Perkinsus Species Research Articles

Differences in phagosome acidification of Manila clam hemocytes in response to two Perkinsus species with contrasting proliferation dynamics in the host: P. olseni and P. mediterraneus

Understanding the factors determining the host ranges of Perkinsus spp., a significant group of pathogenic protozoans affecting shellfish, is essential for preventing their spread and designing effective control measures. Considering that differences in the ability to proliferate within the host may influence the determination of host range, we first injected six Perkinsus spp. into Manila clams Ruditapes philippinarum and monitored the variations of trophozoite numbers. Although all six species were detected in the challenged clams 28 days post infection, the infection intensities varied among species, and particularly two species showed contrasting infection trends: P. mediterraneus showed a decreasing trend of infection, declining to the lowest intensity, whereas that of P. olseni continuously increased, reaching the highest intensity. In vitro exposure to Manila clam hemocytes revealed that the survival of P. mediterraneus trophozoites was suppressed, in contrast to P. olseni, which maintained their viability. Despite similar phagocytic indices for both species, the rate of phagosome acidification was significantly higher for hemocytes phagocytizing P. mediterraneus than those targeting P. olseni. Notably, phagosome acidification was significantly suppressed in hemocytes phagocytizing live P. olseni trophozoites, suggesting that P. olseni may secrete a substance that modulates phagosome acidification, and thereby evades intracellular digestion by the host’s hemocytes. Conversely, P. mediterraneus, with a lower affinity for infecting Manila clams, did not exhibit such modulation. Based on these results, we consider that the ability to modulate phagosome acidification in host hemocytes might be at least one factor in determining the host range of Perkinsus species.

Survey on Perkinsus species in two economic mussels (Mytilus coruscus and M. galloprovincialis) along the coast of the East China Sea and the Yellow Sea.

Perkinsus, a parasitic pathogen of marine bivalves, is widely distributed among various mollusks in numerous countries. However, the prevalence and diversity of Perkinsus species in the two economically important mussels, Mytilus coruscus and M. galloprovincialis, in China remain unknown. The presence of the Perkinsus species was identified in the two mussels sampled along the coast of the East China Sea and the Yellow Sea, using both the alternative Ray's fluid thioglycolate medium (ARFTM) and conventional polymerase chain reaction (PCR). The ARFTM test indicated the presence of Perkinsus-like hypnospores in the two mussels. The diameter of the hypnospores in M. coruscus was significantly smaller than that in M. galloprovincialis. The prevalence of Perkinsus in M. galloprovincialis and M. coruscus ranged from 0 to 37.5% and 0 to 25%, respectively. The mean intensity of Perkinsus in M. galloprovincialis and M. coruscus ranged from 0 to 5.14 and 0 to 4.92, respectively. The PCR assay showed that the prevalence of Perkinsus spp. in M. galloprovincialis and M. coruscus was 0 to 25.0% and 0 to 12.5%, respectively. The homology analysis of the newly obtained internal transcribed spacer (ITS) sequences of Perkinsus revealed the highest identity of 100% with P. beihaiensis. The phylogenetic analysis indicated that the Perkinsus isolates from the two mussels were clustered with P. beihaiensis. The results of the molecular biology indicated that only P. beihaiensis was detected in the two mussels. The highest prevalence of P. beihaiensis was observed in Liaoning province (Dalian, 20.83%), followed by Shandong province, Zhejiang province and Fujian province. Consequently, it is recommended that surveillance should be conducted in Dalian, where the prevalence and mean intensity of P. beihaiensis in M. galloprovincialis are the highest.

Co-infection of two eukaryotic pathogens within clam populations in Arcachon Bay.

