Perkinsus mediterraneus n. sp., a protistan parasite of the European flat oyster Ostrea edulis from the Balearic Islands, Mediterranean Sea

Casas, Sandra M.; Grau, Amàlia; Reece, Kimberly S.; Apakupakul, Kathleen; Azevedo, Carlos; Villalba, António

doi:10.3354/dao058231

Cited by 61 publications

(35 citation statements)

References 45 publications

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“…Recently, the new species Perkinsus mediterraneus (Casas et al 2004) has been described parasitising European oysters Ostrea edulis from Balearic Islands (Spain, Mediterranean Sea).…”

Section: Historical Perspectivementioning

confidence: 99%

Perkinsosis in molluscs: A review

Villalba

Reece

Ordás

et al. 2004

Aquat. Living Resour.

303

243

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-The genus Perkinsus includes protistan parasites infecting marine molluscs throughout the world, some of which are associated with mass mortalities. Life cycle involves vegetative proliferation within the host, by which a cell named trophozoite undergoes successive bipartitioning. Other stages have been observed in vitro or in vivo, depending on the species: hypnospore, zoosporangium and zoospore. Molecular taxonomy supports a close affinity between dinoflagellates and Perkinsus spp. Six species of Perkinsus are currently considered valid: P. marinus, P. olseni, P. qugwadi, P. chesapeaki, P. andrewsi and P. mediterraneus. Histology and, above all, incubation of host tissues in Ray's fluid thioglycollate medium (RFTM) are classic diagnostic methods. In addition, more sensitive and quicker molecular diagnostic techniques based on either immunoassays or PCR have been developed for Perkinsus spp. Epizootiological studies have shown a marked influence of water temperature and salinity on P. marinus infection in oysters Crassostrea virginica, thus determining parasite geographical range and temporal disease dynamics (seasonality). In vitro cultures have been established for four Perkinsus spp. Immune response to infection varies depending on host and involves phagocytosis or encapsulation of the parasite cells by host haemocytes. A polypeptide is secreted by clamTapes philippinarum haemocytes that could kill the parasite. In vitro cultured P. marinus cells secrete proteases that are likely involved in degradation of host tissues. P. marinus can suppress the toxic oxygen radicals produced by host haemocytes. In addition to host death, sublethal effects caused by Perkinsus spp. (reduction of fecundity, growth, and condition) may have significant ecological and economic implications. Various strategies have been assayed to mitigate the consequences of P. marinus epizootics on the oyster industry: modifications of management/culture procedures, selective breeding to obtain resistant oyster strains, and the use of triploid oysters and allochthonous oyster species. Some chemotherapeutants have been proved to inhibit or kill parasite cells in vitro.

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“…Recently, the new species Perkinsus mediterraneus (Casas et al 2004) has been described parasitising European oysters Ostrea edulis from Balearic Islands (Spain, Mediterranean Sea).…”

Section: Historical Perspectivementioning

confidence: 99%

Perkinsosis in molluscs: A review

Villalba

Reece

Ordás

et al. 2004

Aquat. Living Resour.

303

243

View full text Add to dashboard Cite

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“…AF441207 to AF441218 (11); and accession no. AY487834 to AY487843 (10). Additional sequences available in the laboratory of K. S. Reece (unpublished data) were also added to the alignment and used to identify regions of unique sequences in the P. marinus ITS region.…”

Section: Culturedmentioning

confidence: 99%

“…The specificity of the standard and real-time PCR assays was analyzed by testing DNA extracted with the tissue kit from cultured cells of P. marinus, P. chesapeaki, P. andrewsi, and P. atlanticus. DNA samples extracted from flat oysters, Ostrea edulis, infected with P. mediterraneus (10) and from several dinoflagellate species (Hematodinium perezi, Amyloodinium occelatum, Pfiesteria piscicida, Pfiesteria shumwayae, Amphidinium carterae, Crypthecodinium cohnii, Karlodinium micrum, Peridinium foliaceum, and Prorocentrum micans) were also tested.…”

Section: Culturedmentioning

confidence: 99%

Real-Time PCR for Detection and Quantification of the Protistan Parasite Perkinsus marinus in Environmental Waters

Audemard

Reece

Burreson

2004

Appl Environ Microbiol

115

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The protistan parasite Perkinsus marinus is a severe pathogen of the oyster Crassostrea virginica along the east coast of the United States. Very few data have been collected, however, on the abundance of the parasite in environmental waters, limiting our understanding of P. marinus transmission dynamics. Real-time PCR assays with SybrGreen I as a label for detection were developed in this study for quantification of P. marinus in environmental waters with P. marinus species-specific primers and of Perkinsus spp. with Perkinsus genusspecific primers. Detection of DNA concentrations as low as the equivalent of 3.3 ؋ 10 ؊2 cell per 10-l reaction mixture was obtained by targeting the multicopy internal transcribed spacer region of the genome. To obtain reliable target quantification from environmental water samples, removal of PCR inhibitors and efficient DNA recovery were two major concerns. A DNA extraction kit designed for tissues and another designed for stool samples were tested on environmental and artificial seawater (ASW) samples spiked with P. marinus cultured cells. The stool kit was significantly more efficient than the tissue kit at removing inhibitors from environmental water samples. With the stool kit, no significant difference in the quantified target concentrations was observed between the environmental and ASW samples. However, with the spiked ASW samples, the tissue kit demonstrated more efficient DNA recovery. Finally, by performing three elutions of DNA from the spin columns, which were combined prior to target quantification, variability of DNA recovery from different samples was minimized and more reliable real-time PCR quantification was accomplished.Perkinsus species are parasites of marine molluscs that can have a severe pathogenic effect on their hosts and cause significant economic losses. One of the most detrimental species in this group is Perkinsus marinus (27), a parasite of the eastern oyster, Crassostrea virginica (Gmelin). Since the 1950s, P. marinus has been responsible for severe mortalities among C. virginica populations along the mid-Atlantic region of the United States (1, 7).P. marinus is waterborne and directly transmitted from oyster to oyster (26). Experimentally, transmission of P. marinus is dose dependent and all three known parasite life stages, trophozoite, prezoosporangia, and zoospore, have been shown to induce infection in oysters (1,26,31,41). However, studies of transmission dynamics in the environment have been hindered by the inability to detect free-living stages of the parasite in water. Traditionally, detection of P. marinus in oysters has involved histology or culture of oyster tissues in fluid thioglycolate medium (FTM) (33, 34), and the latter is still the most commonly used diagnostic test to determine infection prevalence and intensity in oysters. The major drawbacks of the FTM assay are its lack of species specificity and its inability to detect low-intensity infections corresponding to fewer than 1,000 P. marinus cells per g of wet oyster tissue (9)....

