First record of Perkinsus olseni, a protozoan parasite infecting the commercial clam Ruditapes decussatus in Spanish Mediterranean waters

We present the first record in Spanish Mediterranean waters of the protozoan parasite Perkinsus olseni infecting the clam Ruditapes decussatus. Perkinsus infection was detected all year around albeit at a low level of infection intensity. Histological analysis, induction of zoospores and in situ hybridisation assay confirmed the presence of Perkinsus sp. The identity of the parasite was determined by species-specific PCR assay in DNA samples obtained from infected clams. Sequencing of amplified fragments showed 100% identity to the ITS region of P. olseni. We confirmed for the first time the presence of P. olseni in Spanish Mediterranean waters.     

Perkinsosis in the Manila clam Ruditapes philippinarum affects responses to the harmful-alga, Prorocentrum minimum

The dinoflagellate Prorocentrum minimum is increasingly recognized as a harmful algal bloom (HAB) species that affects filter-feeding shellfish. An experiment was done to investigate possible interactions between parasitic diseases and exposure to P. minimum in Manila clams, Ruditapes philippinarum. Manila clams, with variable levels of infection with Perkinsus olseni, were exposed for three or six days to the benign phytoplankton species Chaetoceros neogracile or a mixed diet of C. neogracile and P. minimum. After three or six days of exposure, clams were assessed individually for condition index, parasite status, and plasma and hemocyte parameters (morphological and functional) using flow-cytometry. Histological evaluation was also performed on individual clams to assess prevalence and intensity of parasitic infection, as well as other pathological conditions.Prorocentrum minimum caused several changes in Manila clams, especially after six days of exposure, such as decreased hemocyte phagocytosis and size and clam condition index. Pathological conditions observed in Manila clams exposed to P. minimum were hemocyte infiltration in the intestine and gonad follicles, myopathy, and necrosis of the intestine epithelial cells. The parasite P. olseni alone had no significant effect on Manila clams, nor did it modulate the hemocyte variables in clams exposed to P. minimum; however, the parasite did affect the pathological status of Manila clams exposed to the P. minimum culture, by causing atrophy and degeneration of residual ova in the gonadal follicles and hyaline degeneration of the muscle fibers, indicating synergistic effects of both stressors on the host over a short period of time. Additionally, an in vitro experiment also demonstrated detrimental effects of P. minimum and exudates upon P. olseni cells, thus suggesting HAB antagonistic suppression of transmission and proliferation of the parasite in the natural environment over a longer period of time. The results of this experiment demonstrate the complexity of interactions between host, parasite, and HAB.     

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).     

Pathology survey of the short-neck clam Ruditapes philippinarum occurring on sandy tidal flats along the coast of Ariake Bay, Kyushu, Japan

The pathological condition of the short-neck clam Ruditapes philippinarum was surveyed along the coast of Kumamoto, Japan, in June 2004. DNA sequences of the non-transcribed spacer region and internal transcribed spacer region flanking 5.8S rRNA identified Perkinsus olseni among the clams. Ray’s fluid thioglycollate medium assay indicated that 96.7% of the clams surveyed from the Kiguchi River tidal flat (native clams, Stn KR-N) and 96.7% of the clams surveyed from the Midori River tidal flat (Stn MR) were infected with P. olseni with an infection intensity of 464,278 and 199,937 Perkinsus cells/gram tissue wet weight (gWW), respectively. In contrast, 66.7% of the clams imported from China and stored along the Kiguchi River tidal flat (Stn KR-I) and 20.2% of clams from the Arao tidal flat (Stn AT) were infected with P. olseni with an infection intensity of 37,547 and 3382 Perkinsus cells/gWW, respectively. Brown ring disease was detected in the clam population from Stn KR-I at a prevalence of 90.0%. Polymerase chain reaction and the 16S rRNA sequence suggested that the agents of brown ring disease observed at Stn KR-I were Vibrio tapetis-like bacteria. Sporocysts and metacercariae of unidentified trematodes were also observed in the gonads and mantle of the clams from Stn KR-I, Stn MR, and Stn AT, at prevalences of 7.1-42.9%. Metacestodes (larval tapeworms) were found in the foot and digestive gland at a prevalence of 52.5%, 30.0%, and 14.3% in clams from Stns MR, AT, and KR-N, respectively. Histology also showed massive hemocyte infiltration and inflammation among clams heavily infected with P. olseni. Castration of the follicle was typical among clams infected with the trematode. The data indicate that most of the clams along the coast of Kumamoto are infected with various pathogens at various rates of infection, and these pathogens could have negative effects on the clam population in the long term.     