The parasitic species Perkinsus olseni (= atlanticus) (Perkinsea, Alveolata) infects a wide range of mollusc species and is responsible for mortality events and economic losses in the aquaculture industry and fisheries worldwide. Thus far, most studies conducted in this field have approached the problem from a "one parasite-one disease" perspective, notably with regards to commercially relevant clam species, while the impact of other Perkinsus species should also be considered as it could play a key role in the disease phenotype and dynamics. Co-infection of P. olseni and P. chesapeaki has already been sporadically described in Manila clam populations in Europe. Here, we describe for the first time the parasitic distribution of two Perkinsus species, P. olseni and P. chesapeaki, in individual clam organs and in five different locations across Arcachon Bay (France), using simultaneous in situ detection by quantitative PCR (qPCR) duplex methodology. We show that P. olseni single-infection largely dominated prevalence (46-84%) with high intensities of infection (7.2 to 8.5 log-nb of copies. g-1of wet tissue of Manila clam) depending on location, suggesting that infection is driven by the abiotic characteristics of stations and physiological states of the host. Conversely, single P. chesapeaki infections were observed in only two sampling stations, Ile aux Oiseaux and Gujan, with low prevalences 2 and 14%, respectively. Interestingly, the co-infection by both Perkinsus spp., ranging in prevalence from 12 to 34%, was distributed across four stations of Arcachon Bay, and was detected in one or two organs maximum. Within these co-infected organs, P. olseni largely dominated the global parasitic load. Hence, the co-infection dynamics between P. olseni and P. chesapeaki may rely on a facilitating role of P. olseni in developing a primary infection which in turn may help P. chesapeaki infect R. philippinarum as a reservoir for a preferred host. This ecological study demonstrates that the detection and quantification of both parasitic species, P. olseni and P. chesapeaki, is essential and timely in resolving cryptic infections and their consequences on individual hosts and clam populations.

Occurrence and seasonal variation of Perkinsus sp. Infection in wild mollusk populations from coastal waters of Qingdao, northern China

Perkinsosis has been recognized as one of the major threats to natural and farmed bivalve populations, many of which are of commercial as well as environmental significance. Three Perkinsus species have been identified in China, and the Manila clam (Ruditapes philippinarum) was the most frequently infected species in northern China. Although the occurrence and seasonal variation of Perkinsus spp. have previously been examined, the pathological characteristics of these infections in wild Manila clams and sympatric species in China have seldom been reported. In the present study, the prevalence and intensity of Perkinsus infection in wild populations of Manila clams and 10 sympatric species from three sites were investigated by Ray’s fluid thioglycolate medium (RFTM) assay seasonally across a single year. Perkinsus infection was only identified in Manila clams, with a high prevalence (274/284 = 96.48 %) and low intensity (89.8 % with a Mackin value ≤ 2, suggesting generally low-intensity infections) throughout the year. Heavily infected clams were mainly identified in Tianheng in January, which displayed no macroscopic signs of disease. An overview of the whole visceral mass section showed that the trophozoites mostly aggregated in gills and connective tissue of the digestive tract, to a lesser extent in the mantle and foot, and even less frequently in adductor muscle and connective tissues of the gonad. PCR and ITS-5.8S rRNA sequencing of 93 representative RFTM-positive samples revealed a 99.69 to 100 % DNA sequence identity to Perkinsus olseni. Unexpectedly, significantly higher infection intensities were usually identified in January and April when the Condition Index (CI) was relatively high. We propose that factors associated with the anthropogenic harvesting pressure and irregular disturbances should be responsible for the uncommon seasonal infection dynamics of perkinsosis observed in the present study.

Identification of glycine betaine as a host-derived molecule required for the vegetative proliferation of the protozoan parasite Perkinsus olseni.

Perkinsus olseni is an industrially significant protozoan parasite of Manila clam, Ruditapes philippinarum. So far, various media, based on Dulbecco's Modified Eagle Medium and Ham's F-12 nutrient mixture with supplementation of fetal bovine serum (FBS), have been developed to proliferate the parasitizing trophozoite stage of P. olseni. The present study showed that P. olseni did not proliferate in FBS-deficient Perkinsus broth medium (PBMΔF), but proliferated well in PBMΔF supplemented with tissue extract of host Manila clams, indicating that FBS and Manila clam tissue contained molecule(s) required for P. olseni proliferation. Preliminary characterization suggested that the host-derived molecule(s) was a heat-stable molecule(s) with a molecular weight of less than 3 kDa, and finally a single molecule required for the proliferation was purified by high-performance liquid chromatography processes. High-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance analyses identified this molecule as glycine betaine (=trimethylglycine), and the requirement of this molecule for P. olsseni proliferation was confirmed by an assay using chemically synthesized, standard glycine betaine. Although glycine betaine was required for the proliferation of all examined Perkinsus species, supplementation of glycine betaine precursors, such as choline and betaine aldehyde, enhanced the proliferation of 4 Perkinsus species (P. marinus, P. chesapeaki, P. mediterraneus and P. honshuensis), but not of 2 others (P. olseni and P. beihaiensis). Thus, it was concluded that the ability to biosynthesise glycine betaine from its precursors varied among Perkinsus species, and that P. olseni and P. beihaiensis lack the ability required to biosynthesize glycine betaine for proliferation.

