Franck Vandenbulcke

Original involvement of antimicrobial peptides in mussel innate immunity.

Recently, the existence and extended diversity of antimicrobial peptides has been revealed in two mussel species. These molecules are classified into four groups according to common features of their primary structure: defensins, mytilins, myticins and mytimycin. In Mytilus galloprovincialis, gene structure reveals synthesis as precursors in circulating hemocytes. Synthesised even in absence of challenge, the precursors mature and the peptides are stored in granules as active forms. The different peptides are engaged in the destruction of bacteria inside phagocytes, before being released into hemolymph to participate in systemic responses. Such involvement in anti‐infectious responses is unique, and apparently more related to those of mammalian phagocytes than to those of insects.

Cathepsin L and cystatin B gene expression discriminates immune cœlomic cells in the leech Theromyzon tessulatum

Previous studies evidenced that cystatin B-like gene is specifically expressed and induced in large circulating coelomic cells following bacterial challenge in the leech Theromyzon tessulatum. In order to understand the role of that cysteine proteinase inhibitor during immune response, we investigated the existence of members of cathepsin family. We cloned a cathepsin L-like gene and studied its tissue distribution. Immunohistochemical studies using anti-cathepsin L and anti-cystatin B antibodies and ultrastructural results demonstrated the presence of three distinct coelomic cell populations: (1) the chloragocytes, which were initially defined as large coelomocytes, (2) the granular amoebocytes and (3) small coelomic cells. Among those cells, while chloragocytes contain cystatin B and cathepsin L, granular amoebocytes contain only cathepsin L and the third cell population contains neither cathepsin nor inhibitor. Finally, results evidenced that cathepsin L immunopositive granular amoebocytes are chemoattracted to the site of injury and phagocyte bacteria.

A structural equation model of soil metal bioavailability to earthworms: confronting causal theory and observations using a laboratory exposure to field-contaminated soils

Structural equation models (SEM) are increasingly used in ecology as multivariate analysis that can represent theoretical variables and address complex sets of hypotheses. Here we demonstrate the interest of SEM in ecotoxicology, more precisely to test the three-step concept of metal bioavailability to earthworms. The SEM modeled the three-step causal chain between environmental availability, environmental bioavailability and toxicological bioavailability. In the model, each step is an unmeasured (latent) variable reflected by several observed variables. In an exposure experiment designed specifically to test this SEM for Cd, Pb and Zn, Aporrectodea caliginosa was exposed to 31 agricultural field-contaminated soils. Chemical and biological measurements used included CaC12-extractable metal concentrations in soils, free ion concentration in soil solution as predicted by a geochemical model, dissolved metal concentration as predicted by a semi-mechanistic model, internal metal concentrations in total earthworms and in subcellular fractions, and several biomarkers. The observations verified the causal definition of Cd and Pb bioavailability in the SEM, but not for Zn. Several indicators consistently reflected the hypothetical causal definition and could thus be pertinent measurements of Cd and Pb bioavailability to earthworm in field-contaminated soils. SEM highlights that the metals present in the soil solution and easily extractable are not the main source of available metals for earthworms. This study further highlights SEM as a powerful tool that can handle natural ecosystem complexity, thus participating to the paradigm change in ecotoxicology from a bottom-up to a top-down approach.

Determination of the performance of vermicomposting process applied to sewage sludge by monitoring of the compost quality and immune responses in three earthworm species: Eisenia fetida, Eisenia andrei and Dendrobaena veneta

The aim of this study was to assess the effectiveness of vermicomposting process applied on three different sewage sludge (precomposted with grass clippings, sawdust and municipal solid wastes) using three different earthworm species. Selected immune parameters, namely biomarkers of stress and metal body burdens, have been used to biomonitor the vermicomposting process and to assess the impact of contaminants on earthworms physiology. Biotic and abiotic parameters were also used in order to monitor the process and the quality of the final product. Dendrobaena veneta exhibited much lower resistance in all experimental conditions, as the bodyweight and the total number of circulating immune cells decreased in the most contaminated conditions. All earthworm species accumulated heavy metals as follows Cd>Co>Cu>Zn>Ni>Pb>Cr: Eisenia sp. worms exhibited the highest ability to accumulate several heavy metals. Vermicompost obtained after 45days was acceptable according to agronomic parameters and to compost quality norms in France and Poland.

Ultrastructural and autometallographic studies of the nephrocytes of Lithobius forficatus L. (Myriapoda, Chilopoda): role in detoxification of cadmium and lead

Abstract Ultrastructural and autometallographic investigations of the nephrocytes of the centipede Lithobius forficatus (Myriapoda : Chilopoda) and their role in detoxification of cadmium and lead are presented. This cell-type exhibits the characteristic features of podocytes, i.e. the typical pedicel-basal lamina complex and the well-developed lysosomal vacuolar system. Ultrastructural study demonstrated that heavy metals, such as cadmium and lead, may cause intracellular injuries. The autometallographic procedure showed that large electron-dense granules, referred to as cytosomes in the ultrastructural study, are the main organelles of the nephrocytes to accumulate heavy metals. Consequently, these cells may play an important role in the detoxification process.

Insight into the expression variation of metal-responsive genes in the seedling of date palm (Phoenix dactylifera)

Phytochelatin synthase and metallothionein gene expressions were monitored via qPCR in order to investigate the molecular mechanisms involved in Cd and Cr detoxification in date palm (Phoenix dactylifera). A specific reference gene validation procedure using BestKeeper, NormFinder and geNorm programs allowed selection of the three most stable reference genes in a context of Cd or Cr contamination among six reference gene candidates, namely elongation factor α1, actin, aldehyde dehydrogenase, SAND family, tubulin 6 and TaTa box binding protein. Phytochelatin synthase (pcs) and metallothionein (mt) encoding gene expression were induced from the first days of exposure. At low Cd stress (0.02 mM), genes were still up-regulated until 60th day of exposure. At the highest metal concentrations, however, pcs and mt gene expressions decreased. pcs encoding gene was significantly up-regulated under Cr exposure, and was more responsive to increasing Cr concentration than mt encoding gene. Moreover, exposure to Cd or Cr influenced clearly seed germination and hypocotyls elongation. Thus, the results have proved that both analyzed genes participate in metal detoxification and their expression is regulated at transcriptional level in date palm subjected to Cr and Cd stress. Consequently, variations of expression of mt and pcs genes may serve as early-warning biomarkers of metal stress in this species.