Elvira Abollo

Genetic evidence for the existence of sibling species within Contracaecum rudolphii (Hartwich, 1964) and the validity of Contracaecum septentrionale (Kreis, 1955) (Nematoda: Anisakidae)

Specimens of Contracaecum rudolphii sensu lato (s.l.) (Nematoda: Anisakidae) from Phalacrocorax carbo sinensis from northeastern and central Italy were characterised genetically and compared with those from Phalacrocorax aristotelis from Galician coasts, Spain (identified as C. rudolphii A by multilocus enzyme electrophoresis) and with specimens of C. septentrionale from Alca torda from the Galician coasts, Spain. The first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of ribosomal DNA (rDNA) were amplified by polymerase chain reaction (PCR) from individual nematodes and the amplicons subjected to single-strand conformation polymorphism (SSCP) analysis and/or sequencing. For each ITS region, C. septentrionale specimens were distinct from those of C. rudolphii (s.l.) and C. rudolphii A based on SSCP profiles and ITS sequences. Some specimens of C. rudolphii (s.l.) had the same SSCP profiles and ITS sequences as C. rudolphii A, whereas the others had distinct SSCP profiles and ITS sequences and were suggested to represent C. rudolphii B based on host and geographical origins and genetic similarity to C. rudolphii A. While no length or nucleotide variation in the ITS-1 and ITS-2 sequences was detected within each taxon, nucleotide differences of 1.8–5.5% (ITS-1) and 5.1–12.2% (ITS-2) were detected among them. The results support the hypothesis that C. rudolphii represents a complex of at least two sibling species and provide support for the validity of C. septentrionale as a separate species. The definition of genetic markers in the ITS rDNA provides opportunities for investigating the life cycles, transmission patterns and ecology of the anisakid nematodes studied herein.

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.

Cockle Cerastoderma edule fishery collapse in the Ría de Arousa (Galicia, NW Spain) associated with the protistan parasite Marteilia cochillia.

The highest shellfishery catch in Galicia (NW Spain) has traditionally been cockle Cerastoderma edule. The shellfish bed located in Lombos do Ulla (Ría 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 Ría de Arousa, whereas it was not found in other rías; 2 mo later, the cockle catch in the Ría 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.

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.

Nyctiphanes couchii as intermediate host for the acanthocephalan Bolbosoma balaenae in temperate waters of the NE Atlantic

Cystacanths of the acanthocephalan Bolbosoma balaenae (Gmelin, 1790) were found encapsulated in the cephalothorax of the euphausiid Nyctiphanes couchii (Bell, 1853) from temperate waters in the NE Atlantic Ocean. Euphausiids were caught in locations outside the Ría de Vigo in Galicia, NW Spain, and prevalence of infection was up to 0.1%. The parasite was identified by morphological characters. Cystacanths were 8.09 ± 2.25 mm total length (mean ± SD) and had proboscises that consisted of 22 to 24 longitudinal rows of hooks, each of which had 8 or 9 hooks per row including 2 or 3 rootless ones in the proboscis base and 1 field of small hooks in the prebulbar part. Phylogenetic analyses of 18S rDNA and cytocrome c oxidase subunit I revealed a close relationship with other taxa of the family Polymorphidae (Meyer, 1931). The results extend northwards ot the known distribution of B. balaenae. Taxonomic affiliation of parasites and trophic ecology in the sampling area suggest that N. couchii is the intermediate host for B. balenae, and we suggest that the whales Balaenoptera physalus (Linnaeus, 1758) and B. acutorostrata (Lacepède, 1804) are its definitive hosts. This life cycle is probably completed with or without paratenic hosts.