Diversity and distribution of Perkinsus spp. along the coast of China: Implications for widespread transmission of Perkinsus spp. in mollusks

Perkinsus species, which are parasitic pathogens of mollusks, have been transmitted and dispersed to various molluscan species along the coastal waters of many countries. However, few studies have addressed the diversity and distribution of Perkinsus spp. along the coast of China. Here we used conventional PCR amplification and sequencing techniques, combined with a qPCR assay as a confirmatory test, to evaluate the prevalence of Perkinsus species in molluscan species among different sea regions of China. Three Perkinsus species—P. olseni, P. beihaiensis, and P. chesapeaki—were detected, with P. chesapeaki reported for the first time along the Chinese coast. Seven of eight molluscan species carried Perkinsus species, including Crassostrea gigas, C. hongkongensis, Sinonovacula constricta, Ruditapes philippinarum, Scapharca subcrenata, Meretrix lyrate, and Haliotis diversicolor. Perkinsus olseni was prominent in the Yellow and Bohai Sea and East China Sea, while P. beihaiensis was prominent in the South China Sea. Most of the molluscan species carried Perkinsus spp. with the medium or low levels of PCR-prevalence (<30%). The three Perkinsus species possess high levels of internal transcriber spacer haplotypes, some of which are shared among many countries. The much higher PCR-prevalence of Perkinsus spp. in the clam Ruditapes philippinarum and the oyster Crassostrea hongkongensis suggests that Perkinsus species may be transmitted and dispersed to other mollusks through the transportation of Perkinsus-carried R. philippinarum and C. hongkongensis. Perkinsus carrying tended to be generally linked with a broader geographic range, lower prevalence, more diversified molluscan hosts, and more diversified Perkinsus haplotypes.

Mitochondrial Genomes in Perkinsus Decode Conserved Frameshifts in All Genes.

Mitochondrial genomes of apicomplexans, dinoflagellates, and chrompodellids that collectively make up the Myzozoa, encode only three proteins (Cytochrome b [COB], Cytochrome c oxidase subunit 1 [COX1], Cytochrome c oxidase subunit 3 [COX3]), contain fragmented ribosomal RNAs, and display extensive recombination, RNA trans-splicing, and RNA-editing. The early-diverging Perkinsozoa is the final major myzozoan lineage whose mitochondrial genomes remained poorly characterized. Previous reports of Perkinsus genes indicated independent acquisition of non-canonical features, namely the occurrence of multiple frameshifts. To determine both ancestral myzozoan and novel perkinsozoan mitochondrial genome features, we sequenced and assembled mitochondrial genomes of four Perkinsus species. These data show a simple ancestral genome with the common reduced coding capacity but disposition for rearrangement. We identified 75 frameshifts across the four species that occur as distinct types and that are highly conserved in gene location. A decoding mechanism apparently employs unused codons at the frameshift sites that advance translation either +1 or +2 frames to the next used codon. The locations of frameshifts are seemingly positioned to regulate protein folding of the nascent protein as it emerges from the ribosome. The cox3 gene is distinct in containing only one frameshift and showing strong selection against residues that are otherwise frequently encoded at the frameshift positions in cox1 and cob. All genes lack cysteine codons implying a reduction to 19 amino acids in these genomes. Furthermore, mitochondrion-encoded rRNA fragment complements are incomplete in Perkinsus spp. but some are found in the nuclear DNA suggesting import into the organelle. Perkinsus demonstrates further remarkable trajectories of organelle genome evolution including pervasive integration of frameshift translation into genome expression.