Isolation and identification of Perkinsus olseni from feces and marine sediment using immunological and molecular techniques

Molecular and immunological probes were used to identify various life stages of Perkinsus olseni, a protozoan parasite of the Manila clam Ruditapes philippinarum, from a marine environment and decomposing clam tissue. Western blotting revealed that the antigenic determinants of the rabbit anti-P. olseni antibody developed in this study were peptides with molecular masses of 55.9, 24.0, and 19.2 kDa. Immunofluorescent assay indicated that the rabbit anti-P. olseni IgG was specific to all life stages, including the prezoosporangium, trophozoite, and zoospore. Perkinsus olseni prezoosporangium-like cells were successfully isolated from marine sediment collected from Hwangdo on the west coast of Korea, where P. olseni-associated clam mortality has recurred for the past decade. Purified cells were positively stained with the rabbit anti-P. olseni antibody in an immunofluorescence assay, confirming for the first time the presence of P. olseni in marine sediment. Actively replicating zoospores inside the prezoosporangia were observed in the decomposing clam tissue collected from Hwangdo. P. olseni was also isolated from the feces and pseudofeces of infected clams and confirmed by PCR. The clams released 1-2 prezoosporangia per day through feces. The data suggested that the fecal discharge and decomposition of the infected clam tissue could be the two major P. olseni transmission routes.     

Monoclonal antibody analysis of Perkinsus marinus extracellular products

The protozoan oyster parasite Perkinsus marinus releases a complex set of extracellular products (ECP) during in vitro culture. These products have been previously implicated in parasite virulence, and their expression can be altered by medium supplementation with oyster tissue homogenate. Little is known regarding ECP function, regulation, or mechanism of storage and release. Perkinsus marinus ECP were purified from a protein-free medium and used to produce a panel of five monoclonal antibodies. Several of the antibodies recognised series of proteins implying that the ECP may originate from comparatively few parental molecules. The ECP are secreted by several pathways, including the release of one product from an external cell layer, and two other products from two morphologically distinct intracellular compartments. Antibodies against separate epitopes on one protein provided information about possible protein structure. A sandwich ELISA format allowed sensitive quantification of that protein and showed significantly reduced protein expression in oyster tissue homogenate supplemented cultures. Immunopurification allowed tandem mass spectroscopic amino acid sequencing of that protein. Another antibody was used to characterise the P. marinus cell wall. This antibody specifically bound to trophozoite and tomont walls, and was used to investigate the morphological and antigenic changes in these walls during Ray's fluid thioglycollate medium-induced formation of hypnospores. It was also used to confirm that oyster tissue homogenate supplementation could induce formation of hypnospores. This antibody labeled P. marinus cells in fixed oyster tissue in a species-specific manner.     

Experimental evaluation of the pathogenicity of Perkinsusolseni in juvenile Manila clams Ruditapes philippinarum

We evaluated the pathogenicity of Perkinsus olseni towards the Manila clam, Ruditapes philippinarum, by an experimental challenge. For production of prezoosporangia of P. olseni, we injected uninfected Manila clams with cells of a pure strain of P. olseni and reared them for 7 d. Prezoosporangia were isolated from the soft tissue of the injected clams after culturing in Ray’s fluid thioglycollate medium. Hatchery-reared, uninfected juvenile clams (3-10 mm shell length) were challenged by immersion in one of two concentrations of a prezoosporangial suspension of P. olseni for 6 d. The challenged clams had significantly higher mortality at both the concentrations than the unchallenged clams. The mortality due to infection dose-dependently began approximately 4 weeks and 7 weeks after challenge in the higher and lower concentrations, respectively. This is the first experimental evidence that P. olseni causes direct mortality in Manila clams. The lethal level of infection was estimated at approximately 10⁷ pathogen cells/g soft tissue weight.     

