Spatial and temporal disease dynamics of the parasite Hematodinium sp. in shore crabs, Carcinus maenas.

BACKGROUND: The parasitic dinoflagellates of the genus Hematodinium represent the causative agent of so-called bitter or pink crab disease in a broad range of shellfish taxa. Outbreaks of Hematodinium-associated disease can devastate local fishing and aquaculture efforts. The goal of our study was to examine the potential role of the common shore (green) crab Carcinus maenas as a reservoir for Hematodinium. Carcinus maenas is native to all shores of the UK and Ireland and the North East Atlantic but has been introduced to, and subsequently invaded waters of, the USA, South Africa and Australia. This species is notable for its capacity to harbour a range of micro- and macro-parasites, and therefore may act as a vector for disease transfer. METHODS: Over a 12-month period, we interrogated 1191 crabs across two distinct locations (intertidal pier, semi-closed dock) in Swansea Bay (Wales, UK) for the presence and severity of Hematodinium in the haemolymph, gills, hepatopancreas and surrounding waters (eDNA) using PCR-based methods, haemolymph preparations and histopathology. RESULTS: Overall, 13.6% were Hematodinium-positive via PCR and confirmed via tissue examination. Only a small difference was observed between locations with 14.4% and 12.8% infected crabs in the Dock and Pier, respectively. Binomial logistic regression models revealed seasonality (P < 0.002) and sex (P < 0.001) to be significant factors in Hematodinium detection with peak infection recorded in spring (March to May). Male crabs overall were more likely to be infected. Phylogenetic analyses of the partial ITS and 18S rRNA gene regions of Hematodinium amplified from crabs determined the causative agent to be the host generalist Hematodinium sp., which blights several valuable crustaceans in the UK alone, including edible crabs (Cancer pagurus) and langoustines (Nephrops norvegicus). CONCLUSIONS: Shore crabs were infected with the host generalist parasite Hematodinium sp. in each location tested, thereby enabling the parasite to persist in an environment shared with commercially important shellfish.

Pathology and Case Definition of Severe Perkinsea Infection of Frogs.

Severe Perkinsea infection (SPI) is an emerging disease of frogs responsible for mass mortalities of tadpoles across the United States. It is caused by protozoa belonging to the phylum Perkinsozoa that form a distinct group referred to as the Pathogenic Perkinsea Clade of frogs. In this work, we provide detailed description of gross and histologic lesions from 178 naturally infected tadpoles, including 10 species from 22 mortality events and 6 amphibian health monitoring studies from diverse geographic areas. On external examination, we observed abdominal distension (10, 5.6%), cutaneous erythema and petechia (3, 1.7%), subcutaneous edema (3, 1.7%), and areas of white skin discoloration (3, 1.7%). On macroscopic examination of internal organs, we found hepatomegaly (68, 38.2%), splenomegaly (51, 28.7%), nephromegaly (47, 26.4%), ascites (15, 8.4%), segmental irregular thickening and white discoloration of the intestine (8, 4.5%), pancreatomegaly (4, 2.2%), and pancreatic petechia (1, 0.6%). Histologically, over 60% of the liver (148/165, 89.7%), kidney (113/147, 76.9%), spleen (96/97, 99%), and pancreas (46/68, 67.6%) were invaded by myriad intracellular and extracellular Perkinsea hypnospore-like and trophozoite-like organisms. Numerous other tissues were affected to a lesser extent. Mild histiocytic inflammation with fewer lymphocytes or eosinophils was commonly observed in areas of infection that were not obscured by lympho-granulocytic hematopoietic tissue. In light of these observations, we suggest a logical pathogenesis sequence. Finally, we propose a "case definition" for SPI to promote standardized communication of results and prevent misdiagnosis with epidemiological and pathologically overlapping diseases such as ranavirosis.

Field Studies on Amyloodiniosis in Red Sea Cultured Asian Seabass (Lates calcarifer) and Hamour (Epinephelus polyphekadion).