Molecular and histological identification of the protozoan parasite Perkinsus olseni in the blood cockle Anadara kagoshimensis (Tokunaga, 1906) occurring on the south coast of Korea

A recent report on co-infection by two Perkinsus species in mud arks Anadara trapezia (Bivalvia Arcidae) in Australia implies possible Perkinsus infection in other Arcidae species. Blood cockle Anadara kagoshimensis has been exploited since the 1960s on the south coast of Korea, and the landings have decreased due to mass mortality in recent years. During a histopathology survey of blood cockle on the south coast, we identified a Perkinsus-like pathogen in the visceral mass. Accordingly, we applied Perkinsus species-specific PCR and subsequent sequencing to identify the pathogen to species level using internal transcribed spacer (ITS) rDNA. Ray's fluid thioglycollate medium (RFTM) and immunofluorescence assays (IFA) were also used to quantify and visualize the infection in this novel host. Perkinsus-like organisms were mainly found in the hemal space of visceral mass connective tissue. The Bayesian inference tree based on ITS rDNA sequences is strongly affiliated with P. olseni, suggesting that the Perkinsus-like organism in A. kagoshimensis is P. olseni, commonly found in Manila clams in Korean waters. RFTM and subsequent 2 M NaOH digestion assay revealed that the infection intensity ranged from 8000 to 1,481,000 cells/g gill, with a mean of 1.2 × 105 ± 2.5 × 105, and the prevalence was higher in September (fall) with 13.5% (101/750) than in June (spring) (0.4%, 3/750). Histology revealed that heavily infected clams exhibited tissue inflammation suggesting that P. olseni infection could damage the structure and physiological function of A. kagoshimensis; although we could not find a significant difference in condition index between the infected and uninfected individuals.

Molecular detection of a novel perkinsid associated with the deep-sea clam Phreagena okutanii.

Based on environmental DNA surveys, it is widely held that phylogenetically diverse protists exist in chemosynthetic ecosystems. However, knowledge regarding the protists associated with the endemic animals inhabiting these environments is still very limited. In the present study, utilizing polymerase chain reaction (PCR) techniques, we detected fragments of the small subunit ribosomal RNA (SSU rRNA) gene and the internal transcribed spacer (ITS) region of the ribosomal RNA genes from a particular protist in the gills of the vesicomyid clam Phreagena okutanii (formerly described as Calyptogena okutanii), a representative animal in chemosynthetic ecosystems. Based on the phylogeny of the SSU rRNA gene, the organism in question belongs to the genus Perkinsus, which is exclusively composed of protistan parasites infecting mollusks. Intriguingly, based on the ITS phylogeny, this protist was not related to any known Perkinsus species and was deeply branched within the radiation of this genus, thus represents an undescribed species. In addition, the protist detected by PCR was localized to the intercellular spaces in the gills of the host clam with fluorescence in situ hybridization. Although the ecological significance of this novel deep-sea perkinsid remains unclear, our present findings may provide important insights into the diversity of the genus Perkinsus.

Supplementation with lipids enhances zoosporulation of Perkinsus species

The protozoan parasite Perkinsus olseni is a major pathogen of marine mollusks. A large number of zoospores (the most infective stage) is required to experimentally examine the transmission and invasion processes of this parasite, but the conventional zoosporulation method in seawater is inefficient. A recent study found that P. olseni zoosporulation was highly enhanced in a nutrient-rich medium which was developed for trophozoite proliferation in Perkinsus. Here, we examined the effect of each medium component on P. olseni zoosporulation and found that lipids enhanced zoosporulation. Moreover, zoospores failed to develop into trophozoites in seawater supplemented with lipids, indicating that this zoosporulation method using seawater supplemented with lipids is ideal for preparing a large number of P. olseni zoospores for various biological assays. Lipid supplementation also significantly enhanced zoosporulation in P. honshuensis, P. chesapeaki, and P. marinus, although the enhancing effect of the lipid supplementation varied by species. Considering the limited availability of lipids in the sea water column, our findings suggest that Perkinsus zoosporulation likely occurs mainly in the vicinity of hosts (not in the sea water column), such as in the mantle cavity or on the body surface of mollusk hosts.

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.

Development of a simple host-free medium for efficient prezoosporulation of Perkinsus olseni trophozoites cultured in vitro.

