Search

14 pages, 8 figures, 4 tables.-- Under a Creative Commons license, The analysis of the natural variability of seawater (Ta, Ωaragonite and pCO2) at Rodas Beach (NW Iberian Peninsula, Spain) revealed an increase of acidification. However, such pH change was not linked to any detrimental effect of the shell taphonomic characteristics of live razor clams harvested during distinct temporal series (length, thickness, organic content or strength). Temperature affected negatively shell strength and thickness, although the large correlation between the environmental variables would limit the individual characterization. Modelled trends in pH (and Ωaragonite) showed a significant decrease in the last 20 years, despite Ω > 1. Therefore, more recent shells are being secreted in a progressively less saturated carbonate environment and, consequently, more prone to suffer dissolution (and weakening) in projected climatic scenarios. When shells of harvested razor clams were exposed to projected climatic scenarios in the laboratory, dissolution rates were significantly greater for cold-acidic scenarios (more corrosive) as compared to warm-acidic. The median dissolution time (DT50) for shells under the cold-acidic scenario was reduced by half (15 years) when compared to the values observed for shells under current water chemistry conditions (30 years). Galician coastline, often characterised by pCO2-rich and cold waters due to upwelling system, would represent the most corrosive scenario for the shells according to the responses monitored in our survey which highlight future compromise for the ecosystem services supplied by these hard skeletons. Future climate scenarios might condition performance of bivalves but also more complex processes related to carbonate structures. Local biodiversity may be lowered which may reduce the possibility that many species find shelter and feeding grounds, diminishing the optimal substrate for other organisms as needed elements for optimal services in the ecosystems, JMFB acknowledges the project ARIOS (CTM2016-76146-C3-2-R/CTM2016-76146-C3-1-R) funded by the Spanish government through the Ministerio de Economía y Competitividad that included European FEDER funds and the project Atlazul (0755_ATLAZUL_6_E) co-financed by the Interreg-POCTEP Programme through the European Regional Development Fund. AV and FFP were supported by BOCATS2 (PID2019-104279 GB-C21) project funded by MCIN/AEI/10.13039/501100011033 and contributing to 628 WATER:iOS CSIC PTI. LGP was supported by a Talento Senior Grant (16_IN585A_2020_986937) from Xunta de Galicia (Spain)

During a histopathological survey of Mytilus galloprovincialis in Galicia (NW Spain), microcells were observed infecting several organs of the symbiont copepod Mytilicola intestinalis. Positive results of PCR assay with specific primers for genus Mikrocytos and a clear signal of in situ hybridization with MACKINI-1 digoxigenin- labelled DNA probe (DIG-ISH) indicated a protozoan parasite of Mikrocytos genus. The ultrastructural study revealed intra and extracellular locations, polymorphic nuclei, intracellular round vesicles in the cytoplasm and absence of mitochondria. The present paper reports the characterization of the Mikrocytos sp. infecting M. intestinalis and proposes a novel species in the genus: Mikrocytos mytilicoli n. sp. A sequence of 18S-28S rDNA was obtained with 95.6% maximum identity (query cover 100%) with Mikrocytos mackini. Phylogenetic analysis showed that M. mytilicoli n. sp. and M. mackini share a common ancestor. However, comparison of the ITS1 rDNA region showed low similarity (75.8%) with M. mackini, which, combined with differences in ultrastructural details, host and geographic location, support the designation of a new species. This is the first description of a microcytid parasite of the genus Mikrocytos from a non-bivalve host.

Outbreaks of Marteilia cochillia have caused massive mortalities of common cockle, Cerastoderma edule, in some natural beds in Galicia (NW Spain) since 2012. The life cycle of Marteilia spp. is still unresolved and the most accepted hypothesis suggests that an additional host is involved. Researchers have assumed that sporangia are shed into the environment in the faeces, but details about this process have not been reported previously. Here, we report the massive liberation of Marteilia cochillia sporangia through the exhalant siphon into the environment, packaged as faeces. Using light microscopy observations on fresh samples, imprints and histology, we also describe a thick (ca. 5 µm) transparent envelope covering the sporangia that has not been reported previously. The massive release of encapsulated sporangia reported here ensures that millions of infective stages of M. cochillia cycle through the environment and become available for infection. The elucidation of the role played by the sporangia envelope would be of utmost importance for the understanding M. cochillia life cycle.

