Marteilia refringens and Marteilia pararefringens sp. nov. are distinct parasites of bivalves and have different European distributions.

Marteilia refringens causes marteiliosis in oysters, mussels and other bivalve molluscs. This parasite previously comprised two species, M. refringens and Marteilia maurini, which were synonymized in 2007 and subsequently referred to as M. refringens 'O-type' and 'M-type'. O-type has caused mass mortalities of the flat oyster Ostrea edulis. We used high throughput sequencing and histology to intensively screen flat oysters and mussels (Mytilus edulis) from the UK, Sweden and Norway for infection by both types and to generate multi-gene datasets to clarify their genetic distinctiveness. Mussels from the UK, Norway and Sweden were more frequently polymerase chain reaction (PCR)-positive for M-type (75/849) than oysters (11/542). We did not detect O-type in any northern European samples, and no histology-confirmed Marteilia-infected oysters were found in the UK, Norway and Sweden, even where co-habiting mussels were infected by the M-type. The two genetic lineages within 'M. refringens' are robustly distinguishable at species level. We therefore formally define them as separate species: M. refringens (previously O-type) and Marteilia pararefringens sp. nov. (M-type). We designed and tested new Marteilia-specific PCR primers amplifying from the 3' end of the 18S rRNA gene through to the 5.8S gene, which specifically amplified the target region from both tissue and environmental samples.

A Minchinia mercenariae-like parasite infects cockles Cerastoderma edule in Galicia (NW Spain).

The cockle Cerastoderma edule fishery has traditionally been the most important shellfish species in terms of biomass in Galicia (NW Spain). In the course of a survey of the histopathological conditions affecting this species in the Ria of Arousa, a haplosporidan parasite that had not been observed in Galicia was detected in one of the most productive cockle beds of Galicia. Uni- and binucleate cells and multinucleate plasmodia were observed in the connective tissue mainly in the digestive area, gills and gonad. The parasite showed low prevalence, and it was not associated with abnormal cockle mortality. Molecular identification showed that this parasite was closely related to the haplosporidan Minchinia mercenariae that had been reported infecting hard clams Mercenaria mercenaria from the Atlantic coast of the United States. The molecular characterization of its SSU rDNA region allowed obtaining a fragment of 1,796 bp showing 98% homology with M. mercenariae parasite. Phylogenetic analysis supported this identification as this parasite was clustered in the same clade as M. mercenariae from the United States and other M. mercenariae-like sequences from the UK, with bootstrap value of 99%. The occurrence of M. mercenariae-like parasites infecting molluscs outside the United States is confirmed.

Long-term epidemiological study of disseminated neoplasia of cockles in Galicia (NW Spain): temporal patterns at individual and population levels, influence of environmental and cockle-based factors and lethality.

The dynamics of disseminated neoplasia (DN) affecting cockles Cerastoderma edule (L.) in Galicia was addressed at individual and population levels. Early stage of DN was characterized by isolated neoplastic cells occurring in branchial vessels or in the connective tissue of gills, mantle, gonad or digestive gland. As disease progressed, the neoplastic cells appeared loose in foci and became widely distributed throughout the organs. In advanced stages, the connective tissue of most organs was infiltrated by neoplastic cells, which displaced normal cells, leading to the loss of the normal tissue/organ architecture. Host defence reaction was occasionally observed. A field survey performed for 7 years, in two cockle beds located in different Galician Rías, showed that DN is a hyperendemic disease usually present all year-round at high prevalence in adult cockles but with annual prevalence minima in spring likely due to the death of heavily affected cockles, concurrently with gonad ripeness-spawning. DN was detected in the cockles ranging from 10 to 39 mm in size; the highest DN prevalence and severity corresponded to the cockles of intermediate size/age (22-29 mm/0.7-1 year old). Sex did not appear to influence DN occurrence. An inhibitory effect of DN on cockle gametogenesis was detected.

Disseminated neoplasia causes changes in ploidy and apoptosis frequency in cockles Cerastoderma edule.