The parasitizing stage (trophozoite) of the protozoan parasite Perkinsus olseni progresses to the dormant stage (prezoosporangium) immediately after the death of the host through physiologically and morphologically drastic changes. This development is reproducible in Ray's fluid thioglycollate medium (RFTM). In this study, supplementation with tissue extract from a host, the Manila clam, significantly improved the efficiency of development, as determined by the numbers and sizes of developed prezoosporangia. Similar results were seen following supplementation with boiled host tissue extract, which indicates that a thermally stable component of the host is required for the parasite's development. Subsequently, we found that a commercially available lipid concentrate significantly increased prezoosporulation without host tissue, suggesting that the lipids in host tissue enhance prezoosporangia development. Moreover, we determined that yeast extract, sodium thioglycollate, and sodium chloride were the only components of RFTM required for prezoosporulation. Based on these findings, we prepared a simple, host-free medium for P. olseni prezoosporulation-Lipid concentrate Yeast extract Medium (LpcYM)-consisting of yeast extract, lipid concentrate, sodium thioglycollate, and sodium chloride. We confirmed that the prezoosporangia developed in LpcYM produce zoospores that are infectious to Manila clams and that trophozoites of other Perkinsus species (P. marinus, P. honshuensis, and P. chesapeaki) also develop to prezoosporangia in this host-free medium. As LpcYM has the simplest composition of prezoosporulation media available thus far, it enables us to conduct molecular and biochemical studies examining the drastic transformation process of this parasite.

Biochemical Characterization of Oyster and Clam Galectins: Selective Recognition of Carbohydrate Ligands on Host Hemocytes and Perkinsus Parasites.

Both vertebrates and invertebrates display active innate immune mechanisms for defense against microbial infection, including diversified repertoires of soluble and cell-associated lectins that can effect recognition and binding to potential pathogens, and trigger downstream effector pathways that clear them from the host internal milieu. Galectins are widely distributed and highly conserved lectins that have key regulatory effects on both innate and adaptive immune responses. In addition, galectins can bind to exogenous (“non-self”) carbohydrates on the surface of bacteria, enveloped viruses, parasites, and fungi, and function as recognition receptors and effector factors in innate immunity. Like most invertebrates, eastern oysters (Crassostrea virginica) and softshell clams (Mya arenaria) can effectively respond to most immune challenges through soluble and hemocyte-associated lectins. The protozoan parasite Perkinsus marinus, however, can infect eastern oysters and cause “Dermo” disease, which is highly detrimental to both natural and farmed oyster populations. The sympatric Perkinsus chesapeaki, initially isolated from infected M. arenaria clams, can also be present in oysters, and there is little evidence of pathogenicity in either clams or oysters. In this review, we discuss selected observations from our studies on the mechanisms of Perkinsus recognition that are mediated by galectin-carbohydrate interactions. We identified in the oyster two galectins that we designated CvGal1 and CvGal2, which strongly recognize P. marinus trophozoites. In the clam we also identified galectin sequences, and focused on one (that we named MaGal1) that also recognizes Perkinsus species. Here we describe the biochemical characterization of CvGal1, CvGal2, and MaGal1 with focus on the detailed study of the carbohydrate specificity, and the glycosylated moieties on the surfaces of the oyster hemocytes and the two Perkinsus species (P. marinus and P. chesapeaki). Our goal is to gain further understanding of the biochemical basis for the interactions that lead to recognition and opsonization of the Perkinsus trophozoites by the bivalve hemocytes. These basic studies on the biology of host-parasite interactions may contribute to the development of novel intervention strategies for parasitic diseases of biomedical interest.

Perkinsus mediterraneus infection induces oxidative stress in the mollusc Mimachlamys varia.

Perkinsus mediterraneus is a protozoan parasite that can cause marine mollusc diseases known as perkinsosis being a serious threat for clam cultures worldwide. The aim of the present study was first to determine the Perkinsus species infecting the variegated scallop Mimachlamys varia and then to evaluate the existence of oxidative stress in gills of M.varia according to different degrees of infection. DNA sequencing confirmed that P.mediterraneus was the species infecting M.varia. ROS production was progressively increasing with the degree of infection although the differences were only significant in the high-infected group. Low degree of infection significantly increased superoxide dismutase (SOD) and glutathione S-transferase (GST) activities and nitrite levels with respect to the control group. In the high-infected group, a significant increase was evidenced in all analysed enzymes, catalase, SOD, glutathione reductase and GST. Non-significant differences in MDA levels were observed between the control and low-infected groups; however, a significant increase in MDA levels was observed in the high-infected group. In conclusion, the infection by Perkinsus mediterraneus in M.varia induces oxidative stress and an antioxidant response directly related to the infection degree that can contribute to the pathogenicity of the infection.

