Asunción Cao

Observations raise the question if the Pacific oyster, Crassostrea gigas, can act as either a carrier or a reservoir for Bonamia ostreae or Bonamia exitiosa

This study investigated the ability of the Pacific oyster, Crassostrea gigas, to act as a carrier or reservoir of the protistan Bonamia ostreae. Studies were carried out independently in Ireland and in Spain. Naïve C. gigas were exposed to B. ostreae both in the field and in the laboratory via natural exposure or experimental injection. Naïve flat oysters, Ostrea edulis, were placed in tanks with previously exposed C. gigas. Oysters were screened for B. ostreae by examination of ventricular heart smears and by polymerase chain reaction (PCR) screening of tissue samples (gill and/or heart) and shell cavity fluid. PCR-positive oysters were further screened using histology and in situ hybridization (ISH). B. ostreae DNA was detected in the tissues and/or shell cavity fluid of a small number of C. gigas in the field and in the laboratory. B. ostreae-like cells were visualized in the haemocytes of 1 C. gigas and B. ostreae-like cells were observed extracellularly in the connective tissues of 1 other C. gigas. When C. gigas naturally exposed to B. ostreae were held with naïve O. edulis, B. ostreae DNA was detected in O. edulis; however, B. ostreae cells were not visualized. In Spain, B. exitiosa DNA was also detected in Pacific oyster tissues. The results of this study have important implications for C. gigas transfers from B. ostreae-endemic areas to uninfected areas and highlight B. ostreae and B. exitiosas ability to survive extracellularly and in other non-typical hosts.

First report of the protozoan parasite Perkinsus marinus in South America, infecting mangrove oysters Crassostrea rhizophorae from the Paraíba River (NE, Brazil).

The present work aimed to study the infection by Perkinsus sp. in the mangrove oysters Crassostrea rhizophorae from the estuary of the Paraíba River (Paraíba State, Brazil). Perkinsosis was detected by incubation of oyster gill pieces in Rays fluid thioglycollate medium. The monthly prevalence values were all above 70%, thus infection was not likely to be a transient event. Perkinsus sp. parasites isolated from eight oysters were propagated in vitro. PCR-RFLP analysis of in vitro cultured cells as well as the sequences of the rDNA ITS region allowed the identification of the in vitro propagated parasites as Perkinsus marinus. Phylogenetic analyses using rDNA ITS region sequences strongly supported the Perkinsus sp. from Paraíba in a monophyletic group with P. marinus. Thus, the results confirmed the species affiliation of Paraíba Perkinsus sp. as P. marinus. This is the first report of P. marinus in Brazil and South America and the first report of P. marinus naturally infecting C. rhizophorae.

Very low microsatellite polymorphism and large heterozygote deficits suggest founder effects and cryptic structure in the parasite Perkinsus olseni

Twelve microsatellite markers were used to characterize 130 clonal cultures of Perkinsus olseni derived from 30 clams from six different geographic locations. Only two loci were polymorphic in the four populations studied from Spanish coast (mean sample size = 31.2), and a third locus was variable in only two populations. In contrast, five parasites isolated from five clams from Japan and New Zealand showed variation at nine loci. Low genetic variation (2.08 ± 0.64 alleles per locus; mean genetic diversity: 0.101 ± 0.022), and very high F(IS) values (0.857 on average) were observed in Spanish populations. A total of 39 multilocus genotypes (MLGs) were identified in the whole sample (121 clonal isolates after excluding incomplete MLGs due to missing data). A three-level hierarchical analysis of molecular variance found significant levels of genetic variation within infrapopulations (all the parasites in a single host; Φ(IS) = 0.679) and among infrapopulations within the component population (all the parasites among a host population; Φ(SC) = 0.579). Differences among the component population from different geographic locations were not significant (Φ(CT) = 0.057). These results suggest that an important fraction of F(IS) is explained by the Wahlund effect, but also strong inbreeding within infrapopulations. Another explanation for the high F(IS) within infrapopulations is the presence of haploid and diploid stages in the clam. Although fully aquatic system provides many opportunities for mixing of parasites from different clams, results are consistent with the consideration of all P. olseni in a clam as a cohesive genetic unit (i.e., deme). If the parasite was introduced into the Spanish coast with the importation of infected clams from Asia and Oceania, the low microsatellite polymorphism could be reflecting founder effects in the recent evolutionary history of P. olseni. The loss of alleles would be intensified in a scenario structured in numerous demes because of recurrent founder effects at microgeographic level.