BACKGROUND AND OBJECTIVE: Amyloodinium ocellatum infects the gills and skin of both marine and brackish water fishes. The aim of the present study was to examine pathogenesis, prevalence, trials for treatment and histopathological alterations of Amyloodinosis in naturally infested Asian Seabass Barramundi Lates calcarifer and Hamour Epinephelus polyphekadion in Ismailia Governorate, Egypt. MATERIALS AND METHODS: A total number of 1447 Red Sea cultured Seabass (Lates calcarifer) broadstock and a total number of 53 Red Sea cultured Hamour, Epinephelus polyphekadion broadstock were collected and subjected for the study. Fishes showed symptoms of sudden death and respiratory distress besides Amyloodiniosis on gills and skin. All fishes were treated with various treatment protocols while gills of naturally infected fishes were examined histopathologically. RESULTS: The clinical signs of infested fishes were flashing, surfacing, off food and respiratory distress. The intensity of infestation of Amyloodiniosis was more sever in Asian Seabass than Epinephelus polyphekadion while treatment of choice was copper sulphate (prolonged bath), freshwater bath and formalin consequently. CONCLUSION: Treatment of choice for Amyloodinium ocellatum infestation in Asian Seabass was copper sulphate (prolonged bath) followed by freshwater bath then formalin.

Effect of antiprotozoal molecules on hypnospores of Perkinsus spp. parasite.

Perkinsus protozoan parasites have been associated with high mortality of bivalves worldwide, including Brazil. The use of antiproliferative drugs to treat the Perkinsosis is an unusual prophylactic strategy. However, because of their environment impact it could be used to control parasite proliferation in closed system, such as hatchery. This study evaluated the anti-Perkinsus activity potential of synthesized and commercial compounds. Viability of hypnospores of Perkinsus spp. was assessed in vitro. Cells were incubated with three 2-amino-thiophene (6AMD, 6CN, 5CN) and one acylhydrazone derivatives (AMZ-DCL), at the concentrations of 31.25; 62.5; 125; 250 and 500 µM and one commercial chlorinated phenoxy phenol derivative, triclosan (2, 5, 10 and 20 µM), for 24-48 h. Two synthetic molecules (6CN and AMZ-DCL) caused a significant decline (38 and 39%, respectively) in hypnospores viability, at the highest concentration (500 µM), after 48 h. Triclosan was the most cytotoxic compound, causing 100% of mortality at 20 µM after 24 h and at 10 µM after 48 h. Cytotoxic effects of the compounds 6CN, AMZ-DCL, and triclosan were investigated by measuring parasite's zoosporulation, morphological changes and metabolic activities (esterase activity, production of reactive oxygen species and lipid content). Results showed that zoosporulation occurred in few cell. Triclosan caused changes in the morphology of hypnospores. The 6CN and AMZ-DCL did not alter the metabolic activities studied whilst Triclosan significantly increased the production of reactive oxygen species and changed the amount and distribution of lipids in the hypnospores. These results suggest that three compounds had potential to be used as antiprotozoal drugs, although further investigation of their mechanism of action must be enlightened.

Perkinsus beihaiensis (Perkinsozoa) in oysters of Bahia State, Brazil / Perkinsus beihaiensis (Perkinsozoa) em ostras do Estado da Bahia

Abstract 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.

Resumo Este estudo relata o patógeno Perkinsus beihaiensis em ostras do gênero Crassostrea no litoral do Estado da Bahia (Brasil), sua prevalência, intensidade de infecção e correlação com a salinidade. As ostras (n = 240) foram coletadas entre outubro e dezembro de 2014 em oito estações amostrais entre as latitudes 13°55'S e 15°42'S. Os procedimentos laboratoriais incluíram análise macroscópica, histologia, cultivo em meio de tioglicolato de Ray (RFTM), reação em cadeia da polimerase (PCR) e sequenciamento de DNA. PCR e sequenciamento foram também utilizados para a identificação genética das ostras. Foram identificadas duas espécies de ostras: Crassostrea rhizophorae e C. brasiliana. Em ambas as espécies, P. beihaiensis foi a única espécie de Perkinsus detectada. Em C. rhizophorae, a prevalência média foi de 82,8% por histologia e de 65,2% por RFTM. Em C. brasiliana, as prevalências foram de 70,5% e 35,7%, respectivamente. A maior prevalência de P. beihaiensis em C. rhizophorae foi provavelmente influenciada pela salinidade, com a qual este apresentou correlação positiva (r>0,8). Em ambas as espécies, P. beihaiensis esteve localizada principalmente no epitélio gástrico. A infecção foi geralmente leve ou moderada, sem danos aparentes aos hospedeiros, mas em casos de infecção severa, houve reação hemocitária e desorganização de tecidos. As prevalências geralmente altas na região sugerem que as ostras devam ser monitoradas com relação a este patógeno, principalmente em áreas de cultivo.