This study provides morphological and molecular characterization of a new species, Haplosporidium pinnae), very likely responsible for mass mortality of fan mussels, Pinna nobilis, in the Western Mediterranean Sea. The parasite was found in dead or moribund P. nobilis but did not occur in healthy fan mussels from locations that were not affected by abnormal mortality. Histological examination of infected fan mussels showed uninucleate cells of a haplosporidan parasite throughout the connective tissue and hemolymph sinuses of the visceral mass and binucleate cells and, rarely, multinucleate plasmodia were also detected in the connective tissue. Additionally, stages of sporulation occurred in the epithelium of the host digestive gland tubules. Spores were slightly ellipsoidal with a hinged operculum in one pole. Typical haplosporosomes were not found with TEM but vesicles with two concentric membranes resembling haplosporosomes were abundant in the cytoplasm of the multinucleate plasmodia occurring in host digestive gland tubules. SEM analysis showed multiple structures on the spore surface; some spores had two or four long tape-like filaments attached to the spore wall. Phylogenetic analysis based on the SSU rDNA sequence placed this parasite within a large clade including species of the order Haplosporida, not in the Bonamia/Minchinia subclade or the subclade containing most Haplosporidium species, but within a subclade of Haplosporidium sp. from Penaeus vannamei. Our results suggested that H. pinnae and the parasite of P. vannamei may represent a distinct new genus within the order Haplosporida.

This paper reports the results of the survey developed after the first detection of protozoan Marteilia sp. infection of the grooved razor shell Solen marginatus (Pulteney, 1799) from Galicia (NW Spain) in 2006. Furthermore, we analysed other parasites and pathological conditions found in grooved razor shell populations throughout this survey, such as metacercariae of trematodes, prokaryotic infections and disseminated neoplasms, some of which could cause moderate or severe damage to the host depending on the intensity of infection. A total of 17 natural beds distributed along the Galician coast were analysed, and Marteilia sp. was detected in 6 of them with low prevalence, moderate intensity and no negative effects over the populations.

Several stages of a haplosporidan parasite, including spores, were detected infecting three out of four specimens of the Pen Shell Pinna nobilis from the coast of Alicante (Western Mediterranean). A mortality event initiated few weeks before the sampling. The infection was systemic in the connective tissue, with free uni-nucleate stages and early plasmodia, whereas sporulation process took place in the digestive tubules disrupting them. Morphological details, by light and transmission electron microscopy, and PCR amplification confirmed that the parasite belongs to the haplosporidan group. Spores were pleomorphic, usually elongated ovoid, with round to elongated haplosporosomes-like in the sporoplasma. The operculum was situated in the apical zone of the wall, with an external lid, and the nucleus tended to be eccentric in the basal zone. Spore ornamentation was not observed. The single uninfected specimen appeared to be healthy. This is the first report of a haplosporidan parasite infecting a member of the Superfamily Pinnoidea and this is the first histopathological study of a mortality event in the endangered and protected P. nobilis.

The effectiveness of depuration for the removal of hepatitis A virus (HAV), norovirus (NoV) genogroups I (GI) and II (GII), and F+RNA bacteriophage (F+RNA) was evaluated for pullet carpet shell clams (Venerupis pullastra) and Mediterranean mussels (Mytilus galloprovincialis). The objective was to compare the behaviour of the different pathogens under commercial depuration conditions during 7 days in an authorized plant. Standard double agar overlay method (ISO 10705-1) was employed for F+RNA quantification. Recently developed ISO/TS 15216:2013 standard method, based on RT-real time PCR, were employed for the quantification of HAV and NoV. The reduction of F+RNA showed a two-phase depuration kinetic. The average reduction rates were 1-log units for clams and 2-log units for mussels, with residual levels after the process of 6.3 × 103 and 8.3 × 101 F+RNA/100 g, respectively. HAV, NoV GI and GII were detected intermittently throughout the entire process, ranging mostly from 103 to 105 RNA copies/g digestive tissue (DT). NoV GI showed the higher viral levels followed by NoV GII and HAV. All of them were detected in clams after seven days of depuration, however, in mussels only NoV GI was detected after the process. Generally, clams showed slower depuration rates and higher contamination levels for all viruses analysed.

