Objective The overall objectives of the project are to characterise the immune cells of the developmental stages of the mussel Mytilus edulis (galloprovincialis) and to develop nucleotide probes to antimicrobial peptides for investigating the expression of these antimicrobial peptides in the developmental stages of Mytilus. The project will also quantify the susceptibility of larvae to specific bacterial pathogens. The effects of environmental stressors on humoral and cellular immune function in developmental stages will be investigated and the cell/tissue specificity of the stressors identified. Finally, the project will establish whether environmental factors (temperature, specific environmental contaminants) increase the susceptibility of larvae to specific pathogens.STATE OF PROGRESSImmunocytochemical labelling with antibodies to adult haemocytes, total actin and Mytilus fibronectin has been carried out on larval and post larval mussels. Biochemical characterisation of actin and fibronectin in larval and post larval mussels has been carried out. The release of reactive oxygen metabolites by disaggregated trochophore and veliger larvae has been investigated. Nucleotide probes for the defence peptides, MGD-1, Mytilin, Myticin and 18S-ribosomal probe have been constructed and used to detect antimicrobial peptides in M. edulis at different developmental stages. Protocols for determining and quantifying the viability of larvae have been investigated using both staining and morphological criteria. Protocols for investigating the effects of bacterial pathogens on mussel larvae have been investigated.ACHIEVEMENTESThe results showed that actin from larvae and postlarvae was separated by SDS-PAGE and the Western immunoblot was labelled with anti-total actin, with the antibody recognising a 43 kDa protein band. The Western blotting analysis of larval and postlarval FN showed labelling with autologous anti-FN antiserum with a 220 kDa band which was coincident with the adult Mytilus haemolymph FN used as standard. Immunocytochemical labelling identified sites for actin and fibronectin in larvae and postlarvae but failed to detect adult haemocyte antigens in the larvae. The results for release of oxyradicals showed for the NBT assay that PMA had an inhibitory effect on larval cells from both trochophore and veliger stages. LPS did appear to stimulate larval cells. With the cytochrome c assay, PMA and PLS increased the superoxide generation in the larval cells. All individual mussels expressed mytilin and myticin genes whereas only 3 out of 8 expressed MGD-1 gene. There was no hybridisation signal on RNAs from eggs and trochophore larvae, strongly suggesting the absence of antimicrobial gene expression during the first stages of development. RNAs from postlarvae as early as 1 mm showed a hybridisation signal with mytilin B and myticin A probes, within the same size range as thoseDESCRIPTION OF THE WORKCharacterisation of the immune cells of the developmental stages of Mytilus will involve light and electron microscopy, immunocytochemistry, together with assays for phagocytosis and release of reactive oxygen metabolites. The development of nucleotide probes to antimicrobial peptides will involve total RNA extraction and electrophoresis and probe constructions according to the amino acid sequences. Northern blot validation will be done on untreated adults and detection of transcripts will be carried out by reverse-transcription PCR. The susceptibility of larvae to specific bacterial pathogens will be undertaken using laboratory exposure to specific concentrations of bacteria. The effects of environmental stressors on humoral and cellular immune function in developmental stages will also involve laboratory exposures. It is proposed that for all the Tasks outlined the following developmental stages will be investigated:Trochophore larvae - no shelled ciliated planktonic stage (approximately 24-48 hours post fertilization)Veliger larvae - shelled planktonic stage, actively swims and has functional gut (approximately 1-4 weeks post fertilization)Postlarval stages - successfully completed settlement and metamorphosis (> 6 weeks post fertilization). Fields of science natural sciencesphysical sciencesopticsmicroscopyelectron microscopymedical and health sciencesbasic medicineimmunologynatural sciencesbiological sciencesgeneticsnucleotidesnatural sciencesbiological sciencesgeneticsRNAnatural scienceschemical sciencesorganic chemistryamines Programme(s) FP4-FAIR - Specific research, technological development and demonstration programme in the field of agriculture and fisheries (including agro-industry, food technologies, forestry, aquaculture and rural development), 1994-1998 Topic(s) 5.3.2 - Health of aquacultured species Call for proposal Data not available Funding Scheme CSC - Cost-sharing contracts Coordinator NERC Centre of Coastal and Marine Sciences EU contribution No data Address Prospect Place West Hoe PL1 3DH Plymouth United Kingdom See on map Total cost No data Participants (3) Sort alphabetically Sort by EU Contribution Expand all Collapse all IFREMER - CNRS - Universite de Montpellier II France EU contribution No data Address 2, Place Eugene Bataillon 34095 Montpellier See on map Total cost No data UNIVERSITY OF WALES, SWANSEA United Kingdom EU contribution No data Address Singleton Park SWANSEA See on map Total cost No data Universita degli Studi di Perugia Italy EU contribution No data Address Via A. Pascoli 06123 Perugia See on map Total cost No data