Transepithelial migration of mucosal hemocytes in Crassostrea virginica and potential role in Perkinsus marinus pathogenesis.

We have recently described the presence of hemocytes associated with mucus covering the pallial organs (mantle, gills, and body wall) 3 of the eastern oyster Crassostrea virginica. These hemocytes, hereby designated "pallial hemocytes" share common general characteristics with circulating hemocytes but also display significant differences particularly in their cell surface epitopes. The specific location of pallial hemocytes as peripheral cells exposed directly to the marine environment confers them a putative sentinel role. The purpose of this study was to gain a better understanding of the source of these pallial hemocytes by evaluating possible exchanges between circulatory and pallial hemocyte populations and whether these exchanges are regulated by pathogen exposure. Bi-directional transepithelial migrations of hemocytes between pallial surfaces and the circulatory system were monitored using standard cell tracking approaches after staining with the vital fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) in conjunction with fluorescent microscopy and flow cytometry. Results showed bi-directional migration of hemocytes between both compartments and suggest that hemocyte migration from the pallial mucus layer to the circulatory system may occur at a greater rate compared to migration from the circulatory system to the pallial mucus layer, further supporting the role of pallial hemocytes as sentinel cells. Subsequently, the effect of the obligate parasite Perkinsus marinus and the opportunistic pathogen Vibrio alginolyticus on transepithelial migration of oyster hemocytes was investigated. Results showed an increase in hemocyte migration in response to P. marinus exposure. Furthermore, P. marinus cells were acquired by pallial hemocytes before being visible in underlying tissues and the circulatory system suggesting that this parasite could use pallial hemocytes as a vehicle facilitating its access to oyster tissues. These results are discussed in light of new evidence highlighting the role of oyster pallial organs as a portal for the initiation of P. marinus infections in oysters.

New insights into the entrance of Perkinsus olseni in the Manila clam, Ruditapes philippinarum.

In order to understand interactions between Perkinsus olseni and its host mollusk species Manila clam Ruditapes philippinarum, this study focused on invasion processes of the parasite, particularly the mechanisms of zoospore transformation to trophozoites in its portal entry into the host. We exposed Manila clam to P. olseni zoospores, then periodically quantified parasite intensity in various host organs and tissues. We detected large numbers of parasite cells within gills and labial palps of the host clam from the early to the final stages, moderately within mantle and digestive organs but low numbers within hemolymph, foot and adductor muscles. Our results suggest that P. olseni first invades the gills and labial palps of the host clam with limited translocation throughout the host body via the host's circulatory system until 12 days post exposure to zoospores. P. olseni zoospores exposed to extracts of gills and labial palps transformed into trophozoites more efficiently than they did when exposed to other tissues; this transformation was not observed when zoospores were exposed to heated organ extracts. Our results suggest the involvement of a host molecule in the transformation of P. olseni zoospores, leading to initial infection primarily within gills and labial palps of the host clam.

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.

De novo transcriptome assembly of Perkinsus olseni trophozoite stimulated in vitro with Manila clam (Ruditapes philippinarum) plasma.

The protistan parasite Perkinsus olseni is a deadly causative agent of perkinsosis, a molluscan disease affecting Manila clam (Ruditapes philippinarum), having a significant impact on world mollusc production. Deciphering the underlying molecular mechanisms in R. philippinarum-P. olseni interaction is crucial for controlling this parasitosis. The present study investigated the transcriptional expression in the parasite trophozoite using RNA-seq. Control and treatment (in vitro challenged with Manila clam-plasma) P. olseni trophozoite RNA were extracted and sequenced on the Illumina HiSeq 2000 instrument using a 100-bp paired-end sequencing strategy. Paired reads (64.7 million) were de novo assembled using Trinity, and the resultant transcripts were further clustered using CAP3. The re-constructed P. olseni transcriptome contains 47,590 unique transcripts of which 23,505 were annotated to 9764 unique proteins. A large number of genes were associated with Gene Ontology terms such as stress and immune-response, cell homeostasis, antioxidation, cell communication, signal transduction, signalling and proteolysis. Among annotated transcripts, a preliminary gene expression analysis detected 679 up-regulated and 478 down-regulated genes, linked to virulence factors, anti-oxidants, adhesion and immune-response molecules. Genes of several metabolic pathways such as DOXP/MEP, FAS II or folate biosynthesis, which are potential therapeutic targets, were identified. This study is the first description of the P. olseni transcriptome, and provides a substantial genomic resource for studying the molecular mechanisms of the host-parasite interaction in perkinsosis. In this sense, it is also the first evaluation of the parasite gene expression after challenge with clam extracellular products.