Bonamiosis constrains the flat oyster industry worldwide. The protistan species Bonamia ostreae had been considered solely responsible for this disease in Europe, but the report of B. exitiosa infecting Ostrea edulis 5 yr ago in Galicia (NW Spain), and subsequently in other European countries, raised the question of the relevance of each species in bonamiosis. The spatial distribution of B. exitiosa and B. ostreae in Galicia was addressed by sampling 7 natural O. edulis beds and 3 culture raft areas, up to 3 times in the period 2009 to 2010. B. ostreae infected flat oysters in every natural bed and every raft culture area. True B. exitiosa infections (histological diagnosis) were detected in every raft culture area but only in 2 natural beds, i.e. in 4 rías. PCR-positive results for B. exitiosa were recorded in 4 out of 5 beds where true infections were not found, thus the occurrence of B. exitiosa in those 4 beds cannot be ruled out. Additionally, 4 cohorts of hatchery-produced oyster spat were transferred to a raft to analyse Bonamia spp. infection dynamics through oyster on-growing. The highest percentages of oysters PCR-positive for both Bonamia spp. were recorded in the first months of on-growing; other peaks of PCR-positive diagnosis were successively lower. Differences in the percentage of PCR-positive cases and in the prevalence of true infection between B. exitiosa and B. ostreae through on-growing were not significant. Our results support that B. exitiosa is adapted to infect O. edulis in the Galician marine ecosystem.

The aim of the present study was to carry out a survey of parasites and other conditions affecting pod razor clam populations, Ensis siliqua, in two beds from Galicia (NW Spain). In Galicia, the production of E. siliqua has increased in recent years due to the development of specific plans for its exploitation, however few and quite recent pathological studies have been carried out in this species. The results of this study showed the presence of different protozoa as the more prevalent group, especially Nematopsis sp. gregarines, unidentified branchial protozoa, renal coccidia and Trichodina sp. ciliates. Larval stages of trematodes and neoplastic disorders were also observed with lower prevalences. Furthermore, an ultrastructural analysis of two types of unidentified basophilic inclusions, both found in the digestive gland, revealed the presence of icosahedral viral particles and prokaryotic organisms, respectively. None of the parasites detected in E. siliqua from this study was notifiable to the World Organisation for Animal Health (OIE) and the majority of the symbionts and conditions observed in their tissues did not cause host damage. Nevertheless, parasites like bucephalid digenean sporocysts, viral inclusions, prokaryotic infections, disseminated neoplasm or germinoma detected in some samples could cause moderate or severe damage to the host depending on the intensity of infection.

16 pages, 4 figures, 2 tables, A histopathological survey revealed parasites and pathological conditions affecting lagoon cockles Cerastoderma glaucum along the Galician coast; serious pathological threats were not detected because the potentially pathogenic conditions (infections with a Marteilia-like parasite and bucephalid sporocysts, disseminated neoplasia and a condition involving large foci of heavy haemocytic reaction) were rare, while more prevalent parasites had negligible or limited pathogeny. Considering that C. edule and C. glaucum are sympatric in some Galician rias, it is remarkable that C. glaucum was not seriously affected by Marteilia cochillia while C. edule suffered an intense outbreak of this parasite associated with massive mortality. Comparison of the digestive gland between cockle species showed co-occurrence of digestive tubules in different phases, with abundant disintegrated tubules, in the case of C. glaucum, while C. edule showed synchronicity and absence of fully disintegrated tubules; these differences could influence their susceptibility to M. cochillia because the main location of this parasite in common cockles is the epithelia of the digestive gland. Moreover, the observation of histological sections through the digestive gland easily allows differentiating the 2 cockle species., This study was partly funded by the Consellería do Mar da Xunta de Galicia, through the project PGIDIT-CIMA 13/03

Basophilic intracellular prokaryotic-like colonies were observed in the gills of banded carpet shell Polititapes virgineus (= Tapes rhomboides) (Linnaeus, 1767) from a natural bed in Galicia (NW Spain). Light microscope observations suggested the presence of 2 types of colonies, but transmission electron microscopy revealed that these were the same Rickettsiales-like colonies, one infected and the other uninfected by phage particles. This is the first report of the presence of phage particles in Rickettsiales-like organisms in the gills of P. virgineus.