First report of three protozoan parasites (a haplosporidian, Marteilia sp. and Marteilioides sp.) from the Manila clam, Venerupis (=Ruditapes) philippinarum in Japan

Recently, natural stocks of the Manila clam, Venerupis (=Ruditapes) philippinarum, have been drastically reduced in Japan. To clarify the reason for this decline in number, clams were sampled monthly from Yamaguchi and processed for histological observations, during which three protozoan parasites were discovered. Transmission electron microscopy revealed that these parasites were unidentified haplosporidian in the connective tissues, Marteilia sp. in the digestive gland and Marteilioides sp. in the oocytes. Histopathological observations suggest that Marteilia sp. and Marteilioides sp. were not pathogenic to the host. However, infection with a haplosporidian may have a negative impact on the clams. The prevalence of these parasites was low and further investigations should be undertaken to clarify their taxonomic status and establish any pathogenicity to clams.     

Taxonomic review of the subgenus Uroleucon (Uromelan) (Hemiptera: Aphididae) in the Korean peninsula

Until now, five species of the subgenus Uroleucon (Uromelan) have been recognized in Korea. This is the first report of Uroleucon (Uromelan) adenophorae (Matsumura, 1918) occurring on Adenophora triphylla (Campanulaceae) in Gangwon-do, South Korea. Host plants are reviewed and an identification key to species is presented for six Uroleucon (Uromelan) species from the Korean Peninsula.     

In vitro effects of temperature and salinity on fatty acid synthesis in the oyster protozoan parasite Perkinsus marinus

The effects of temperature and salinity on fatty acid synthetic activities in the oyster protozoan parasite, Perkinsus marinus, were tested in vitro at 10, 18 and 28 °C in a salinity of 28 psu and 14, 20 and 28 psu at a temperature of 28 °C using ¹³C sodium acetate as a substrate. Salinity treatments exhibited few treatment effects, but temperature significantly affected cell proliferation, fatty acid content and fatty acid synthesis rates. Fatty acid synthesis rates increased approximately two-fold for every 10 °C increase in temperature; however, the predominant fatty acid synthesized differed between treatments. At 10 °C, the synthesis rate for 18:1(n−9) was not significantly different from the 18 °C treatment and weight percent of 18:1(n−9) was higher at 10 than 18 and 28 °C. In contrast, the synthesis rate for 20:4(n−6) was over five times lower at 10 than at 18 and 28 °C, and the percent fatty acid content of 20:4(n−6) was over two-fold lower at 10 than at 18 and 28 °C. Results suggest that further elongation and desaturation of 18:1(n−9) to 20 carbon polyunsaturated fatty acids may be inhibited at low temperatures. These findings may be relevant to field observations that disease progression and virulence of this parasite are correlated to high water temperatures.     

Presence of Marteilia sp. (Paramyxea) in the razor clam Solen marginatus (Pennántt, 1777) in Galicia (NW Spain)

Protistan parasites of the genus Marteilia, phylum Paramyxea, cause the molluscs disease named Marteiliosis. Histological observations and transmission electron microscopy revealed the presence of life cycle stages of a Marteilia sp. in the bivalve mollusc Solen marginatus (Solenidae). Parasites occurred in epithelial cells of the digestive ducts and tubules. Early stages (primary cells) presented one or several nuclei while advances stages formed a complex of cells-within-cells (secondary and tertiary cells) culminating in spores. Refringent bodies were present inside the presporangia. This is the first report of a Marteilia sp. in S. marginatus.     