Cryptic infection of a broad taxonomic and geographic diversity of tadpoles by Perkinsea protists.

The decline of amphibian populations, particularly frogs, is often cited as an example in support of the claim that Earth is undergoing its sixth mass extinction event. Amphibians seem to be particularly sensitive to emerging diseases (e.g., fungal and viral pathogens), yet the diversity and geographic distribution of infectious agents are only starting to be investigated. Recent work has linked a previously undescribed protist with mass-mortality events in the United States, in which infected frog tadpoles have an abnormally enlarged yellowish liver filled with protist cells of a presumed parasite. Phylogenetic analyses revealed that this infectious agent was affiliated with the Perkinsea: a parasitic group within the alveolates exemplified by Perkinsus sp., a "marine" protist responsible for mass-mortality events in commercial shellfish populations. Using small subunit (SSU) ribosomal DNA (rDNA) sequencing, we developed a targeted PCR protocol for preferentially sampling a clade of the Perkinsea. We tested this protocol on freshwater environmental DNA, revealing a wide diversity of Perkinsea lineages in these environments. Then, we used the same protocol to test for Perkinsea-like lineages in livers of 182 tadpoles from multiple families of frogs. We identified a distinct Perkinsea clade, encompassing a low level of SSU rDNA variation different from the lineage previously associated with tadpole mass-mortality events. Members of this clade were present in 38 tadpoles sampled from 14 distinct genera/phylogroups, from five countries across three continents. These data provide, to our knowledge, the first evidence that Perkinsea-like protists infect tadpoles across a wide taxonomic range of frogs in tropical and temperate environments, including oceanic islands.

Pallial mucus of the oyster Crassostrea virginica regulates the expression of putative virulence genes of its pathogen Perkinsus marinus.

Perkinsus marinus is a pathogen responsible for severe mortalities of the eastern oyster Crassostrea virginica along the East and Gulf coasts of the United States. When cultivated, the pathogenicity of this microorganism decreases significantly, hampering the study of its virulence factors. Recent investigations have shown a significant increase of the in vivo virulence of P. marinus exposed to oyster pallial mucus. In the current study, we investigated the effect of pallial mucus on P. marinus gene expression compared with cultures supplemented with oyster digestive extracts or with un-supplemented cultures. In parallel, parasite cells cultured under these three conditions were used to challenge oysters and to assess virulence in vivo. Perkinsus marinus mRNA sequencing was performed on an Illumina GAIIX sequencer and data were analysed using the Tuxedo RNAseq suite for mapping against the draft P. marinus genome and for differential expression analysis. Results showed that exposure of P. marinus to mucus induces significant regulation of nearly 3,600 transcripts, many of which are considered as putative virulence factors. Pallial mucus is suspected to mimic internal host conditions, thereby preparing the pathogen to overcome defense factors before invasion. This hypothesis is supported by significant regulation in several antioxidant proteins, heat shock proteins, protease inhibitors and proteasome subunits. In addition, mucus exposure induced the modulation of several genes known to affect immunity and apoptosis in vertebrates and invertebrates. Several proteases (proteolysis) and merozoite surface proteins (cell recognition) were also modulated. Overall, these results provide a baseline for targeted, in depth analysis of candidate virulence factors in P. marinus.

Early host-pathogen interactions in a marine bivalve: Crassostrea virginica pallial mucus modulates Perkinsus marinus growth and virulence.

