Isotopic niche provides an insight into the ecology of a symbiont during its geographic expansion.

The study of the recent colonization of a symbiont and its interaction with host communities in new locations is an opportunity to understand how they interact. The use of isotopic ratios in trophic ecology can provide measurements of a species' isotopic niche, as well as knowledge about how the isotopic niches between symbiont and host species overlap. Stable isotope measurements were used to assess the sources of carbon assimilated by the host species (the bivalves Mytilus galloprovincialis and Scrobicularia plana) and their associated symbiont pea crab Afropinnotheres monodi, which occurs within these bivalves' mantle cavities. The mixing model estimates suggest that all of them assimilate carbon from similar sources, particularly from pseudofaeces and particulate organic matter in this symbiotic system based on filter feeding. The symbiotic species occupy comparable trophic levels and its association seems to be commensal or parasitic depending on the duration of such association. The pea crab A. monodi reflects a sex-specific diet, where males are more generalist than the soft females because the latter's habitat is restricted to the host bivalve. The high isotopic overlap between soft females and M. galloprovincialis may reflect a good commensal relationship with the host.

Pseudomonas baetica: pathogenicity for marine fish and development of protocols for rapid diagnosis.

Pseudomonas baetica is a pathogen isolated in 2012 from wedge sole (Dicologoglossa cuneata). The aims of this study were (i) to determine the influence of temperature on its virulence, and (ii) to develop specific protocols for rapid diagnosis. Virulence assays carried out by bath using Senegalese sole fry showed that virulence is strongly influenced by temperature: LD50 at 14°C was 8.5 × 105 cfu ml-1 while at 20°C no mortalities were recorded. On the other hand, the high mortality rates observed in virulence assays involving intraperitoneal injection of 2.5 × 105 cfu g-1 suggest that P. baetica may be pathogenic for the five fish species tested (wedge sole, Senegalese sole, sea bream, European sea bass and meagre). Two PCR protocols, using specific primers targeting the gyrB and rpoD genes, were developed for rapid diagnosis from pure cultures. An additional protocol, using both primer sets, was also optimized for detection from fish tissue samples. Specificity was tested using 81 strains from 66 bacterial species, taxonomically and/or ecologically related; only the P. baetica strains showed the expected DNA amplicons. A specific dot-blot assay using polyclonal antibodies was also developed for differentiation of P. baetica from related species. Altogether, the protocols described here will constitute useful tools for diagnosis and clarify the relevance of this pathogen.

Evidence of vertical transmission of ostreid herpesvirus 1 in the Portuguese oyster Crassostrea angulata.

In order to determine if ostreid herpesvirus 1 (OsHV-1) can be vertically transmitted, 9 full-sib families of the Portuguese oyster Crassostrea angulata were produced using a factorial mating design with 3 males and 3 females. The parents were survivors from an OsHV-1 mortality outbreak. OsHV-1 DNA was not detected by conventional PCR in the mantle of parents, gametes or 3day-old larvae. However, viral DNA was detected by real-time PCR in all gametes and larvae samples. These results show that C. angulata that have survived an OsHV-1 mortality outbreak can carry the virus and vertically transmit it to their offspring.

Experimental infection of European flat oyster Ostrea edulis with ostreid herpesvirus 1 microvar (OsHV-1µvar): Mortality, viral load and detection of viral transcripts by in situ hybridization.

Ostreid herpesvirus 1 (OsHV-1) infections have been reported in several bivalve species. Mortality of Pacific oyster Crassostrea gigas spat has increased considerably in Europe since 2008 linked to the spread of a variant of OsHV-1 called µvar. In the present study we demonstrated that O. edulis juveniles can be infected by OsHV-1µvar when administered as an intramuscular injection. Mortality in the oysters injected with OsHV-1µvar was first detected 4 days after injection and reached 25% mortality at day 10. Moreover, the high viral load observed and the detection of viral transcripts by in situ hybridization in several tissues of dying oysters suggested that OsHV-1µvar was the cause of mortality in the O. edulis juveniles. This is therefore the first study to provide evidence about the pathogenicity of OsHV-1µvar in a species that does not belong to the Crassostrea genus. Additionally, we present a novel method to detect OsHV-1 transcripts in infected individuals' using in situ hybridization.

Detection of Marteilia refringens infecting the European flat oyster Ostrea edulis and the dwarf oyster Ostrea stentina in southern Portugal and Spain.

In the present study, Marteilia sp. was detected by histological examination and in situ hybridisation in Ostrea edulis and Ostrea stentina collected in southern Iberian Peninsula. Marteilia refringens DNA was detected by PCR in O. edulis (collected in southern Portugal) and O. stentina (collected in southern Spain and Portugal). Sequencing analysis revealed the presence of M. refringens type O in O. edulis, and type O and M in O. stentina. This is the first confirmed detection of M. refringens in Portugal and the first report on the occurrence of M. refringens infecting O. stentina in Europe.

Pseudomonas baetica sp. nov., a fish pathogen isolated from wedge sole, Dicologlossa cuneata (Moreau).