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“…Current practices in the Ebro Delta include the importation of seeds and juveniles for fattening, and with the planned intensification of clam cultivation in this area, this activity might be expected to increase the risk of introducing exotic species of Perkinsus. However, to date, only P. olseni and P. mediterraneus have been described in Mediterranean waters (Casas et al 2004, Abollo et al 2006, Elandaloussi et al 2009). …”

Section: Introductionmentioning

confidence: 99%

Phylogenetic relationship of Perkinsus olseni from the Ebro Delta, Spain, to other Perkinsus species, based on ribosomal DNA sequences

Elandaloussi¹,

Carrasco²,

Furones³

et al. 2009

Dis. Aquat. Org.

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The phylogenetic relationship of Perkinsus olseni originating from the Ebro Delta, Spain, to other Perkinsus spp. was investigated using the nontranscribed spacer (NTS) region and the internal transcribed spacer (ITS) region (including ITS1, 5.8S and ITS2) of the ribosomal DNA sequences. These 2 molecular markers (NTS and ITS) were sequenced from prezoosporangia of Perkinsus sp. originating from Manila clam Ruditapes philippinarum from the Ebro Delta. The sequence of the 5.8S ITS region of the ribosomal RNA gene was 100% similar to that of P. olseni. Higher genetic variability was found for the NTS sequence, with 80.7 to 81.8% similarity to P. olseni. The NTS sequence of a P. olseni isolate previously detected in R. decussatus from the same area was also obtained and showed 81% identity with our isolate. Evidence obtained from phylogenetic analysis of the 5.8S ITS and NTS aligned sequences appears to indicate that P. olseni strains group together according to their host rather than their geographic origins within a well-resolved P. olseni clade. KEY WORDS: Perkinsus olseni · Ruditapes philippinarum · Manila clam · Ebro Delta · Spain · Nontranscribed spacer · NTS · ITS rRNA Resale or republication not permitted without written consent of the publisherDis Aquat Org 86: [135][136][137][138][139][140][141][142] 2009 (Siddall et al. 2001). The development of molecular methods has facilitated the taxonomic identification of Perkinsus spp., and analysis of such molecular sequences has permitted the study of the phylogenetic relationship of Perkinsus spp. with other protists. The most common molecular markers for Perkinsus spp. discrimination are the 5.8S sequence of the internal transcribed spacer (ITS) region and the nontranscribed spacer (NTS) region of ribosomal DNA (rDNA); the comparison of ribosomal sequences with reference sequences constitutes the only confirmatory method for the identification of Perkinsus spp. (Reece et al. 1997, Kotob et al. 1999, Robledo et al. 1999, Coss et al. 2001, Casas et al. 2002, Murrell et al. 2002, Burreson et al. 2005, Park et al. 2005.In the present study, we report the species identity of Perkinsus sp. found in Ruditapes philippinarum from Spanish Mediterranean waters based on an analysis of NTS and ITS (including ITS1, 5.8S and ITS2) sequences, and the phylogenetic relationship of P. olseni strains from this area to other Perkinsus spp. MATERIALS AND METHODSIsolation of prezoosporangia. Clams were collected from the shallow areas of the Fangar Bay located on the Ebro Delta in June 2006. For induction of prezoosporangia, 24 whole clams were individually incubated in 20 ml FTM supplemented with streptomycin (500 µg ml -1 ) and penicillin G (500 U ml -1 ) (Ray 1963) for 7 d at room temperature in the dark. Hypnospores formed in the FTM were harvested by centrifuging at 1500 × g for 10 min and washing 3 times using sterilised seawater.DNA extraction, PCR amplification and sequencing. Genomic DNA was extracted from prezoosporangia using the DNeasy tissue kit (Qiagen). E...

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Perkinsus mediterraneus n. sp., a protistan parasite of the European flat oyster Ostrea edulis from the Balearic Islands, Mediterranean Sea

Cited by 61 publications

References 45 publications

Perkinsosis in molluscs: A review

Perkinsosis in molluscs: A review

Real-Time PCR for Detection and Quantification of the Protistan Parasite Perkinsus marinus in Environmental Waters

Phylogenetic relationship of Perkinsus olseni from the Ebro Delta, Spain, to other Perkinsus species, based on ribosomal DNA sequences

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