Deep transcriptome sequencing of Pecten maximus hemocytes: a genomic resource for bivalve immunology.

Pecten maximus, the king scallop, is a bivalve species with important commercial value for both fisheries and aquaculture, traditionally consumed in several European countries. Major problems in larval rearing, however, still limit hatchery-based seed production. High mortalities during early larval stages, likely related to bacterial pathogens, represent the most relevant bottleneck. To address this issue, understanding host defense mechanisms against microbes is extremely important. In this study next-generation RNA-sequencing was carried on scallop hemocytes. To enrich for immune-related transcripts, cDNA libraries from hemocytes challenged in vivo with inactivated-Vibrio anguillarum and in vitro with pathogen-associated molecular patterns, as well as unchallenged controls, were sequenced yielding 216,444,674 sequence reads. De novo assembly of the scallop hemocyte transcriptome consisted of 73,732 contigs (31% annotated). A total of 934 contigs encoded proteins with a known immune function, grouped into several functional categories. Particular attention was reserved to Toll-like receptors (TLRs), a family of pattern recognition receptors (PRRs) involved in non-self recognition. Through mining the scallop hemocyte transcriptome, at least four TLRs could be identified. The organization of canonical TLR domains demonstrated that single cysteine cluster and multiple cysteine cluster TLRs co-exist in this species. In addition, preliminary data concerning their mRNA level following bacterial challenge suggested that different members of this family could exhibit opposite responses to pathogenic stimuli. Finally, a global analysis of differential expression comparing gene-expression levels in in vitro and in vivo stimulated hemocytes against controls provided evidence on a large set of transcripts involved in the great scallop immune response.

Functional immune response in Pecten maximus: combined effects of a pathogen-associated molecular pattern and PAH exposure.

Pathogen-associated molecular patterns (PAMPs) enable recognition of structures present in microorganisms such as lipopolysaccharides (LPS). LPS are an essential constituent of the outer membrane of Gram-negative bacteria, stimulating the innate immune system of invertebrates. Here, LPS from Escherichia coli (055:B5) were used to investigate the functional immune response of Pecten maximus after stimulation with a PAMP and to determine the combined effect of a phenanthrene exposure and LPS challenge. Organisms were exposed to 200 mug l(-1) phenanthrene and after 7 d were injected with either physiological saline (injection controls) or LPS solution, and returned to their respective exposure tanks. Haemolymph was sampled from the scallops 48 h post-injection and immune function was assessed using a combination of cellular biological responses. The LPS challenge significantly altered the immune response in P. maximus with increased cell counts and phagocytic activity. An immunosuppressive effect of phenanthrene was also observed in this study; however, exposure to phenanthrene did not significantly impair the organism's ability to respond to a PAMP challenge. The overall level of phagocytosis and cytotoxic capability following the LPS challenge was lower in phenanthrene exposed scallops and may have consequences for disease resistance in this commercially-exploited species.

Immunohistochemistry of great scallop Pecten maximus larvae experimentally challenged with pathogenic bacteria.

Three challenge experiments were carried out on larvae of the great scallop Pecten maximus. Larvae were bath-challenged with Vibrio pectenicida and 5 strains resembling Vibrio splendidus and one Pseudoalteromonas sp. Unchallenged larvae were used as negative controls. The challenge protocol was based on the use of a multidish system, where the scallop larvae (10, 13 and 15 d post-hatching in the 3 experiments, respectively) were distributed to 2 ml wells with stagnant seawater and exposed to the bacterial cultures by bath challenge. Presence of the challenge bacteria in the wells was verified by polymerase chain reaction (PCR). A significantly increased mortality was found between 24 and 48 h in most groups challenged with V. pectenicida or V. splendidus-like strains. The exception was found in larval groups challenged with a Pseudoalteromonas sp. LT 13, in which the mortality rate fell in 2 of the 3 challenge experiments. Larvae from the challenge experiments were studied by immunohistochemistry protocol. Examinations of larval groups challenged with V. pectenicida revealed no bacterial cells, despite a high degree of positive immunostaining. In contrast, intact bacterial cells were found in larvae challenged with V. splendidus. In the case of larvae challenged with the Pseudoalteromonas sp., positive immuno-staining was limited to visible bacteria inside the digestive area and cells of the mucosa. The experiments confirm that V. splendidus and V. pectenicida are pathogenic to scallop larvae, and that the Pseudoalteromonas strain is probably not a primary pathogen, although it cannot be ruled out as a secondary pathogen.