Five Gram-negative bacterial isolates, recovered from an outbreak that occurred in March 2006 in Huelva, Spain, affecting adult diseased cultured wedge sole [Dicologlossa cuneata (Moreau)], were characterized phenotypically and genotypically in order to clarify their taxonomic position. On the basis of 16S rRNA gene sequence analysis, the isolates were included in the genus Pseudomonas, within the Pseudomonas fluorescens-related species group, their closest relatives being the Pseudomonas jessenii and Pseudomonas koreensis subgroups. The highest sequence similarities were recorded with the type strains of Pseudomonas reinekei, P. moorei, P. umsongensis, P. jessenii and P. mohnii (99.4-99.3 % similarity). Sequence analysis of the housekeeping genes gyrB and rpoD clearly differentiated the isolates from currently described Pseudomonas species, the highest sequence similarities recorded to type strains being below 95 % for both genes. Phylogenetic analysis using concatenated sequences of the three genes showed Pseudomonas moraviensis DSM 16007T and P. koreensis DSM 16610T as the closest reference strains. DNA-DNA hybridization assays with related strains confirmed that these isolates belong to a novel species of the genus Pseudomonas, for which the name Pseudomonas baetica sp. nov. is proposed. The type strain is strain a390T (=CECT 7720T=LMG 25716T). The novel species could be easily distinguished from phylogenetically related species by several phenotypic characteristics, including gelatin hydrolysis, acid production from glucose and growth at 6 % NaCl. Virulence assays revealed that the novel species is pathogenic for wedge sole.

Characterization of ISR region and development of a PCR assay for rapid detection of the fish pathogen Tenacibaculum soleae.

The aims of this work were to characterize the 16S-23S internal spacer region of the fish pathogen Tenacibaculum soleae and to develop a PCR assay for its identification and detection. All T. soleae strains tested displayed a single internal spacer region class, containing tRNA(I) (le) and tRNA(A) (la) genes; nevertheless, a considerable intraspecific heterogeneity was observed. However, this region proved to be useful for differentiation of T. soleae from related and non-related species. Species-specific primers were designed targeting the 16S rRNA gene and the internal spacer region region, yielding a 1555-bp fragment. Detection limit was of 1 pg DNA per reaction (< 30 bacterial cells) when using pure cultures. The detection level in the presence of DNA from fish or other bacteria was lower; however, 10 pg were detected at a target/background ratio of 1 : 10(5) . The PCR assay proved to be more sensitive than agar cultivation for the detection of T. soleae from naturally diseased fish, offering a useful tool for diagnosis and for understanding the epidemiology of this pathogen.

Identification of Vibrio harveyi isolated from diseased cultured wedge sole Dicologoglossa cuneata.

We report the first isolation of Vibrio harveyi from wedge sole Dicologoglossa cuneata. The pathogen was recovered from ulcers and internal organs of ailing cultured fish, from 7 different outbreaks between 2004 and 2006. The 15 isolates found were phenotypically characterized using biochemical tests and BIOLOG GN plates, which revealed high phenotypic diversity. Diagnosis was confirmed with PCR using V harveyi specific primers and partial 16S and 23S rRNA gene sequencing. A virulence evaluation of the isolates was also performed using fry and juvenile wedge sole. Significant mortalities were recorded by intraperitoneal injection; however, no mortalities were recorded by bath immersion.

Detection of Marteilia refringens using nested PCR and in situ hybridisation in Chamelea gallina from the Balearic Islands (Spain).

In the course of a histopathological survey performed to discover the cause of mass mortality of the striped clam Chamelea gallina in the Balearic Islands (Spain, Mediterranean Sea), we detected a Marteilia-like parasite in 3 clams. Molecular methods were applied to identify the parasite. DNA extracted from a paraffin block was used to carry out a PCR assay for Marteilia refringens detection based on a rDNA sequence of the parasite (the intergenic spacer of ribosomal genes, IGS). The nucleotide sequence of the IGS amplified fragment and the positive signal obtained by in situ hybridisation analysis with a M. refringens-specific probe allowed us to confirm the presence of this parasite in the digestive gland tissue of C. gallina.

Identification of Marteilia refringens infecting the razor clam Solen marginatus by PCR and in situ hybridization.

Marteilia refringens is a protozoan parasite recognized as a significant pathogen of the European flat oyster Ostrea edulis. It is believed to have a complex life-cycle involving several hosts. In this study, we applied molecular approaches to identify this parasite in samples of the razor clam Solen marginatus from the south west coast of Spain. We used a PCR assay to amplify a fragment of the IGS rDNA region. PCR products were sequenced and the phylogenetic affinity of the sequences was determined. In situ hybridization analysis showed tissue distribution and presence of different developmental stages of the parasite in the digestive diverticula epithelium, which suggested a true parasitism in these individuals. This is the first report of the occurrence of M. refringens in the razor clam S. marginatus in the south Atlantic. The methodology described herein may be useful for accurate identification of the parasite strain in different hosts and thus provide valuable information for marteiliosis control programmes.