Resistance of the Manila clam (Venerupis philippinarum) to infection with Mikrocytos mackini

Manila clams (Venerupis philippinarum) challenged in laboratory trials via bath exposure proved to be resistant to infections with Mikrocytos mackini (protistan parasite of unknown taxonomic affiliation), while Pacific oysters (Crassostrea gigas) challenged simultaneously using identical conditions developed infections. Although M. mackini was detected by a nucleic acid pathogen specific (PCR) assay in 10-30% of the challenged V. philippinarum that were sampled soon after exposure (0-48 h, n = 40), all of the subsequent V. philippinarum (n = 62) sampled 9-17 weeks post-exposure tested negative for M. mackini by PCR assay. Prevalence of infection for the exposed C. gigas (n = 100) during this same period ranged from 50% to 100% by PCR assay. Infection was confirmed in the oysters (58%, n = 60) by a digoxigenin-labelled DNA probe designed to detect M. mackini by in situ hybridization, but M. mackini was not found in any of the exposed Manila clams (n = 63) using this technique.     

Superoxide dismutases from the oyster parasite Perkinsus marinus: purification,biochemical characterization,and development of a plate microassay for activity

We have isolated and biochemically characterized superoxide dismutase (SOD) activity in cell extracts of clonally cultured Perkinsus marinus, a facultative intracellular parasite of the Eastern oyster, Crassostrea virginica. In order to assess the SOD activity throughout the purification, we developed and optimized a 96-well-plate microassay based on the inhibition of pyrogallol oxidation. The assay was also adapted to identify SOD activity type (Cu/Zn-, Mn-, or FeSOD), even in mixtures of more than one type of SOD. All SOD activity detected in the cell extracts was of the FeSOD type. Most of the SOD activity in P. marinus trophozoites resides in a major component of subunit molecular weight 24 kDa. The protein was purified by affinity chromatography on an anti-SOD antibody-Sepharose column. Amino-terminal peptide sequence of the affinity-purified protein corresponds to the predicted product of the PmSOD1 gene and indicates that amino-terminal processing has taken place. The results are discussed in the context of processing of mitochondrially targeted SODs.     

Occurrence, hosts, morphology, and molecular characterisation of Pasteuria bacteria parasitic in nematodes of the family Plectidae

Parasitic bacteria of the genus Pasteuria are reported for three Anaplectus and four identified and several unidentified Plectus species found in eight countries in various habitats. The pasteurias from plectids agree in essential morphological characters of sporangia and endospores as well as in developmental cycle with those of the Pasteuria species and strains described from tylenchid nematodes, but appear to be mainly distinguished from these by absence of a distinct perisporium in the spores and the endospores obviously not being cup- or saucer-shaped. The wide range of measurements and morphological peculiarities of sporangia and endospores suggest that probably several Pasteuria species have to be distinguished as parasites in Plectidae. From an infected juvenile of an unidentified plectid species the 16S rRNA gene sequence of Pasteuria sp. was obtained. Substantial sequence divergence from described Pasteuria species and its phylogenetic position on molecular trees indicate that this Pasteuria sp. could be considered as a new species. Preliminary results of the analysis of DNA phylogeny of Pasteuria spp. and their nematode hosts provide evidence for incongruence of their phylogenetic history and of host switching events during evolution of the bacterial parasites.     

A case of gastric strongyloidiasis in a Korean patient

A 69-year-old Korean man was admitted to emergency room with complaints of abdominal pain, vomiting, and diarrhea. Laboratory tests revealed eosinophilia, anemia, hypoproteinemia, and hyponatremia. The gastric mucosa showed whitish mottled and slightly elevated lesions on the body angle of antrum. Microscopically, chronic gastritis with incomplete intestinal metaplasia was observed. Many adult worms, larvae, and eggs in cross sections were located in the crypts. Furthermore, the filariform larvae of Strongyloides stercoralis with a notched tail were detected through the culture.     

