Toll signal transduction pathway in bivalves: complete cds of intermediate elements and related gene transcription levels in hemocytes of immune stimulated Mytilus galloprovincialis.

Based on protein domain structure and organization deduced from mRNA contigs, 15 transcripts of the Toll signaling pathway have been identified in the bivalve, Mytilus galloprovincialis. Identical searches performed on publicly available Mytilus edulis ESTs revealed 11 transcripts, whereas searches performed in genomic and new transcriptome sequences of the Pacific oyster, Crassostrea gigas, identified 21 Toll-related transcripts. The remarkable molecular diversity of TRAF and IKK coding sequences of C.

Toll-like receptors and MyD88 adaptors in Mytilus: complete cds and gene expression levels.

TLR- and MyD88-related sequences have been previously investigated in Mytibase and then in new transcript reads obtained by Illumina technology from the mussel, Mytilus galloprovincialis. Based on full cds and domain organizations of virtual translations, we identified 23 Toll-like receptors (TLRs) and 3 MyD88 adaptors. MgTLRs can be arranged in 4 clusters according to extra-cellular LRR domain content.

Individual variability of mytimycin gene expression in mussel.

The antifungal peptide mytimycin (MytM) is synthesized by hemocytes of the Mediterranean mussel, Mytilus galloprovincialis. In addition to sequence and gene structure diversities previously reported from pooled hemocytes, the present report focused on the expression of mytm gene in individual M. galloprovincialis, before and after challenge.

Massively parallel amplicon sequencing reveals isotype-specific variability of antimicrobial peptide transcripts in Mytilus galloprovincialis.

Background: Effective innate responses against potential pathogens are essential in the living world and possibly contributed to the evolutionary success of invertebrates. Taken together, antimicrobial peptide (AMP) precursors of defensin, mytilin, myticin and mytimycin can represent about 40% of the hemocyte transcriptome in mussels injected with viral-like and bacterial preparations, and unique profiles of myticin C variants are expressed in single mussels. Based on amplicon pyrosequencing, we have ascertained and compared the natural and Vibrio-induced diversity of AMP transcripts in mussel hemocytes from three European regions.

MIF from mussel: coding sequence, phylogeny, polymorphism, 3D model and regulation of expression.

Three macrophage migration inhibitory factor (MIF)-related sequences were identified from a Mytilus galloprovincialis EST library. The consensus sequence included a 5'-UTR of 32 nucleotides, the complete ORF of 345 nucleotides, and a 3'-UTR of 349 nucleotides. As for other MIFs, M.

Gene expression specificity of the mussel antifungal mytimycin (MytM).

We previously reported the nucleotide sequences and diversity of mytimycin (MytM) from the Mediterranean mussel, Mytilus galloprovincialis. Using real-time PCR (q-PCR), we observed that the MytM gene was mainly expressed in circulating hemocytes and to a less extent in the mantle. In vivo challenge with bacteria or with the yeast, Candida albicans, did not increase the expression as measured by q-PCR in hemocytes.

Insights into the innate immunity of the Mediterranean mussel Mytilus galloprovincialis.

Background: Sessile bivalves of the genus Mytilus are suspension feeders relatively tolerant to a wide range of environmental changes, used as sentinels in ecotoxicological investigations and marketed worldwide as seafood. Mortality events caused by infective agents and parasites apparently occur less in mussels than in other bivalves but the molecular basis of such evidence is unknown. The arrangement of Mytibase, interactive catalogue of 7,112 transcripts of M.

Diversity of coding sequences and gene structures of the antifungal peptide mytimycin (MytM) from the Mediterranean mussel, Mytilus galloprovincialis.

Knowledge on antifungal biomolecules is limited compared to antibacterial peptides. A strictly antifungal peptide from the blue mussel, Mytilus edulis named mytimycin (MytM) was reported in 1996 as partial NH(2) 33 amino acid sequence. Using back-translations of the previous sequence, MytM-related nucleotide sequences were identified from a normalized Mytilus galloprovincialis expressed sequence tag library.

Effects of vibrio challenge on digestive gland biomarkers and antioxidant gene expression in Mytilus galloprovincialis.

In bivalve molluscs, responses to bacterial infection have been largely characterized in terms of both functional responses and gene expression in the immune cells, the hemocytes. The effects of bacterial challenge at the tissue level, where bacterial infection may cause stressful conditions, have not been so far specifically investigated. Biomarkers are widely utilised to evaluate the health status of bivalves, from the molecular to the organism level, in response to both natural and anthropogenic stressors.

Specificity of anti-Vibrio immune response through p38 MAPK and PKC activation in the hemocytes of the mussel Mytilus galloprovincialis.