Comparison of haemocytic parameters among flat oyster Ostrea edulis stocks with different susceptibility to bonamiosis and the Pacific oyster Crassostrea gigas.

Farming of the flat oyster Ostrea edulis in Europe is severely constrained by the protozoan Bonamia ostreae. The introduction of the resistant species Crassostrea gigas has been a relief for the farmers, while the pilot programmes to select O. edulis strains resistant to bonamiosis performed in various countries can be seen as a promising strategy to minimise the effects of bonamiosis. However, the physiological bases of this differential susceptibility remain unknown. A search for an explanation of the intra and interspecific differences in oyster susceptibility to bonamiosis was accomplished by comparing some immune parameters among various O. edulis stocks and C. gigas. On December 2003, naïve and Bonamia-relatively resistant flat oysters from Ireland, Galician flat oysters and Pacific oysters C. gigas were deployed in a Galician area affected by bonamiosis; haemolymph samples were taken in February and May 2004. A new oyster deployment at the same place was carried out on June 2004 and haemolymph sampling was performed on April 2005. On November 2004, new sets of Irish flat oysters and C. gigas were deployed in Ireland and haemolymph sampling was performed in June 2005. Various haemocytic parameters were measured: total and differential haemocyte count, phagocytic ability, respiratory burst (superoxide anion [O(2)(-)] and hydrogen peroxide [H(2)O(2)]) and nitric oxide [NO] production. The comparison of the parameters was carried out at 3 levels: (1) between O. edulis and C. gigas, (2) among O. edulis stocks with different susceptibility to bonamiosis, and (3) between Bonamia-infected and non infected O. edulis. In addition, haemocyte-B. ostreaein vitro encounters were performed to analyse interspecific differences in the haemocytic respiratory burst, using flow cytometry. Significant differences associated with total and differential haemocyte count, and respiratory burst between O. edulis and C. gigas were detected, which could be linked to differences in susceptibility to bonamiosis between both species. Additionally, significant changes in total and differential haemocyte count, and respiratory burst of O. edulis associated with B. ostreae infection were found. However, no consistent difference in any haemocyte parameter between the O. edulis stocks involved in the study was recorded.

Expression of mutant protein p53 and Hsp70 and Hsp90 chaperones in cockles Cerastoderma edule affected by neoplasia

High prevalence of disseminated neoplasia has been found in cockles Cerastoderma edule of Galicia (NW Spain). Disseminated neoplasia has been associated with high mortalities of various bivalve species. In vertebrates, proteins such as p53 and heat shock proteins (HSPs) play important roles in carcinogenesis. The protein p53 has been detected in neoplastic cells of bivalve molluscs such as Mytilus edulis, Mytilus trossulus, Mya arenaria, Spisula solidissima, Crassostrea rhizophorae and Crassostrea gigas. In this study, western blotting analyses were used to test the expression of Hsp70, Hsp90 and mutant p53 proteins in the cells and plasma of the haemolymph of cockles showing various intensities of neoplasia. Disseminated neoplasia was previously diagnosed by examination of stained haemolymph monolayers with light microscopy. In the present study, mutant p53 was detected in haemolymph cells of cockles diagnosed as affected by moderate and heavy neoplasia intensity, whereas it was not detected in cockles with either no or light neoplasia. The higher the neoplasia intensity, the higher the levels of Hsp70 and Hsp90. These proteins were not found in plasma. The results reveal the possible association between p53 and HSPs in neoplastic cells of cockles, which could prevent p53 from carrying out its functions, as occurs in human cancers.

