Microarray-based identification of differentially expressed genes in families of turbot (Scophthalmus maximus) after infection with viral haemorrhagic septicaemia virus (VHSV).

Viral haemorrhagic septicaemia virus (VHSV) is one of the major threats to the development of the aquaculture industry worldwide. The present study was aimed to identify genes differentially expressed in several turbot (Scophthalmus maximus) families showing different mortality rates after VHSV. The expression analysis was conducted through genome-wide expression profiling with an oligo-microarray in the head kidney. A significant proportion of the variation in the gene expression profiles seemed to be explained by the genetic background, indicating that the mechanisms by which particular species and/or populations can resist a pathogen(s) are complex and multifactorial. Before the experimental infections, fish from resistant families (low mortality rates after VHSV infection) showed high expression of different antimicrobial peptides, suggesting that their pre-immune state may be stronger than fish of susceptible families (high mortality rates after VHSV infection). After infection, fish from both high- and low-mortality families showed an up-modulation of the interferon-induced Mx2 gene, the IL-8 gene and the VHSV-induced protein 5 gene compared with control groups. Low levels of several molecules secreted in the mucus were observed in high-mortality families, but different genes involved in viral entrance into target cells were down-regulated in low-mortality families. Moreover, these families also showed a strong down-modulation of marker genes related to VHSV target organs, including biochemical markers of renal dysfunction and myocardial injury. In general, the expression of different genes involved in the metabolism of sugars, lipids and proteins were decreased in both low- and high-mortality families after infection. The present study serves as an initial screen for genes of interest and provides an extensive overview of the genetic basis underlying the differences between families that are resistant or susceptible to VHSV infection.

Gene expression analysis of clams Ruditapes philippinarum and Ruditapes decussatus following bacterial infection yields molecular insights into pathogen resistance and immunity.

The carpet shell clam (Ruditapes decussatus) and Manila clam (Ruditapes philippinarum), which are cultured bivalve species with important commercial value, are affected by diseases that result in large economic losses. Because the molecular mechanism of the immune response of bivalves, especially clams, is scarce and fragmentary, we have examined all Expressed Sequence Tags (EST) resources available in public databases for these two species in order to increase our knowledge on genes related with the immune function in these animals. After automatic annotation and classification of the 3784 not-annotated ESTs of R. decussatus and 4607 of R. philippinarum found in GenBank, 424 ESTs of R. decussatus and 464 of R. philippinarum were found to be putatively involved in immune response. These were carefully reviewed and reannotated. As a result, 13 immune-related ESTs were selected and studied to compare the immune response of R. decussatus and R. philippinarum following a Vibrio alginolyticus challenge. Quantitative PCR was performed, and the expression of each EST was determined. The results showed that, in R. philippinarum, the immune response seems to be faster than that in R. decussatus. Additionally, expression of NF-κB activating genes in R. decussatus did not seem to be sufficient to promote an immune response after Vibrio infection. R. philippinarum, however, was able to trigger and efficiently regulate the transcriptional activity of NF-κB, even when low expression values were reported.

Morphological characterization and functional immune response of the carpet shell clam (Ruditapes decussatus) haemocytes after bacterial stimulation.

The morphology and functionality of Ruditapes decussatus haemocytes have been characterized by light microscopy and flow cytometry, leading to the identification of three different cellular subpopulations. Granulocytes were the largest cells, the hyalinocytes were smaller and contained fewer granules and the intermediate cells showed a size similar to hyalinocytes and a higher number of granules. The phagocytosis of different particles and the associated production of oxygen radicals were measured by flow cytometric methods. Granulocytes were the most active cells, followed by the intermediate cells and hyalinocytes. The effect of stimulation of haemocytes with lipopolysaccharide (LPS), with a heat inactivated bacterial mixture or with the infection of Vibrio splendidus on the cell viability and the expression of selected immune-related genes were studied. While significant low levels of damaged cells were registered in LPS-stimulated cells, the treatment with dead bacteria or V. splendidus reduced cell viability 1 h, 3 h and 6 h after treatment. The stimulation of haemocytes with LPS and dead bacteria induced changes in the expression of defender against cell death (DAD-1), thrombin, prosaposin, inhibitor of apoptosis (IAP), factor B and C3 complement component.

Molecular detection of a haplosporidian parasite in carpet shell clam Ruditapes decussatus from Spain.

A protozoan parasite identified by histology as a haplosporidian was detected in carpet shell clam Ruditapes decussatus from Spain during routine samplings. Analysis based on the sequence of the small subunit ribosomal RNA gene revealed that this organism was related to the haplosporidian group, mainly to Urosporidium. A specific probe was used for in situ hybridization studies to show the specificity of the amplified sequence.