Antonio Villalba

Gametogenic cycle of cultured mussel, Mytilus galloprovincialis, in the bays of Galicia (N.W. Spain)

Abstract The patterns of gonad and adipogranular (ADG) cell development were studied throughout the culture process in mussels cultured in Vigo, Arousa, Muros and Ares-Betanzos Bays (Galicia, Spain). These patterns were similar in Vigo, Arousa and Muros Bays. Gametogenesis progressed in autumn and early winter, using reserves stored during the previous months. Mussels remained ripe during winter and mass spawning took place in spring. A rapid gonad restoration occurred after spawning and there was a new mass spawning later in spring. New sequences of gonad restoration and spawning occurred again in some mussels, but most of them reabsorbed their gonads concurrently with development of storage tissue. A new gametogenic cycle started by late summer. In Ares-Betanzos Bay, mussels did not become ripe until spring and only one mass spawning took place, in summer.

Widespread transmission of independent cancer lineages within multiple bivalve species

Most cancers arise from oncogenic changes in the genomes of somatic cells, and while the cells may migrate by metastasis, they remain within that single individual. Natural transmission of cancer cells from one individual to another has been observed in two distinct cases in mammals (Tasmanian devils and dogs), but these are generally considered to be rare exceptions in nature. The discovery of transmissible cancer in soft-shell clams (Mya arenaria) suggested that this phenomenon might be more widespread. Here we analyse disseminated neoplasia in mussels (Mytilus trossulus), cockles (Cerastoderma edule), and golden carpet shell clams (Polititapes aureus) and find that neoplasias in all three species are attributable to independent transmissible cancer lineages. In mussels and cockles, the cancer lineages are derived from their respective host species; however, unexpectedly, cancer cells in P. aureus are all derived from Venerupis corrugata, a different species living in the same geographical area. No cases of disseminated neoplasia have thus far been found in V. corrugata from the same region. These findings show that transmission of cancer cells in the marine environment is common in multiple species, that it has originated many times, and that while most transmissible cancers are found spreading within the species of origin, cross-species transmission of cancer cells can occur.

Growth, mortality, pathological conditions and protein expression of Mytilus edulis and M. galloprovincialis crosses cultured in the Rı́a de Arousa (NW of Spain)

Abstract In this article, the growth, mortality, pathological conditions and protein expression of hatchery obtained mussels from pure and hybrid crosses between individuals from three genetically divergent European populations were evaluated in the Ria de Arousa (NW Spain) under raft-suspended cultivation conditions. Progenitors for the crosses were obtained from a Mytilus edulis population from The Netherlands and from two M. galloprovincialis populations located on each of the two sides of a major genetic break, associated with the Almeria–Oran oceanographic front in the Iberian Peninsula. Results indicated that mussels from crosses between individuals of M. galloprovincialis populations have a significantly higher biomass production than those from hybrid crosses between individuals of M. galloprovincialis and M. edulis populations. This different performance was not due to differences in growth rate but rather to the considerably higher mortality, during the warmer season, of the mussels from hybrid crosses. This strong viability selection operating against hybrid mussels with respect to pure M. galloprovincialis crosses under raft cultivation conditions in the Ria de Arousa was very similar to that operating in natural populations of the M. edulis / M. galloprovincialis hybrid zone in SW England, reported in previous studies. Our results also show that the lower viability of hybrid mussels is clearly associated with both higher parasitization by the protistan Marteilia refringens and lower levels of the stress proteins calreticulin and heat shock protein 70. Among the mussels from the different M. galloprovincialis crosses, those from crosses between males and females of autochthonous (Galician) origin are the ones which show a better performance.

The mussel Xenostrobus securis: a well-established alien invader in the Ria de Vigo (Spain, NE Atlantic)

Biological and habitat characterization of the non-indigenous invasive species Xenostrobus securis was undertaken in the Ria de Vigo. This study included genetic identification of mussel samples collected from introduced and endemic areas, and the assessment of mussel size, population abundance, geographic distribution, pathological condition, and sediment composition of substrata type. The mussel had a marked patchy distribution, being more abundant in brackish sites with fine sediments and high organic matter content. Pathological analysis revealed that X. securis does not play any role as vector for introducing allochthonous pathogens in the Ria de Vigo. Nevertheless, depending on its invasiveness potential, the mussel could be a key host favouring spreading and epizootic outbreaks of marteliosis which is known to be harmful for local bivalve populations. Phylogenetic analyses of the COI gene placed all the resulting sequences in a clade within the genus Xenostrobus and its phylogeny congruent with an Australian/Pacific origin. The COI tree suggests two historical introductions in European waters. One of these invasions seems to have started in Galicia, moving from there towards Italy and France, while the geographical spread of the second invasion cannot be deciphered, although the Australian/Pacific origin of this invasion seems very possible. The 18S network is congruent with one invasion starting in Galicia or in Italy, as the Australian haplotype is closely related to the haplotype found in these areas. Several hypotheses accounting for the colonization history of this species in Galician waters are discussed.