Perkinsus marinus is an important protistan parasite of the eastern oyster Crassostrea virginica. Recent findings showed that oyster pallial organs (mantle, gills) are a major portal of entry for the parasite. Therefore, mucus covering these organs represents the first host effectors encountered by P. marinus. This study consisted of several experiments designed to investigate the effect of oyster pallial mucus on the growth, protease production and infectivity of P. marinus. In each experiment, P. marinus performance in cultures supplemented with pallial mucus (mantle, gill, or both) was compared to that of parasite cells grown in unsupplemented media or in cultures supplemented with oyster plasma or digestive extracts. P. marinus grown in media supplemented with C. virginica mantle mucus showed a significantly higher growth rate than cultures enriched with the other supplemental extracts, while cultures grown in gill mucus promoted higher protease production. Conversely, P. marinus grown in cultures supplemented with pallial mucus of the non-compatible host Crassostrea gigas (Pacific oyster) were dramatically inhibited. Challenge experiments showed a significant increase in P. marinus virulence in cultures supplemented with C. virginica pallial mucus as compared to unsupplemented cultures or to those supplemented with digestive extract or plasma. These results suggest that C. virginica mucus plays a significant role in the pathogenesis of P. marinus by enhancing the proliferation and the infectivity of this devastating parasite. The contrasting results obtained with both oyster species indicate that P. marinus host specificity may begin in the mucus.

Early host-pathogen interactions in marine bivalves: evidence that the alveolate parasite Perkinsus marinus infects through the oyster mantle during rejection of pseudofeces.

Parasites have developed myriad strategies to reach and infect their specific hosts. One of the most common mechanisms for non-vector transmitted parasites to reach the internal host environment is by ingestion during feeding. In this study, we investigated the mechanisms of oyster host colonization by the alveolate Perkinsus marinus and focused on how oysters process infective waterborne P. marinus cells during feeding in order to determine the portal(s) of entry of this parasite to its host. We also compared the infectivity of freely-suspended cells of P. marinus with that of cells incorporated into marine aggregates to link changes in particle processing by the feeding organs with infection success and route. Finally, we evaluated the effect of oyster secretions (mucus) covering the feeding organs on P. marinus physiology because these host factors are involved in the processing of waterborne particles. The ensemble of results shows a unique mechanism for infection by which the parasite is mostly acquired during the feeding process, but not via ingestion. Rather, infection commonly occurs during the rejection of material as pseudofeces before reaching the mouth. The pseudofeces discharge area, a specialized area of the mantle where unwanted particles are accumulated for rejection as pseudofeces, showed significantly higher parasite loads than other host tissues including other parts of the mantle. Aggregated P. marinus cells caused significantly higher disease prevalence and infection intensities when compared to freely-suspended parasite cells. Mucus covering the mantle caused a quick and significant increase in parasite replication rates suggesting rapid impact on P. marinus physiology. A new model for P. marinus acquisition in oysters is proposed.

Characterization and molecular epidemiology of a fungal infection of edible crabs (Cancer pagurus) and interaction of the fungus with the dinoflagellate parasite Hematodinium.

This study reports on an emerging fungal disease of the edible crab, Cancer pagurus. Juvenile (prerecruit) crabs were found to be subject to this disease condition during the months of May to September at two intertidal sites in South Wales, United Kingdom. Histopathology revealed that the fungi overwhelm the host response in the tissues, leading to progressive septicemia. The causative agent of this infection was isolated and grown in pure culture and was identified as a member of the Ophiocordyceps clade by sequencing of the small subunit of the fungal ribosomal DNA (rDNA). Of the crabs naturally infected with the fungus, 94% had a coinfection with the parasitic dinoflagellate Hematodinium species. To determine if there was any interaction between the two disease-causing agents, apparently fungus-free crabs, both with and without natural Hematodinium infections, were challenged with the fungal isolate. The presence of Hematodinium caused a significant reduction in fungal multiplication in the hemocoel of the crabs in comparison to that in Hematodinium-free individuals. Histopathology of coinfected crabs showed a systemic multiplication of Hematodinium within host tissues, leading to a rapid death, while Hematodinium-free crabs experimentally infected with the fungal isolate died due to fungal sepsis (septicemia) with the same characteristic pathology as seen in natural infections.

Experimental challenges of wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams.

Manila clams, Ruditapes philippinarum, are widely harvested in the coastal waters in Japan. However, there have been significant decreases in the populations of Manila clams since the 1980s. It is thought that infection with the protozoan Perkinsus species has contributed to these decreases. A previous study demonstrated that high infection levels of a pure strain of Perkinsus olseni (ATCC PRA-181) were lethal to hatchery-raised small Manila clams, however, the pathogenicity of wild strain Perkinsus species to wild Manila clam is unclear. To address this, we challenged large (30-40 mm in shell length) and small (3-15 mm in shell length) wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams. We report high mortalities among the small clams, but not among the large ones. This is the first report to confirm the pathogenicity of wild isolate of Perkinsus species to wild Manila clams.