First discovery of Perkinsus beihaiensis in Mediterranean mussels (Mytilus galloprovincialis) in Tokyo Bay, Japan

During analyses of the invasive Mediterranean mussel Mytilus galloprovincialis for pathologies in Tokyo Bay, infection by the protozoan parasite Perkinsus beihaiensis was found through histological examination, Ray’s Fluid Thioglycollate Medium assays, and molecular analyses. Specific PCR assays for each Perkinsus species also revealed the presence of an indigenous congeneric species, Perkinsus olseni, but P. beihaiensis was dominant in M. galloprovincialis. Sequences of the ribosomal internal transcribed spacer region I of P. beihaiensis found in Japan were genetically more similar to those found in South American countries (Panama and Brazil) than in Asian countries (China and India). Though Mediterranean mussels have become widespread in Japanese waters since their invasion in the 1930s, epidemiological surveys show that mussels collected outside Tokyo Bay are free of any Perkinsus infections. Based on these results, it was strongly suggested that P. beihaiensis invaded Tokyo Bay by transportation of bivalves originating from South America but has not yet spread to other parts of Japan. The possibility is not ruled out, however, that the parasite is indigenous in Japan but the environment in Tokyo Bay favors its transmission to Mediterranean mussels.

Utilization of recombinase polymerase amplification combined with a lateral flow strip for detection of Perkinsus beihaiensis in the oyster Crassostrea hongkongensis

BackgroundPerkinsosis, a disease caused by the protist Perkinsus, is responsible for mass mortalities of many molluscan species worldwide. The rapid, early and accurate detection of Perkinsus infection is necessary to react to outbreaks, and manage disease transmission. Current methods for diagnosis of Perkinsus spp. are time-consuming or require professional equipment and experienced personnel, rendering them unsuitable for field application. Recombinase polymerase amplification (RPA) assay is a highly sensitive and selective isothermal amplification technique that operates at temperatures of 37–42 °C, requires minimal sample preparation, and is capable of amplifying as low as 1–10 target DNA copies in less than 20 minutes.MethodsWe report a novel RPA assay that amplifies the internal transcriber spacer (ITS) region of P. beihaiensis, which, followed by rapid detection of amplicons using a lateral flow (LF) strip, enables easy visualization of results by the naked eye.ResultsThe LF-RPA assay successfully amplified P. beihaiensis DNA using a set of primers of 20–25 bp in length. After incubation at 37 °C for 25 min, results were read within 5 min by the naked eye on a lateral flow strip. Our LF-RPA assay was comparably sensitive to qPCR assay, and capable of detecting as few as 26 copies of P. beihaiensis DNA. Cross-amplification occurred with other two Perkinsus species, P. olseni and P. chesapeaki, but not with other potential pathogen taxa in culture environments. We compared the performance of LF-RPA, conventional PCR and qPCR assays on 60 oyster samples. While LF-RPA assay results were 86.2% as sensitive, 77.4% as specific, and generally in agreement with those of conventional PCR results, they were more (93.3%) sensitive, (86.7%) specific, and agreed better with qPCR assay results. Future research should focus on developing simple DNA extraction methods that do not require professional laboratories and complicated extraction procedures, to facilitate application of this LF-RPA assay in the field.ConclusionsOur LF-RPA assay provides a rapid and efficient method for detecting species of Perkinsus. This novel assay has potential to be used in field applications.

Stage-specific gene regulation in Perkinsus olseni parasites

Perkinsus species are important marine parasites of molluscs including mussels and oysters with substantial commercial and environmental impact. Perkinsus forms a sister lineage to the apicomplexan parasites (e.g. malaria agent, Plasmodium) and share similar invasion-related structures with these better studied pathogens. However, much less is known of the transmission and invasion biology of Perkinsus spp. We are developing genetic tools to study these parasites, and Perkinsus is emerging as an experimental model for marine parasites. The life cycle of Perkinsus species start with motile zoospores that are ingested by the host via filtration of sea water. After infection of the mollusc, these cells develop into a replicating non-motile vegetative form of the parasite, the trophozoite. Upon host death or morbidity, the parasites are released back into the water column where they differentiate into zoosporangia that release up to 100 motile zoospores. In vitro, Perkinsus species can be maintained as trophozoites in an axenic sea water-based media, and this is the best studied form of the parasite. However, Perkinsus olseni, can be triggered to sporulate in vitro using Ray’s fluid thioglycollate media, and we seek to better study this phase of the lifecycle that is essential for disease spread. To do this, we are determining stage-specific gene expression, during the transformation from trophozoites to zoospores, by RNA-Seq. This will both identify genes that are specific to this growth stage, and also identify zoospore-specific promoters that can be used in experimental manipulation and study of this poorly known cell stage.