Absence of generation of active oxygen radicals coupled with phagocytosis by the hemocytes of the clam, Ruditapes decussatus (Mollusca: Bivalvia)

The production of oxygen radicals by hemocytes of Ruditapes decussatus as a cytotoxic mechanism associated with phagocytosis was studied. In vitro phagocytosis assays of yeast cells (zymosan) by clam hemocytes were performed. Two different methods for oxygen radical detection were tested, based on the technique of luminol-dependent chemiluminescence and on the reduction of nitroblue tetrazolium. Hemocytes of the Pacific oyster, Crassostrea gigas , were used as positive control in the assays, because of their ability to produce oxygen radicals. The results obtained showed that the hemocytes of R. decussatus were not able to produce toxic oxygen radicals during phagocytosis, irrespective of whether yeast cells were opsonized or not. Similar results were obtained with hemocytes of the cockle, Cerastoderma edule .

Identification and expression of immune genes in the flat oyster Ostrea edulis in response to bonamiosis

The European flat Ostrea edulis is highly susceptible to infection by the protozoan Bonamia ostreae and Bonamia exitiosa, intracellular parasites able to survive and proliferate within the oyster haemocytes. The parasite, once phagocytosed by the haemocyte, the main cellular effector of the immune system, appears to have some counter mechanism that turns off the haemocytes metabolic destructive capacity, so that the parasite survives within the cell. To further understand the molecular basis of the immune response of the flat oyster against the bonamiosis, suppression subtractive hybridization and Q-PCR approaches were combined to identify genes involved in the development of the infection both in early and advanced phases. Four subtractive cDNA libraries were constructed and sequenced, obtaining a high number of ESTs that were seen to be up or down-regulated in the infection. A group of ESTs that play a role in the immune response, such as cytokines, stress proteins, eicosanoids, proteins implicated in phagocytosis and cell junction as well as in transcription signalling were identified and their expression was analysed at different infection levels by Q-PCR. The results here reported can help to enrich our understanding about the immune response of O. edulis against bonamiosis and improve our knowledge of the immune mechanisms of oysters.

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.

Variability of haemocyte and haemolymph parameters in European flat oyster Ostrea edulis families obtained from brood stocks of different geographical origins and relation with infection by the protozoan Bonamia ostreae.

A research project to compare productive traits (growth and mortality), disease susceptibility and immune capability between Ostrea edulis stocks was performed. This article reports the results on the immune capability and its relation with infection by the intrahaemocytic protozoan Bonamia ostreae. Four to five oyster spat families were produced from each of four European flat oyster populations (one from Ireland, one from Greece and two from Galicia, Spain) in a hatchery. The spat were transferred to a raft in the Ría de Arousa (Galicia) for on growing for 2 years. Total haemocyte count (THC) and differential haemocyte count (DHC) were estimated monthly through the second year of growing-out. Three types of haemocytes were distinguished: granulocytes (GH), large hyalinocytes (LHH) and small hyalinocytes (SHH). Significant correlations between the mean relative abundance of GH and SHH of the families and the mean prevalence of B. ostreae, the overall incidence of pathological conditions and the cumulative mortality of the families were found; these correlations supported the hypothesis that high %GH and low %SHH would enhance oyster immune ability and, consequently, would contribute to lower susceptibility to disease and longer lifespan. Infection by B. ostreae involved a significant increase of circulating haemocytes, which affected more markedly the LHH type. The higher the infection intensity the higher the %LHH. This illustrates the ability of B. ostreae to modulate the immune responses of the O. edulis to favour its own multiplication. A significant reduction of the phenoloxidase activity in the haemolymph of oysters O. edulis infected by B. ostreae was observed. Nineteen enzymatic activities in the haemolymph of O. edulis and Crassostrea gigas (used as a B. ostreae resistant reference) were measured using the kit api ZYM, Biomerieux. Qualitative and quantitative differences in enzyme activities in both haemocyte and plasma fractions between B. ostreae noninfected O. edulis from different origins were recorded. However, no clear positive association between enzyme activity and susceptibility to bonamiosis was found. The only enzyme detected in the resistant species C. gigas that was not found in the susceptible one O. edulis was beta-glucosidase (in plasma). B. ostreae infected O. edulis showed significant increase of some enzyme activities and the occurrence of enzymes that were not detected in noninfected oysters. These changes could be due to infection-induced enzyme synthesis by the host or to enzyme synthesis by the parasite.

Growth, mortality and parasitization of mussels cultivated in the Ría de Arousa (NW Spain) from two sources of seed: intertidal rocky shore vs. collector ropes.

The cultivation of the mussel, Mytilus galloprovincialis, in the Galician Rias (NW Spain) requires approximately 4600 tonnes of mussel seed every year. Traditionally, this seed is obtained from two different sources: intertidal rocky shore and collector ropes. In this study, the biological performance (growth rate, mortality and parasitization) of mussels from both sources of seed was compared in a cultivation area of the Ria de Arousa. The results from this experiment indicate there is no difference in biological performance between the two types of mussels seed under standard cultivation conditions. Nevertheless, the use of mussel seed from collector ropes is recommended due to their significantly larger size at harvest time. The main deficiency of this type of seed, its high mortality from seeding to thinning out, could be offset by improving the procedure of tying the mussels to culture ropes.