Comparison of protein expression profiles between three Perkinsus spp., protozoan parasites of molluscs, through 2D electrophoresis and mass spectrometry

The genus Perkinsus includes protozoan parasites of a wide range of marine molluscs worldwide, some of which have been responsible for heavy mollusc mortalities and dramatic economic losses. This study was performed with the aim of increasing the knowledge of Perkinsus spp. proteome. Proteins extracted from in vitro cultured cells of three species of this genus, P. marinus, P. olseni and P. chesapeaki, were analysed using 2D electrophoresis. Four gels from each species were produced. Qualitative and quantitative comparisons among gels were performed with Proteamweaver software. Cluster analysis grouped the four gels of each Perkinsus sp.; furthermore, P. marinus and P. olseni gels were grouped in a cluster different from P. chesapeaki. Around 2000 spots of each species were considered, from which 213 spots were common to the 3 species; P. chesapeaki and P. marinus shared 310 spots, P. chesapeaki and P. olseni shared 315 spots and P. marinus and P. olseni shared 242 spots. A number of spots were exclusive of each Perkinsus species: 1161 spots were exclusive of P. chesapeaki, 1124 of P. olseni and 895 of P. marinus. A total of 84 spots, including common and species-specific ones, were excised from the gels and analysed using MALDI-TOF and nESI-IT (MS/MS) techniques. Forty-two spots were successfully sequenced, from which 28 were annotated, most of them clustered into electron transport, oxidative stress and detoxification, protein synthesis, carbohydrate metabolism, signal transduction, metabolic process and proteolysis.

Comparison of antibacterial activity in the hemolymph of marine bivalves from Galicia (NW Spain)

A screening study of in vitro antibacterial activity was conducted in marine bivalves with economical importance and widespread along the coast of Galicia (NW Spain). Hemocyte lysate supernatant (HLS) and plasma of Mytilus galloprovincialis, Ostrea edulis, Crassostrea gigas, Ruditapes decussatus, Ruditapes philippinarum, and Cerastoderma edule were incubated with Vibrio splendidus and Micrococcus sp. HLS and plasma for all the species demonstrated antibacterial activity, and C. edule had the highest activity per unit of protein in these hemolymph fractions. Significant differences were not found between HLS and plasma activities. Furthermore, antibacterial activity against Micrococcus sp. (Gram-positive) was stronger than against V. splendidus (Gram-negative).

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.

Microsatellite marker development in the protozoan parasite Perkinsus olseni.

The analysis of an enriched partial genomic library and of public expressed sequence tag (EST) resources allowed the characterization of the first microsatellite loci in the protozoan parasite Perkinsus olseni. Clonal cultures from laboratory isolates derived from infected clams Ruditapes decussatus (from Spain), R. philippinarum (from Spain and Japan), and Austrovenus stutchburyi (from New Zealand) were used for the characterization of 12 microsatellites. Low variation was detected at most loci, with the number of alleles at polymorphic loci ranging from 2 to 7 (average 3.20 +/- 0.51) and gene diversity from 0.11 to 0.79 (average 0.40 +/- 0.07). Preliminary results show that (1) isolates of P. olseni are diploid cells, and (2) multiple infections can occur within a single host. Eight of the loci analyzed successfully cross-amplified in the congeneric species P. mediterraneus. These microsatellite markers will be useful to analyze in detail the population genetic structure of P. olseni, crucial for the efficient management of this parasitic disease.

Long-term affected flat oyster (Ostrea edulis) haemocytes show differential gene expression profiles from naïve oysters in response to Bonamia ostreae

European flat oyster (Ostrea edulis) production has suffered a severe decline due to bonamiosis. The responsible parasite enters in oyster haemocytes, causing an acute inflammatory response frequently leading to death. We used an immune-enriched oligo-microarray to understand the haemocyte response to Bonamia ostreae by comparing expression profiles between naïve (NS) and long-term affected (AS) populations along a time series (1 d, 30 d, 90 d). AS showed a much higher response just after challenge, which might be indicative of selection for resistance. No regulated genes were detected at 30 d in both populations while a notable reactivation was observed at 90 d, suggesting parasite latency during infection. Genes related to extracellular matrix and protease inhibitors, up-regulated in AS, and those related to histones, down-regulated in NS, might play an important role along the infection. Twenty-four candidate genes related to resistance should be further validated for selection programs aimed to control bonamiosis.