Growth, mortality and susceptibility of oyster Crassostrea spp. to Perkinsus spp. infection during on growing in northeast Brazil.

Crassostrea rhizophorae and C. gasar oysters are cultivated in the northeast region. Perkinsus parasites infect bivalves, and their effects on oysters from tropical regions are poorly understood. This study evaluated the impact of Perkinsus infection on the productive traits of native oysters. Oysters were sampled bimonthly during 7 months, from July 2010 to February 2011, to evaluate growth rate, mortality and shell color patterns (white and dark-gray) (n = 500), and to determine the prevalence and intensity of Perkinsus (n = 152). Perkinsus and Crassostrea species were determined using molecular tools. Results showed that most dark-gray (90%, n = 20) and white (67%, n = 18) oysters were C. gasar and C. rhizophorae, respectively. Oysters showed a high growth rate and moderate cumulative mortality (44%). C. gasar oysters grew better and showed lower mortality and lower incidence of Perkinsus compared to C. rhizophorae. The mean prevalence of Perkinsus was moderate (48%), but the infection intensity was light (2.2). Perkinsosis affected very small oysters (19.4 mm). In conclusion, native oysters, especially C. gasar, have a great potential for culture, mortality is not associated with perkinsosis, and the shell color of oysters can be used to improve selection for spats with better performance.

Perkinsus beihaiensis (Perkinsozoa) in oysters of Bahia State, Brazil.

This study reports the pathogen Perkinsus beihaiensis in oysters of the genus Crassostrea on the coast of the State of Bahia (Brazil), its prevalence, infection intensity and correlation with salinity. Oysters (n = 240) were collected between October and December 2014 at eight sampling stations between latitudes 13°55'S and 15°42'S. The laboratory procedures included macroscopic analysis, histology, culture in Ray's fluid thioglycollate medium (RFTM), Polymerase Chain Reaction (PCR) and DNA sequencing. PCR and sequencing have been used for the genetic identification of oysters as well. Two species of oysters have been identified: Crassostrea rhizophorae and C. brasiliana. In both oyster species P. beihaiensis was the only Perkinsus species detected. In C. rhizophorae, the average prevalence was 82.8% by histology and 65.2% by RFTM. In C. brasiliana, the prevalences were 70.5% and 35.7%, respectively. The higher prevalence of P. beihaiensis in C. rhizophorae was probably influenced by salinity, with which was positively correlated (r> 0.8). In both oysters, P. beihaiensis was located mainly in the gastric epithelium. The infection was generally mild or moderate, without apparent harm to the hosts, but in cases of severe infection, there was hemocytical reaction and tissue disorganization. The generally high prevalence in the region suggests that oysters should be monitored with respect to this pathogen, especially in growing areas.

First report of Perkinsus honshuensis in the variegated carpet shell clam Ruditapes variegatus in Korea.

The recent discovery of Perkinsus honshuensis, a new Perkinsus species infecting Manila clams Ruditapes philippinarum (Sowerby, 1852), in Japan, suggested that, based on proximity, P. honshuensis could also be in Korean waters, where to date, P. olseni was believed to be the only Perkinsus species present. Perkinsus sp. infections consistently occurred among Ruditapes variegatus clams on a pebble beach on Jeju Island, off the south coast of Korea. The typical 'signet ring' morphology of the parasite was observed in the connective tissue of the digestive gland, and infection intensity was comparatively low (3.3 × 103 ± 1.2 × 104 to 1.3 × 104 ± 6.1 × 104 cells g-1 gill weight). Further DNA analyses of internal transcribed spacer (ITS-1, 5.8S and ITS-2) and non-transcribed spacer (NTS) regions of the parasite showed 98.9-99.8 and 98.5-99.5% similarity to those of P. honshuensis from Japan, respectively. Phylogenetic analyses using ITS and NTS sequences indicated that Perkinsus sp. from Jeju formed a highly supported clade with P. honshuensis. This is the first report of P. honshuensis infections in clams in Korean waters and the first report of R. variegatus as a host for that parasite.