Occurrence of Perkinsus olseni (Protozoa: Apicomplexa) and other parasites in the venerid commercial clam Pitar rostrata from Uruguay, southwestern Atlantic coast

A study was conducted into the health status of natural populations of the venerid clam Pitar rostrata from Uruguay. Perkinsus sp. was detected in 22% of the clams. Severe hemocytic infiltration was detected in the tissues parasitized by this protozoan parasite. The sequencing of the ITS-5.8S gene cluster of the parasite confirmed that it belonged to the Perkinsus olseni species. Rickettsia or Chlamidia-like organisms were also found, with a prevalence of 11%, although without apparent host reaction; an unidentified species of Coccidia was found in the nephridia of 78% of the clams, with the intensity of infection ranging from moderate to high. A gregarine, Nematopsis-like organism was observed mainly in the epithelial cells of the intestine, without host response and with a prevalence of 56%. Of the metazoan parasites, trematodes were found in 11% of the individuals analyzed.     

Infestation of the clam Chione fluctifraga by the burrowing worm Polydora sp. nov. in laboratory conditions

Burrowing worms that belong to Polydora spp. infest marine mollusks cultured worldwide, causing problems for production and marketing. The clam Chione fluctifraga is semi-cultured in Bahía Falsa, Baja California, NW Mexico, and some clams harbor burrowing worms. The present study was carried out to determine the identity of the worm species infesting the clam, the infesting process by cohabitation of infested and non-infested clams in aquaria with a variety of substrates (fine sand, gross sand, plastic bag used for clam culture, and aquarium without substrate) and turbulence conditions, and the occurrence of architomy phenomena in connection with infestation of the clam. The burrowing worm was considered as a nova species due to its singular limbate neurosetae and notosetae in the setiger 5, hooks in the setiger 6, eyes not present, and general pigmentation, among other characteristics. Infestation was similar in all substrates and turbulence conditions, but it was more abundant on clams previously infested than on those free of worms, showing a preferential settlement of worm infesting stages on pre-infested clams. Regeneration was observed in all segments of the worm: anterior (metastomium), medium, and posterior (prostomium); the complete regeneration time occurred in 40 days. This is the first record of architomy in a species of Polydora and this phenomenon could account for the increase of infestation intensity in pre-infested clams at the end of the study period. Infestation of clams by settling polichaete in the conditions studied, and the architomy process in this worm species, shows its great infesting capacity.     

The Distribution of Perkinsus marinus in Gulf Coast Oysters: Its Relationship with Temperature,Reproduction, and Pollutant Body Burden

Oysters from 48 Gulf of Mexico sites were examined for presence and infection intensity of the endoparasite, Perkinsus (= Dermocystidium) marinus (Mackin, Owen and Collier, 1950) as part of NOAA's Status and Trends Mussel Watch Program. Prevalence exceeded 75% at 25 sites. Infection intensity did not vary with sex or reproductive stage. Latitude, total polynuclear aromatic hydrocarbon (PAH) content and industrial and agricultural land use significantly affected the parasite's distribution. PAH and pesticide concentrations were latitudinally dependent, suggesting an impact of spawning frequency on uptake and depuration. P. marinus analysis complements the use of pollutant body burden for determining change in environmental quality because it responds differently than pollutant body burden to the biology and ecology of the oyster.     

Tetrahymenidae infection in mosquito populations in a malaria-endemic region of the Amazon

In a field survey performed in a malaria-endemic region of Northern Amazon, Brazil, we encountered ciliate protozoa of the family Tetrahymenidae infecting adults and larvae of the following mosquito species: Culex sp., Anopheles albitarsis l.s., Anopheles strodei, Anopheles mattogrossensis, Anopheles darlingi, and Anopheles oswaldoi l.s. Based on morphological features and life style, we have tentatively identified the parasite as Lambornella sp. The association appears pathogenic for the mosquito. Prevalence of infection in both larvae and adults was higher in the dry season.