Ciona robusta hemocyte populational dynamics and PO-dependent cytotoxic activity.

Hemocyte populations from the ascidian Ciona robusta, separated through a Percoll discontinuous density gradient, are further characterized by May-Grünwald-Giemsa staining and a cytochemical reaction for phenoloxidase. Variability in cell density, acidophilic property and phenoloxidase activity suggest multiple hemocyte type populations, cell lineages and morphotypes that may be involved in distinct cellular responses. Therefore, unilocular refractile granulocytes, typical of this ascidian species, enriched in a fraction separated from the hemolymph show in vitro phenoloxidase-dependent cytotoxic activity against mammalian erythrocytes and a tumor cell lineage, in addition the properties listed above indicate relationships with vacuolated signet ring cells. Finally, bromo-deoxyuridine with, diamino-phenylindole fluorescent reaction and May-Grünwald-Giemsa staining show that in the hemolymph there are hyaline amoebocytes and granulocytes with potential proliferating activity. Present findings and reviewed images of previously reported inflammatory hemocytes in the tunic and pharynx allow us to speculate on theoretical outlines of hemocyte differentiation pathways.

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. Within untreated mussel, MytM mRNA was observed by ISH in about 42% of circulating hemocytes, characterized by large, diffuse nucleus. Injection with Fusarium oxysporum increased such percentage, but in only some of the mussels. Similarly, MytM gene expression increased after injection in only some of the mussels, as measured by qPCR. Responders and not responders are common evidence in any given population of organisms. Nevertheless, even if the use of proper pool size selection has been practised to find out and evaluate the most common response trends, individual analyses must be regarded as optimal.

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. By contrast, injection of the filamentous fungus, Fusarium oxysporum, induced a sudden and strong increase of expression at 9h p.i. (stimulation index of 25.7 ± 2.1). Optimum stimulating dose was 10(4) spores of F. oxysporum per mussel. In the same samples, AMP mytilin and myticin showed no stimulation. Consequently, we hypothesized the existence of 2 different signal transduction pathways, one activated by bacteria and yeast, the other triggered by filamentous fungi. A second challenge performed with F. oxysporum 24 h after the first challenge induced an increase of MytM gene expression (stimulation index of 3.5 ± 1.7). However, this second increase was significantly lower than the first, suggesting less efficient response rather than significant protection.

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. galloprovincialis ORF does not include any signal or C-terminus extensions. The translated sequence of 115 amino acids possesses a molecular mass of 12,681.4, a pI of 6.27 and a stability index of 21.48. Its 3D structure resembles human MIF except for one shorter α-helix. Although evolutionary separated from ticks and vertebrates, Mg-MIF appeared to be closely related to Pinctada fucata and Haliotis, but not to Chlamys farreri and Biomphalaria glabrata. Numerous mutation points were observed within the Mg-MIF ORF, defining 11 amino acid variants within the mussels from Palavas-France and 14 amino acid variants within the mussels from Palermo-Italy. The 2 major variants from Palavas were identical to 2 of the 4 major variants from Palermo. In all the 18 Mg-MIF variants, residues involved in tautomerase and in oxidoreductase activities were conserved. Generally, one mussel expressed 2 Mg-MIF amino acid sequences but with different frequencies of occurrence. Mg-MIF is constitutively expressed principally in hemocytes and in the mantle. In contrast to other animal models, Mg-MIF expression was always down regulated following challenge by bacteria and fungi, confirming previous data obtained with microarray. Down regulation started as soon as 1 h and Mg-MIF expression returned to background 9-48 h after the challenge. Exception was regarding the yeast, Candidaalbicans, down-regulation between 9 and 72 h, suggesting yeast and bacteria-filamentous fungi trigger different mechanisms of elimination.

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. Base mutations were observed at specific locations, mainly within COOH-terminus and 3'UTR, leading to 36 nucleotide sequence variants and 21 different coding sequences (cds) segregating in two different clusters. Most of the base mutations were silent, and the number of pro-peptide variants was restricted to four. Finally, as the two amino acid replacements occurred within COOH-terminus, mature peptide from mytilin B appeared unique. Multiple sequencing of partial mytilin B gene from one mussel revealed that one to four randomly distributed mutation points occurred within intron-3. Only one sequence out of the 91 analyzed contained 16 mutation points. In addition, this sequence was the only one containing four out of the six mutation points occurring within exon-4, that code for most of the COOH-terminus domain, including the unique amino acid replacement. Statistical tests for neutrality indicated negative selection pressure on signal and mature peptide domains, but possible positive selection pressure for COOH-terminus domain.

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. The total hemocyte count (THC) was dramatically lowered by the bacterial injections, as quantified by flow cytometry. V. splendidus induced the strongest decreases with -66% 9h post-injection of living bacteria and -56% 3h post-injection of heat-killed bacteria. Flow cytometry was used to identify three main sub-populations of hemocytes, namely hyalinocytes, small granulocytes and large granulocytes. When THC was minimal, i.e. within the first 9h post-injection, proportions of the three cell categories varied dramatically, suggesting differential involvement according to the targets, but small granulocytes remained the majority. According to a decrease in their number followed by an increase (+90% at 12h with living V. splendidus), hyalinocytes also appeared to be involved as cellular effectors of antibacterial immunity, despite possessing little capacity for phagocytosis and not containing antimicrobial peptides.

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. Injection of V. anguillarum or M. lysodeikticus induced significant up-regulation of lysozyme gene expression, but only 2-3days post-injection, with no change in the total hemocyte counts but an increased percentage of hemocytes expressing lysozyme mRNA. Neither the control injection of PBS-NaCl nor temperature stress modified the lysozyme expression pattern. Consequently, the hemocyte population appears to be capable of discriminating between stress factors, and even between 2 Vibrio species.