In mussel (Mytilus sp.) hemocytes, differential functional responses to injection with different types of live and heat-killed Vibrio species have been recently demonstrated. In this work, responses of Mytilus hemocytes to heat-killed Vibrio splendidus LGP32 and the mechanisms involved were investigated in vitro and the results were compared with those obtained with Vibrio anguillarum (ATCC 19264).

Expression of Mytilus immune genes in response to experimental challenges varied according to the site of collection.

Mussels live in diverse coastal environments experience various physical, chemical and biological conditions, which they counteract with functional adjustments and heritable adaptive changes. In order to investigate possible differences in immune system capabilities, we analyzed by qPCR the expression levels of 4 immune genes (defensin, mytilin B, myticin B, lysozyme) and HSP70 in the Mediterranean mussel, Mytilus galloprovincialis collected in 3 European farming areas {Atlantic Ocean-Ría de Vigo-Spain (RV), French Mediterranean Gulf of Lion-Palavas-Prévost lagoon (PP) and Northern Adriatic Sea-Venice-Italy (VI)} in response to one injection of one of the 3 bacterial species (Vibrio splendidus LGP32, Vibrio anguillarum, Micrococcus lysodeikticus), and to heat shock or cold stress. We confirmed that the 5 genes are constitutively expressed in hemocytes, defensin being the less expressed, myticin B the highest.

Influence of temperature, salinity and E. coli tissue content on immune gene expression in mussel: results from a 2005-2008 survey.

Several bivalves, including mussels, suffered from mortalities particularly in summer. To look for the possible effect of environmental parameters on immune capacities, Mytilus galloprovincialis were collected monthly from August 2005 to July 2008 from the Palavas Laguna, French Mediterranean coast. Q-PCR was used to quantify the expression of three antimicrobial peptide genes (defensin, mytilin B and myticin B), in addition to lysozyme and HSP70.

Functional differential immune responses of Mytilus galloprovincialis to bacterial challenge.

Bivalves are filter-feeders that can accumulate large numbers of bacteria, in particular Vibrio species; these can persist within bivalve tissues largely depending on their sensitivity to the hemolymph bactericidal activity. In this work, functional parameters of the hemolymph of Mytilus galloprovincialis were evaluated in response to in vivo challenge with different bacteria (Gram(-) Vibrio anguillarum and V. splendidus, Gram+ Micrococcus lysodeikticus).

MytiBase: a knowledgebase of mussel (M. galloprovincialis) transcribed sequences.

Background: Although bivalves are among the most studied marine organisms due to their ecological role, economic importance and use in pollution biomonitoring, very little information is available on the genome sequences of mussels. This study reports the functional analysis of a large-scale Expressed Sequence Tag (EST) sequencing from different tissues of Mytilus galloprovincialis (the Mediterranean mussel) challenged with toxic pollutants, temperature and potentially pathogenic bacteria.

Results: We have constructed and sequenced seventeen cDNA libraries from different Mediterranean mussel tissues: gills, digestive gland, foot, anterior and posterior adductor muscle, mantle and haemocytes.

Polymorphism of mytilin B mRNA is not translated into mature peptide.

Diversity of mRNAs from mytilin B, one of the five mytilins identified in the Mediterranean mussel, Mytilus galloprovincialis, has been investigated from circulating hemocytes. One mussel expressed simultaneously two to ten different mytilin B mRNAs as observed in denaturing gradient gel electrophoresis (DGGE), defining 10 individual DGGE patterns (named A to J) within the mussels from Messina, Sicily (Italy). Three patterns accounted for 79% of the individuals whereas other patterns were found in only 2-7% of the 57 analyzed mussels.

Differential involvement of mussel hemocyte sub-populations in the clearance of bacteria.

Mussels are filter-feeders living in a bacteria-rich environment. We have previously found that numerous bacterial species are naturally present within the cell-free hemolymph, including several of the Vibrio genus, whereas the intra-cellular content of hemocytes was sterile. When bacteria were injected into the circulation of the mussel, the number of living intra-hemocyte bacteria dramatically increased in less than an hour, suggesting intense phagocytosis, then gradually decreased, with no viable bacteria remaining 12h post-injection for Micrococcus lysodeikticus, 24h for Vibrio splendidus and more than 48 h for Vibrio anguillarum.

Lysozyme gene expression and hemocyte behaviour in the Mediterranean mussel, Mytilus galloprovincialis, after injection of various bacteria or temperature stresses.