This study investigated razor clam Ensis arcuatus culture from larval rearing to commercial size to assess it's aquaculture potential. Broodstock was spawned several times in hatchery facilities to demonstrate that larval availability is not a constraint for hatchery production. Larval culture lasted 20 days, showing an average survival of 14.35%. The razor clam seed reached a size of 28.5 mm at 4 months. Seed culture is feasible; however, it is constrained by the fact that razor clams need to be buried. Higher survival was observed when culturing razor clam seed with a substrate (82–83%), irrespective of the substrate grain size, while containers held without a substrate showed a significantly lower survival (56.5%). We showed that it is possible to rear E. arcuatus juveniles in bottles, with survival as high as 94–95% being achieved. The length increase of the hatchery-produced E. arcuatus over 600 days was described by the equation Ln L=4.5+(−141.17/t), where L is length in mm and t is time in days. On-growing showed adequate survival rates and it took 2–3 years to reach commercial size.

The razor clam Ensis arcuatus (Jeffreys, 1865) is distributed from Norway to Spain and along the British coast, where it lives buried in sand in low intertidal and subtidal areas. This work is the first study to research the embryology and larval development of this species of razor clam, using light and scanning electron microscopy. A new method, consisting of changing water levels using tide simulations with brief dry periods, was developed to induce spawning in this species. The blastula was the first motile stage and in the gastrula stage the vitelline coat was lost. The shell field appeared in the late gastrula. The trochophore developed by about 19 h post-fertilization (hpf) (198C). At 30 hpf the D-shaped larva showed a developed digestive system consisting of a mouth, a foregut, a digestive gland followed by an intestine and an anus. Larvae spontaneously settled after 20 days at a length of 378 mm.

Protozoan parasites of the genus Marteilia have been detected in marine bivalves and other invertebrates around the world, associated in some cases with mass mortalities. The present paper reports the characterization of the Marteilia sp. protozoan infecting the digestive gland of the Grooved Razor Shell clam (Solen marginatus) from Galicia (NW Spain), proposing a novel species in the genus: Marteilia octospora n. sp. Morphological and molecular techniques were used for the description of this parasite. Tissue imprints were essential in the study to confirm the presence of 8 spores per sporangium, a number never reported in other species from this genus. An ultrastructural study revealed that the size and number of dense granules, free in the mature sporangia, were quite different from granules in other Marteilia spp. Another morphological difference is the absence of a layer of concentric membranes found surrounding the mature spore in other species. In addition, concentric membranous structures observed in the different stages of the parasite have never been mentioned in other species of genus Marteilia. Moreover, molecular analysis of the rDNA intergenic spacer (IGS) and the internal transcribed spacer (ITS-1) showed differences with the sequences available for other Marteilia spp.

The reproductive cycle of Ensis siliqua in the Ria de Corcubion (northwest Spain) was studied using condition indices and histological methods. The gametogenic cycle was annual, with a long sexual rest period during summer and autumn, when the gonadal condition index (GCI) was at minimum levels and sexes were differentiated. Gametogenesis started during November- December and the only period of spawning occurred in April - May. The gonadal condition index (GCI) used showed a clear relationship with the gametogenicstage, rising from zero in stages 0 (sexual rest) and I (start of gametogenesis), to its highest values in stages IIIA (ripe) and IIIB (start of spawning), before decreasing significantly in stage IV (exhaustion). In contrast, the meat condition index (MCI) did not show statistical differences between gametogenic stages. We suggest that the analysis of GCI (based on gonadal weight) may prove a valuable technique in fishery management to recognize the reproductive stage of E. siliqua populations, particularly as it is less expensive and time consuming than histological techniques.

