal leech family Hirudinidae was polyphyletic because Haemadipsidae and Haemopidae each have a hirudinid ancestor. In addition, all but one of the genera within the family Erpobdellidae must be either abandoned or renamed. Unusual findings included compelling evidence of historical plasticity in bloodfeeding behavior, having been lost at least four times in the course of euhirudinean evolution. Biogeographic patterns supported a New World origin for Arhynchobdellida.

Perkinsus marinus tissue distribution and seasonal variation in oysters Crassostrea virginica from Florida, Virginia and New York.

Perkinsus marinus infection intensity was measured in eastern oysters Crassostrea virginica collected in October and December 1993, and March, May, and July 1994 from 3 U.S. sites: Apalachicola Bay (FL), Chesapeake Bay (VA), and Oyster Bay (NY). Gill, mantle, digestive gland, adductor muscle, hemolymph, and remaining tissue (including gonadal material and rectum) were dissected from 20 oysters from each site at each collection time. Samples were separately diagnosed for P. marinus infections by incubation in Ray's Fluid Thioglycollate Medium (RFTM) and subsequent microscopic quantification of purified enlarged hypnospores. At all sampling times and sites, average P. marinus infection intensity (g wet wt tissue-1 or ml hemolymph-1) was lowest in hemolymph samples, and generally highest in the digestive gland. Perkinsus marinus prevalence was 100% at both FL and NY sites for each of the 5 collection times, and, for the VA site, was less than 100% in only 1 month (May 1994). Seasonal intensity patterns and mean total body burdens differed among the sites. Average body burden was highest in VA during October and progressively declined to a minimum in May. This decline was probably due to mortality of heavily infected oysters and diminution of parasite activity associated with colder temperatures and reduced salinities. Intensities varied little during the months of October and December at both the FL and NY sites. Minimum average intensities were observed in March in FL oysters and May in NY oysters. Relatively high P. marinus infection levels that persisted throughout the winter in NY oysters compared with VA oysters could reflect constant high salinity in Long Island Sound which favors parasite activity, and also rapid decline in temperature in the fall that may have prevented epizootic oyster mortalities.

Prevalence and distribution of QPX, Quahog Parasite Unknown, in hard clams Mercenaria mercenaria in Virginia, USA.

In July 1996, the Virginia Institute of Marine Science initiated a sampling program to examine wild and cultured hard clams Mercenaria mercenaria for QPX, Quahog Parasite Unknown, a protistan parasite associated with severe mortalities of hard clams in localized areas in maritime Canada and Massachusetts, USA. The sampling program set out to seasonally monitor wild clams from one site, James River, Virginia, and cultured clams from 2 sites, Chincoteague Bay and Mattawoman Creek, Virginia. Histological examination of initial samples revealed 8% prevalence of the parasite in 1-2 yr old cultured clams in Chincoteague Bay. This is the first documentation of QPX in Virginia. To ascertain the distribution of the parasite in Virginia, the survey was expanded between August 1996 and July 1997 to include 16 additional sites. A total of 1305 wild and cultured clams was sampled from Chesapeake Bay tributaries and coastal areas where harvest and culture occur. QPX was not found in Chesapeake Bay, but was present in cultured clams from 3 coastal embayments--the original Chincoteague Bay site, Burton Bay and Quinby Inlet. The parasite was found in Chincoteague Bay at each sample period at prevalences ranging from 8 to 48%. Infections were generally light to moderate intensity and were most often observed in mantle and gill tissues. The maximum prevalence was observed in May 1997 and coincided with notable clam mortalities. QPX prevalences at the other sites were low, ranging from 4 to 15%. To date QPX has not had a significant impact on Virginia's hard clam fishery and aquaculture industry; however, the presence of the pathogen in 3 of the state's most productive hard clam growout areas warrants continued monitoring and research.

Phylogeny of leeches (Hirudinea) based on mitochondrial cytochrome c oxidase subunit I.

The phylogenetic relationships of leeches were investigated for the first time using molecular data. Twenty-one species were examined representing 7 of the 10 conventionally recognized euhirudinean families. In addition, Acanthobdella peledina, a branchiobdellid, four oligochaetes, and two polychaetes were included. Cladistic analysis of the mitochondrial cytochrome c oxidase subunit I gene yielded one most-parsimonious tree. Contemporary taxonomic groupings of leeches into higher categories were found to be largely consistent with monophyletic groups identified in the analysis. Unusual relationships for which there is some precedent include a sister-group relationship between the piscicolids and Arhynchobdellida, as well as the grouping of the haemopids within Hirudinidae.