Immune response and mechanical stress susceptibility in diseased oysters, Crassostrea virginica.

Eastern oysters, Crassostrea virginica, naturally infected with the parasite Perkinsus marinus were subjected to a mechanical stress by centrifugation, and immune parameters, pathological conditions, and gene expression of selected transcripts were compared to uninfected controls. Immune parameters were assessed by flow cytometry, pathology and parasites by histotechnology and fluid thioglycollate assays, and gene expression by quantitative RT-PCR. Irrespective of mechanical stress, an increased number of hemocytes were observed in P. marinus-infected oysters that corresponded to increased expression of genes that have been shown to be involved in inflammation and apoptosis, two processes associated with regulating immune cell populations. Mechanically stressed, diseased oysters showed histological gill abnormalities and aggregations of hemocytes in tissues not seen in stressed, uninfected oysters. Expression of a high-mobility group protein and hemocyte phagocytosis were significantly upregulated upon mechanical stress only in uninfected oysters. The results of this study demonstrate the role of inflammation in the oyster immune response including possible underlying molecular mechanisms. Furthermore, this study highlights the importance of considering mechanical stressors when characterizing oyster immune function.

The search for the missing link: a relic plastid in Perkinsus?

Perkinsus marinus (Phylum Perkinsozoa) is a protozoan parasite that has devastated natural and farmed oyster populations in the USA, significantly affecting the shellfish industry and the estuarine environment. The other two genera in the phylum, Parvilucifera and Rastrimonas, are parasites of microeukaryotes. The Perkinsozoa occupies a key position at the base of the dinoflagellate branch, close to its divergence from the Apicomplexa, a clade that includes parasitic protista, many harbouring a relic plastid. Thus, as a taxon that has also evolved toward parasitism, the Perkinsozoa has attracted the attention of biologists interested in the evolution of this organelle, both in its ultrastructure and the conservation, loss or transfer of its genes. A review of the recent literature reveals mounting evidence in support of the presence of a relic plastid in P. marinus, including the presence of multimembrane structures, characteristic metabolic pathways and proteins with a bipartite N-terminal extension. Further, these findings raise intriguing questions regarding the potential functions and unique adaptation of the putative plastid and/or plastid genes in the Perkinsozoa. In this review we analyse the above-mentioned evidence and evaluate the potential future directions and expected benefits of addressing such questions. Given the rapidly expanding molecular/genetic resources and methodological toolbox for Perkinsus spp., these organisms should complement the currently established models for investigating plastid evolution within the Chromalveolata.

Copper exposure affects hemocyte apoptosis and Perkinsus marinus infection in eastern oysters Crassostrea virginica (Gmelin).

Dermo disease in the eastern oyster (Crassostrea virginica) is caused by an intracellular protistan parasite Perkinsus marinus. The progression and outcome of this disease is determined by a complex interplay between the host's immunity and parasite's escape mechanisms, both of which can be influenced by environmental pollutants including heavy metals such as copper (Cu). The goal of the present study was to determine the effects of Cu on the levels of apoptosis (which can serve as an important host defense mechanism) in oyster immune cells (hemocytes) in vitro and in vivo as well as on the establishment of P. marinus infections in vivo. Surprisingly, Cu exerted opposing effects on apoptosis levels of hemocytes in vitro and in vivo, stimulating apoptosis in isolated hemocytes but suppressing it during Cu exposure of whole oysters. The mechanisms of this effect are presently unknown and may be related to the different bioavailability of the metal in vitro and in vivo. As expected, Cu accumulated in oyster soft tissues during in vitro exposure. Unexpectedly, this metal also strongly accumulated in hemolymph plasma which is classically considered isoionic with the surrounding seawater, likely reflecting the presence of soluble Cu-binding proteins in oyster plasma. Cu reduced growth of P. marinus in vitro and greatly reduced infection levels of hemocytes in vivo, presumably by direct toxic effects on the parasite. As a possible parasitic counterbalance, Cu accumulation in the hemocytes was reduced by P. marinus infection, although this reduction was not sufficient to prevent the parasiticidal effects of the heavy metal in vivo. This effect of Cu may be useful as a potential therapeutic against Dermo disease in aquaculture conditions. Overall, this study provides important new insights into the potential role of environmental metals in host-parasite relationships and disease dynamics in C. virginica.