Seasonal changes in condition indices and biochemical components of the digestive gland, anterior adductor muscle, foot, and gonad of Ensis arcuatus (Jeffreys, 1865) were analysed from February 1998 to June 1999 in relation to environmental conditions and reproductive events. During summer, E. arcuatus accumulated lipids, particularly triacylglycerols, in the digestive gland and glycogen in the anterior adductor muscle and foot while the gonad was in sexual rest. Phytoplankton blooms caused by the upwelling of cold waters, rich in nutrients, from offshore are responsible for the high availability of food during reserve storage. In autumn, when gametogenesis started, oceanographic conditions changed to a situation with low temperature throughout the water column because of the vertical mixing, and food became scarce. At that point in time, the energy requirements for basal metabolism and the reproductive process were provided by the mobilization of triacylglycerols and glycogen stored in the digestive gland and muscle tissues, respectively. The pattern exhibited by E. arcuatus based on the accumulation of reserves in summer and the subsequent mobilization during gonadal development seems to follow a conservative pattern.

This study presents the first analysis of the reproductive cycle of the razor clam Ensis arcuatus and the effect of oceanographic conditions. From February 1998 to July 2000, samples were taken at 2-week intervals from the natural submerged bank of the Cies Islands in the Ria de Vigo (NW Spain). A follow-up gonadal condition index (GCI) was conducted and calculated as gonad fresh weight/valve dry weight. From histological preparations of the gonad stained with haematoxylin and eosine, a gametogenic scale was drawn up based on seven stages: (0) sexual rest, (I) start of gametogenesis, (II) advanced gametogenesis, (IIIA) ripe, (IIIB) start of spawning, (IIIC) restoration and (IV) exhaustion. E. arcuatus is gonochoric and the gonadal development of the two sexes is highly synchronous. During the summer, they were in the sexual rest stage, coinciding with the upwelling period in the Ria de Vigo when the cold, nutrient-rich waters from the bottom rise to the surface. This phenomenon produces phytoplanktonic blooms which mean an increase in the availability of food. Gametogenesis began at the start of autumn when upwelling ceased and food was less plentiful. During the winter and spring, consecutive spawns occurred interspersed with gonadal restoration periods.

During a histological survey of razor clam Ensis arcuatus (Jeffreys, 1865) from Galicia (NW Spain), basophilic inclusion bodies were observed in epithelial cells of the digestive gland. Transmission electron microscopy revealed the intranuclear position of these inclusions containing viral particles with icosahedral symmetry. Size and symmetry of these unenveloped virus particles suggest similarities to the families Papillomaviridae and Polyomaviridae which have been described as causing a viral gametocytic hypertrophy in oysters Crassostrea virginica and C. gigas. This is the first report of viral particles in E. arcuatus.

The name 'microcells' is frequently used to refer to small-sized unicellular stages of molluscan parasites of the genera Bonamia (Rhizaria, Haplosporidia) and Mikrocytos (Rhizaria). Histological examination of Manila clams Ruditapes philippinarum revealed microcells in the connective tissue of adductor muscle, foot, mantle, gills, siphon and visceral mass. The clams had been collected from 4 beds on the coast of Galicia, Spain. The prevalence of these microcells ranged from 73 to 93% in surface clams and from 3 to 33% in buried clams. However, the detection of brown ring disease signs in clams from every bed prevented us from making the assumption that the microcells alone were responsible for clam mortality. PCR assays using primer pairs designed to detect Bonamia spp. and haplosporidians gave negative results, whereas positive results were obtained with primers for the genus Mikrocytos. A consensus sequence of 1670 bp of the ribosomal gene complex of the microcells was obtained. It contained a section of the 18S region, the whole first internal transcribed spacer, the 5.8S region, the second internal transcribed spacer and a section of the 28S region. Comparison of this sequence with those of M. mackini infecting Crassostrea gigas and Mikrocytos sp. infecting Ostrea edulis showed that the microcells of Galician clams were the most divergent among the compared parasites. This is the first report of a Mikrocytos-like parasite infecting Manila clams. Care must be taken to avoid the spread of this parasite through Manila clam transfers.

