WP4-T2 Effects of pathogens on host defence mechanisms

Participants :

  • 1 - IFREMER
  • 2 - CSIC
  • 7 - UNIGE
  • 8 - UNIPD
  • 10 - CNRS

The innate immune response will be studied in Pacific cupped oysters and mussels collected in the field (WP2 - Task 2) and in Pacific cupped oysters and mussels from experimental trials (WP4 - Task 1) in order to better understand the pathogenesis and pathogen resistance. Contrasted animals (infected versus non-infected individuals, resistant versus susceptible individuals, susceptible versus non-susceptible species) will be compared.

Sub task 2.1 - Available molecular tools and development of new one

The tools currently in use for the study of the immune response of mussels and oysters will first be identified. The expression of immune genes in M. galloprovincialis has been studied using qPCR and also microarrays.Moreover, the expression of immune genes in C. gigas is also well documented. Around 57 000 ESTs and 4 SAGE libraries dedicated to the identification of genes differentially expressed in relation to oyster capabilities to survive Vibrio infections are already available representing a combination of gene markers of health and survival status for monitoring oyster response. On the other hand, reports on gene expression in M. edulis have only been related to pollutants, adding useful information on the structure and regulation of several genes but few of those identified being immune-related.

As a result of previous programmes thousands of EST sequences are now available regarding marine bivalves. To recover genes potentially involved in anti-infectious responses, data mining will be performed in all the available sequences obtained from marine molluscs. Data mining will be based on (i) nucleotide sequences and/or (ii) particular domains.

Partial sequences of new immune-related mRNAs identified from data mining will be used to design PCR primers. Additionally, functional immunology developed in Sub Task 2.2 will release new immune-related proteins, the sequences of which will be used to design primers by back translation according to mussel preferential codons. All the primers will be then tested on M. edulis, M. galloprovincialis and C. gigas mRNAs.
Resulting amplicons will be sequenced to control the obtained sequences corresponded to the expected ones and to validate the primers to be used on expression quantifications.

During the EU grant Imaquanim, qPCR was used on RNA pools from 10 mussels to avoid individual variability bias. In fact, some M. galloprovincialis specimens reacted to the challenge, some others not. Now, measures on micro RNA quantities will be developed to ensure measurements on RNA extracted from individuals.

Sub Task 2.2 - Functional Immunology

The immune system of bivalves consists only of the innate (non specific response). Both haemoytes (phagocytosis, encapsulation) and serum factors (antimicrobial peptides, lectins, complement among others) play a major role in controlling the spread of potential pathogens.
Several immune parameters well established in the different participants laboratories will be measured in the experimental infections described in Task 1: production of intermediate oxygen and nitrogen radicals, phagocytosis assayed by flow cytometry (Xue et al 2001; Gagnaire et al 2007 ; Paillard et al 2006), bactericidal activity of hemocytes and serum; hemocytes lysosomal membrane stability, adherence capacity of hemocytes and lectin detection, haemocyte apoptosis rate, neoplastic cells, DNA repair and enzyme activities such as phenoloxidase, lysozyme, superoxide dismutase. This will provide information on haemocyte immune capacity. The effect of pathogens on p38 MAPK pathway in hemocytes will be tested using Western blot methods (Travers et al 2009).

Bactericidal activity of hemocytes and serum samples against different bacteria will be evaluated by plating samples onto LB agar to enumerate culturable bacteria (CFU/ml). Hemocyte lysosomal membrane stability will be evaluated by the Neutral Red Retention Time assay. The capacity of hemocytes to adhere to culture dishes will be evaluated in supernatants (containing non-adherent cells) by FC analysis using the parameters of relative size (FSC) and granularity (SSC). Lysozyme activity in hemolymph soluble fraction will be evaluated spectrophotometrically as the ability to lyse a standard suspension of M. lysodeikticus. Levels of phosphorylated MAPKs (p38 MAPK) in whole cell extracts from hemocyte monolayers will be determined by electrophoresis and Western blotting using specific antibodies.

Sub task 2.3 - Molecular immunology

The expression of immune genes selected in the Subtask 2.1 (including AMPs, lectines, comlement and signalling pathways) will be studied in infected and non infected bivalves, in susceptible and resistant bivalves and in susceptible and non susceptible species. Particular attention will be paid to comparisons between M. edulis (known to be more resistant) and M. galloprovincialis, also between mussels and oysters (known to be
sensitive) and between juveniles and adults in both mussels and oysters. The most modulated genes as defined from previous objectives will be selected for full mRNA sequencing by classical molecular biology techniques: primer design, PCR, 3’ and 5’ RACE.

One of the purposes of EC Imaquanim project (FOOD-CT-2005-007103) was to study the expression of several antimicrobial peptide genes in M. galloprovincialis according to different challenges, geographical origins or seasons. qPCR on 384 well plates Roche Light Cycler 480 have been routinely used to quantify the expression of defensin, mytilin, myticin and mytimycin.

Major results related to this species are:

  • (i) the same gene was differently expressed according to mussel origin and to the season,
  • (ii) expression of the different genes was differently regulated according to the challenging bacteria, and
  • (iii) E. coli content of the mussel tissues did not correlate with AMP gene expressions.

Same technology will be applied to M. edulis and to C. gigas as soon as corresponding primers will be released by Sub-task 2.1. Gene expression analysis will be performed on hemolymph (and tissues) collected from the same sampled animals by using qPCR, DNA microarrays and advanced sequencing.

Differences regarding immune-related gene expressions. Primers defined under Sub Task 2.1 will be used to quantify background expressions (by qPCR confirmed by in situ hybridization) in non-infected and infected/treated M. edulis and C. gigas, completing the already published data regarding M. galloprovincialis. Particular attention will be paid on comparisons between the 2 mussel species (known to be little sensitive to
infections), between mussels and oysters (known to be sensitive) and between juveniles and adults in both mussels and oysters.

Differences regarding immune-related gene triggering. To understand why and how some immune-related genes are triggered, one genomic library per animal model will be constructed. Screening for plasmids containing immune gene fragments will be performed by hybridization using amplicons previously used for expression quantification. Sequencing will address the upstream region of the considered genes to focus on the promoter regions. To know the structure of the different promoters will enable to design oligonucleotides targeting one or the other gene. Efficacy of the designed oligos will be tested in experimentally infected mussels and oysters.

Hemolymph will be sampled from the posterior adductor muscle (mussels) or from the adductor muscle (Pacific cupped oysters) with a syringe. Total RNA will be extracted from haemocytes pellets and first strand cDNAs will be synthesized using hexaprimers. Infected mussels and Pacific cupped oysters will be obtained from partners under WP2 and WP4 - Task 1, as living animals or purified total RNA.

Forward and reverse primers developed for M. galloprovincialis will be tested on M. edulis and C. gigas mRNA. New primers will be designed according to functional immunity discoveries and data mining. 3’-5’RACE and nested PCR will allow enlarging the sequence to design PCR primers within the UTRs for amplification of full cds. Specificity of the primers will be controlled as suitable for qPCR. qPCR will be performed using the SYBR Green chemistry on a LightCycler 480 384 well-plate (Roche Diagnostics) as used routinely for the EC project Imaquanim.