Nyctiphanes couchii as intermediate host for Rhadinorhynchus sp. (Acanthocephala, Echinorhynchidae) from NW Iberian Peninsula waters

In the mesozooplanktonic community of the coastal upwelling system of the Ría de Vigo (NW Spain), the euphausiid Nyctiphanes couchii has been identified for the first time in temperate waters of the NE Atlantic as the intermediate host for cystacanths of Rhadinorhynchus sp. Parasites were identified using morphological characters described in 20 cystacanths. The hooks of the proboscis were arranged in 14 rows of 26 hooks each, while the hooks of the basal circle were only slightly erected and were longer than remaining spines. A maximum-likelihood estimation (ML) tree inferred from the 18S rRNA data set of Palaeacantocephala revealed that our specimens belong to a highly supported clade with Rhadinorhynchus sp., Pararhadinorhynchus sp. and Transvena annulospinosa. Nonetheless, our morphological and phylogenetic analyses suggested that the status of Rhadinorhynchus pristis should be re-examined. The prevalences of parasites were 0.0019% and 0.0001% for frontal and coastal summer communities, and 0.0068% and 0.0008% for coastal and oceanic autumn communities, respectively. The presence of these cystacanths in different mesozooplankton communities throughout the study suggests that the recruitment of parasites may be affected by the oceanography.

Oyster parasites Bonamia ostreae and B. exitiosa co-occur in Galicia (NW Spain): spatial distribution and infection dynamics

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.

Molecular identification of Anisakis and Hysterothylacium larvae in commercial cephalopods from the Spanish Mediterranean coast.

This study aims to investigate the occurrence of nematode larvae in commercial cephalopods in the Western Mediterranean Sea. A total of 202 animals comprising 123 broadtail shortfin squid (Illex coindetii), 34 European squid (Loligo vulgaris) and 45 common octopus (Octopus vulgaris) were examined using enzymatic digestion. A total of 31 larvae were isolated (prevalence: 14.6%) and identified using molecular analyses which included PCR and sequencing of the ITS (ITS1-5.8S rDNA-ITS2) region. Phylogenetic tree inferred from ITS sequences yielded supported relationships for Anisakis (P: 12.2%) and Hysterothylacium species (P: 4.1%). All parasites were found parasitizing I. coindetii and, as expected, A. pegreffii presented the highest prevalence (11.4%). A. physeteris was also found with a lower prevalence (1.6%) but confirming the role of the broadtail shortfin squid as paratenic host and, its potential host for anisakidosis transmission. A hybrid larva between Anisakis simplex and A. pegreffi was also identified. All Anisakis larvae were found within the visceral cavity; in contrast most of the Hysterothylacium larvae were isolated from the mantle. A significant correlation was found between total nematode prevalence and depth, explained by the presence of larger broadtail shortfin squids inhabiting deeper depths. Therefore, the results obtained in the present study improve the knowledge of the occurrence of Anisakis and Hysterothylacium species in the I. coindetii from the Spanish Mediterranean Sea highlighting the importance of considering I. coindetii as a potential hazard for humans if it is consumed raw or not well cooked and the need of further research in other cephalopods.

Species-specific oligonucleotide probe for detection of Bonamia exitiosa (Haplosporidia) using in situ hybridisation assay.

Bonamiosis is a disease affecting various oyster species and causing oyster mass mortalities worldwide. The protozoans Bonamia exitiosa and B. ostreae (Haplosporidia) are included in the list of notifiable diseases of the World Organisation for Animal Health as the causative agents of this disease. Although the geographic range of both species was considered different for years, both species are now known to co-occur in some European areas affecting the same host, Ostrea edulis, which strengthens the need of species-specific methods to unequivocally identify the species of Bonamia. An oligonucleotide probe for specific detection of B. exitiosa (BEX_ITS) was designed to be used in in situ hybridisation (ISH) assays. ISH assay with BEX_ITS probe showed species-specificity and more sensitivity than traditional histology to visualise the parasite inside host tissue. ISH assay showed that the oyster gonad was the area where the parasite was most frequently located, and was the exclusive organ of infection in some oysters. A recommendation arising from the study is that more than 1 organ (including gonad and gills) should be used for PCR-based diagnosis of B. exitiosa, to maximise the sensitivity.

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.