The highest shellfishery catch in Galicia (NW Spain) has traditionally been cockle Cerastoderma edule. The shellfish bed located in Lombos do Ulla (Ria de Arousa) used to be among those with the highest cockle production; however, cockle mortality rate increased sharply in this bed in April 2012, reaching 100% in May 2012. Salinity and temperature were discounted as potential causes of the mortality. Marteiliosis, which was first detected in February 2012 and reached 100% prevalence in April 2012, was identified as the most probable cause. Marteiliosis had never been detected in Galician cockles, but extensive surveillance of the Galician coast in May to July 2012 detected marteiliosis in most cockle beds of the Ria de Arousa, whereas it was not found in other rias; 2 mo later, the cockle catch in the Ria de Arousa became negligible. Examination of the aetiological agent of marteiliosis with light and transmission electron microscopy supported its assignation to the genus Marteilia; morphological features showed similarity, but not complete identity, with the recently described species M. cochillia Carrasco et al., 2013. Regarding its molecular characterisation, a consensus sequence of 4433 bp containing a partial sequence of the intergenic spacer region, the complete 18S rRNA gene and a partial sequence of the first internal transcribed spacer region was obtained. The obtained sequences were compared with those available for Marteilia spp. and other Paramyxida. Molecular data support that this parasite corresponds to the species M. cochillia, and a PCR assay was designed for its specific diagnosis. The association of huge cockle mortality with M. cochillia infection urges extreme caution to avoid spreading this disease.

The present study assessed the existence of variation in the shell shape of the pod razor shell (Ensis siliqua) throughout its distributional range in the north- eastern Atlantic. Shells of E. siliqua caught at seven collecting sites (three in Portugal, three in Spain and one in Ireland) were studied by geometric morphometric methods, using both landmark- and contour-based methods. Both approaches (landmarks inside the valves and shell outline) discriminated the shells from Aveiro (centre of Portugal) and Strangford Lough (Ireland) from those caught in the nearby localities (remaining Portuguese and Spanish sites,maximum distance of 550 km by sea). Landmark analysis revealed that shells from Aveiro were more similar to shells from Ireland (*1,500 km far away). Contour anal- ysis revealed that shells from Aveiro had a shape with a comparatively larger height-to-width ratio, whereas shells from Ireland showed a slightly more curved outline than in the remaining sites. Landmark- and contour-based methods provided coherent complementary information, confirming the usefulness of geometric morphometric analyses for discerning differences in shell shape among populations of E. siliqua. A brief review of previous applications of geometric morphometric methods to modern bivalve spe- cies is also provided. The authors would like to thank Dr. Dai Roberts and Adele Cromie for providing samples of pod razor shells from Ireland. This study was funded by Community Initiative Programmes (INTERREG-IIIB, Atlantic Area) Sustainable HARvesting of Ensis (090–SHARE) and Towards Integrated Management of Ensis Stocks (206–TIMES) from the European Community. Marta M. Rufino and Paulo Vasconcelos benefited from postdoctoral grants (SFRH/BPD/14935/2004 and SFRH/BPD/26348/2006, respectively) awarded by the Fundação para a Ciência e Tecnologia (FCT—Portugal). Finally, the authors acknowledge three anonymous referees for valuable comments and suggestions that greatly improved the revised manuscript. published

Germinoma is a gonadal neoplasm originating from progenitor cells in germinal epithelium. Over the last four decades it has been diagnosed in several species of marine bivalve molluscs but most consistently in some populations of Mercenaria mercenaria and Mya arenaria in North America. Tissue sections of gonads from Ensis arcuatus (family Pharidae--superfamily Solenacea), collected in Ria de Vigo (Galicia-NW Spain), revealed germinoma in both males and females. Proliferating, undifferentiated, germ cells, with no evidence of maturation, had formed discrete masses in the walls and lumens of gonadal follicles. This is the first report of germinoma in superfamily Solenacea.

Protistan parasites of the genus Marteilia, phylum Paramyxea, cause the molluscs disease named Marteiliosis. Histological observations and transmission electron microscopy revealed the presence of life cycle stages of a Marteilia sp. in the bivalve mollusc Solen marginatus (Solenidae). Parasites occurred in epithelial cells of the digestive ducts and tubules. Early stages (primary cells) presented one or several nuclei while advances stages formed a complex of cells-within-cells (secondary and tertiary cells) culminating in spores. Refringent bodies were present inside the presporangia. This is the first report of a Marteilia sp. in S. marginatus.