A proliferative disease, usually referred as disseminated neoplasia (DN), shows high prevalence in some cockle Cerastoderma edule beds of Galicia (NW Spain). Chromosome counts, examination of chromosome morphology, DNA quantification by flow cytometry and estimation of apoptosis frequency by TUNEL assay and flow cytometry were performed in cockles with different DN severity. Metaphases obtained from gills of DN-affected cockles displayed a chromosome number ranging from 41 to 145, while normal number is 38; changes in chromosome morphology were also evident, with numerous microchromosomes occurring. Haemolymph flow cytometry analysis revealed difference in DNA content between healthy and DN-affected cockles. Aneuploid peaks ranged from 1.3n to 8.9n. Apoptosis frequency was determined on histological sections (TUNEL assay) and haemolymph samples (flow cytometry). Both techniques revealed neoplastic cells in apoptosis. The higher DN severity, the lower the percentage of apoptotic cells. According to flow cytometry results, the negative association between DN severity and apoptosis frequency only affected the neoplastic cells, whereas DN did not significantly affect the percentage of apoptotic hyalinocytes or apoptotic granulocytes.

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.

Disseminated neoplasia and large foci indicating heavy haemocytic infiltration in cockles Cerastoderma edule from Galicia (NW Spain).

A histopathological survey was performed to search for the cause of high mortality in the cockle Cerastoderma edule in northern rias of Galicia (northwestern Spain). From limited sampling, 2 severe pathological conditions with high prevalence were found: disseminated neoplasia and the occurrence of numerous, large foci of heavy haemocytic infiltration. Both conditions were detected in areas affected by high mortality and were absent (foci of haemocytic infiltration) or with lower prevalence (disseminated neoplasia) in an area with unnoticeable mortality that was chosen as a reference. Advanced cases of both pathological conditions were associated with extensive tissue destruction. Because of the severity of the lesions and their high prevalence, both pathological conditions could have contributed to the high mortality.

Parasites and pathologic conditions of the cockle Cerastoderma edule populations of the coast of Galicia (NW Spain).

A survey of pathological conditions affecting cockle populations of the most economically important natural beds of Galician estuaries in NW Spain was performed. Samples of 30 adult cockles were collected from each of 34 natural beds in the spring of 1999 and processed by histological techniques. Disseminated neoplasia were seen in samples from most of the natural beds, in some cases with a high prevalence. The gregarine Nematopsis sp., larval trematode stages, and branchial extracellular large cysts enclosing bacteria-like microorganisms were the most prevalent parasites. Paravortex cardii, intracellular colonies of rickettsiae-like organisms in digestive and gill epithelium, Pseudoklossia sp. coccidians, Trichodina sp., and other ciliates were frequently seen in the samples. Copepods in gills and intestine and unidentified gregarines in intestine epithelia and surrounding connective tissue were less prevalent and were observed in samples of some natural beds. Large foci of heavy hemocytic infiltration were detected in a few sites only. Cysts of Steinhausia sp. and plasmodia and spores of a haplosporidian were seen in cockles from two localities. Inflammation was frequently observed in the samples. Some of the parasites and pathological conditions could be associated with mortality.

Morphological characterization of the hemocytes of the clam, Ruditapes decussatus (Mollusca: Bivalvia).

Hemocytes play an important role in internal defence in molluscs. The morphology of hemolymph cells was studied for the first time in Ruditapes decussatus. Two main types of hemocytes (hyalinocytes and granulocytes) exist in R. decussatus. Three types of granulocytes were identified by light microscopy, in accordance with the presence of basophilic or acidophilic granules or a mixture of both in the cytoplasm. The existence of hyalinocytes and granulocytes was confirmed by electron microscopy. Some monoclonal antibodies (MABs) raised against hemocytes of Crassostrea gigas showed cross-reactivity with the total population of hemocytes of R. decussatus; however, none of the MABs raised against hemocytes of Mytilus edulis showed cross-reactivity. The MABs assayed did not allow us to distinguish hemocyte subpopulations.