The immune response of the scallop Argopecten purpuratus is associated with changes in the host microbiota structure and diversity.

All organisms live in close association with a variety of microorganisms called microbiota. Furthermore, several studies support a fundamental role of the microbiota on the host health and homeostasis. In this context, the aim of this work was to determine the structure and diversity of the microbiota associated with the scallop Argopecten purpuratus, and to assess changes in community composition and diversity during the host immune response. To do this, adult scallops were immune challenged and sampled after 24 and 48 h. Activation of the immune response was established by transcript overexpression of several scallop immune response genes in hemocytes and gills, and confirmed by protein detection of the antimicrobial peptide big defensin in gills of Vibrio-injected scallops at 24 h post-challenge. Then, the major bacterial community profile present in individual scallops was assessed by denaturing gradient gel electrophoresis (DGGE) of 16S rDNA genes and dendrogram analyses, which indicated a clear clade differentiation of the bacterial communities noticeable at 48 h post-challenge. Finally, the microbiota structure and diversity from pools of scallops were characterized using 16S deep amplicon sequencing. The results revealed an overall modulation of the microbiota abundance and diversity according to scallop immune status, allowing for prediction of some changes in the functional potential of the microbial community. Overall, the present study showed that changes in the structure and diversity of bacterial communities associated with the scallop A. purpuratus are detected after the activation of the host immune response. Now, the relevance of microbial balance disruption in the immune capacity of the scallop remains to be elucidated.

Identification and characterization of two variants of the Hfq-sRNA-chaperone in the fish pathogen Piscirickettsia salmonis.

Small RNA and chaperone proteins form synergistic duos that play pivotal roles in controlling gene expression in bacteria. This is the case for Hfq, a highly pleiotropic pretranslational modulator of general protein expression, which responds to harsh environmental conditions and influences fitness and virulence in a wide range of pathogenic Enterobacteria. Given this relevancy, we evaluated the presence and potential role of Hfq in the fish pathogen Piscirickettsia salmonis, a Gram-negative bacterium that threatens the sustainability of Chilean salmon production. Using bioinformatics tools were identified and characterized two variants of Hfq, which share the consensus RNA-binding domains and the active sites described functional Hfq other bacteria. Additionally, we demonstrated that hfq-1 and hfq-2 were transcriptionally active when growing in cell-free media and in infected susceptible fish cell line. Expression of both genes differed under different growth conditions and under stress, suggesting that their roles might be independent and different, depending on the bacterial physiological status. In conclusion, we demonstrate the existence of two different and functional ORF coding for the hfq marker in marine bacteria and a preliminary analysis indicating that these two novel proteins might have relevant roles in the biology and pathogenic potential of P. salmonis.

Molecular characterization of an inhibitor of NF-κB in the scallop Argopecten purpuratus: First insights into its role on antimicrobial peptide regulation in a mollusk.

Inhibitors of nuclear factor kappa B (IκBs) are major control components of the Rel/NF-κB signaling pathway, a key regulator in the modulation of the expression of immune-related genes in vertebrates and invertebrates. The activation of the Rel/NF-κB signaling pathway depends largely in the degradation of IκB proteins and thus, IκBs are a main target for the identification of genes whose expression is controlled by Rel/NF-κB pathway. In order to identify such regulation in bivalve mollusks, the cDNA sequence encoding an IκB protein was characterized in the scallop Argopecten purpuratus, ApIκB. The cDNA sequence of ApIκB is comprised of 1480 nucleotides with a 1086 bp open reading frame encoding for 362 amino acids. Bioinformatics analysis showed that ApIκB displays the conserved features of IκB proteins. The deduced amino acid sequence consists of a 39.7 kDa protein, which has an N-terminal degradation motif, six ankyrin repeats and a C-terminal phosphorylation site motif. Phylogenetic analysis revealed a high degree of identity between ApIκB and other IκBs from mollusks, but also to arthropod cactus proteins and vertebrate IκBs. Tissue expression analysis indicated that ApIκB is expressed in all examined tissues and it is upregulated in circulating hemocytes from scallops challenged with the pathogenic Gram-negative bacterium Vibrio splendidus. After inhibiting ApIκB gene expression using the RNA interference technology, the gene expression of the antimicrobial peptide big defensin was upregulated in hemocytes from non-challenged scallops. Results suggest that ApIκB may control the expression of antimicrobial effectors such as big defensin via a putative Rel/NF-κB signaling pathway. This first evidence will help to deepen the knowledge of the Rel/NF-κB conserved pathway in scallops.