The aim of the present study was to evaluate the expression of the Mytilus galloprovincialis lysozyme gene in different in vivo stress situations, including injection of bacteria Vibrio splendidus LGP32, Vibrio anguillarum or Micrococcus lysodeikticus, as well as heat shock at 30 degrees C and cold stress at 5 degrees C. Injection of V. splendidus LGP32 resulted in: (i) a general down-regulation of lysozyme gene expression, as quantified by Q-PCR; (ii) reduction in the number of circulating hemocytes; (iii) decrease in the percentage of circulating hemocytes expressing lysozyme mRNA which was now restricted to only small cells, as observed by ISH; and (iv) accumulation of hemocytes expressing lysozyme in the muscle sinus where injection took place.

NMR structure of mussel mytilin, and antiviral-antibacterial activities of derived synthetic peptides.

Mytilin is a 34-residue antibacterial peptide from the mussel Mytilus galloprovincialis, which in addition possesses in vitro antiviral activity. The three-dimensional solution structure of the synthetic mytilin was established by using 1H NMR and consists of the common cysteine-stabilized alphabeta motif close to the one observed in the mussel defensin MGD-1. Mytilin is characterized by 8 cysteines engaged in four disulfide bonds (2-27, 6-29, 10-31, and 15-34) only involving the beta-strand II.

cDNA sequence and tissue expression of an antimicrobial peptide, dicentracin; a new component of the moronecidin family isolated from head kidney leukocytes of sea bass, Dicentrarchus labrax.

A 483-bp cDNA was isolated from sea bass (Dicentrarchus labrax) head kidney leukocytes, dicentracin, using PCR primers designed from conserved moronecidin domains. Gene bank analysis revealed that dicentracin cDNA belongs to the moronecidin family. As deduced from alignment with Morone chrysops moronecidin, the precursor of 79 aa appeared to be composed of a signal peptide of 22 aa, followed by the mature AMP (antimicrobial peptide) of 22 aa named dicentracin, and a C-terminal extension of 35 aa.

Deep sea immunity: unveiling immune constituents from the hydrothermal vent mussel Bathymodiolus azoricus.

Marine molluscs are subjected to constant microbial threats in their natural habitats. As a result, they represent suitable models for the study of the molecular mechanisms that govern defense reactions in marine organisms. To understand humoral and cellular defense reactions in animals defying extreme physical and chemical conditions we set out to investigate the deep sea hydrothermal vent mussel Bathymodiolus azoricus found in abundance at the Mid-Atlantic Ridge.

Specific expression of antimicrobial peptide and HSP70 genes in response to heat-shock and several bacterial challenges in mussels.

Defensin, mytilin and myticin are antimicrobial peptides (AMP) involved in mussel innate immunity. Their in vitro antibacterial activity is different according to the targeted bacterial species. To determine if this specificity is correlated to different regulations of gene expressions, adult mussels were challenged in vivo with either Vibrio splendidus LGP32, Vibrio anguillarum, Micrococcus lysodeikticus or by heat shock.

HSP70 gene expression in Mytilus galloprovincialis hemocytes is triggered by moderate heat shock and Vibrio anguillarum, but not by V. splendidus or Micrococcus lysodeikticus.

Complete sequence of HSP70 cDNA from the mussel, Mytilus galloprovincialis was established before quantifying its expression following moderate heat shock or injection of heat-killed bacteria. HSP70 cDNA is comprised of 2378 bp including one ORF of 654 aa, with a predicted 70 bp 5'-UTR and a 343 bp 3'-UTR (GenBank, 18 Jan 05, AY861684). Alignment identity ranged from 89% for Crassostrea ariakensis to 72% for C.

Antiprotozoan and Antiviral Activities of Non-cytotoxic Truncated and Variant Analogues of Mussel Defensin.

We previously reported the crucial role displayed by loop 3 of defensin isolated from the Mediterranean mussel, Mytilus galloprovincialis, in antibacterial and antifungal activities. We now investigated antiprotozoan and antiviral activities of some previously reported fragments B, D, E, P and Q. Two fragments (D and P) efficiently killed Trypanosoma brucei (ID(50) 4-12 μM) and Leishmania major (ID(50) 12-45 μM) in a time/dose-dependent manner.

Key role of the loop connecting the two beta strands of mussel defensin in its antimicrobial activity.

To elucidate the structural features of the mussel defensin MGD1 required for antimicrobial activity, we synthesized a series of peptides corresponding to the main known secondary structures of the molecule and evaluated their activity towards Gram-positive and Gram-negative bacteria, and filamentous fungi. We found that the nonapeptide corresponding to residues 25-33 of MGD1 (CGGWHRLRC) exhibited bacteriostatic activity once it was cyclized by a non-naturally occurring disulfide bridge. Longer peptides corresponding to the amino acid sequences of the alpha-helical part or to the beta-strands of MGD1 had no detectable activity.