Proteomics for novel biomarkers relevant in ecotoxicology from gammarids : challenging the biodiversity and multiplexed immunoassay as diagnostic tool

Informations

Funding country

France

Acronym

ProteoGam

URL

-

Start date

1/1/2015

End date

-

Budget

444,523 EUR

Fundings

Abstract

Hazard assessment of endocrine disruptors raises currently great concerns, especially for invertebrate species. Similarly to scientific researches and tools developed to study endocrine disruptors impacts on vertebrates, national and European expert advisory groups have recently highlighted the need to develop relevant tools for the identification and the assessment of mode of action involved in toxic effects on key physiological functions for invertebrates. Despite the obvious importance of gammarids for aquatic ecosystem functioning, their widespread use in aquatic ecotoxicology (both for laboratory and field experiments) and the different markers of toxicity available, any direct and validated biomarkers are currently available to understand the mode of action of endocrine disruptors and to diagnose their impact. This is the result of the knowledge gap on the endocrine regulation of reproduction in some crustaceans such as amphipods, in relation to the lack of genomic data for non-model species. Recently, a novel approach intimately combining genomic and proteomic methodologies, namely proteogenomics, has emerged for a straightforward strategy for discovering proteins in non-model organisms. For the first time in macro-invertebrates, our research teams have recently conducted a large proteogenomic survey in our sentinel species G. fossarum, allowing a characterization of gammarids’ reproductive function, through the identification and characterisation of novel key proteins. Thanks to new omic techniques, ProteoGam proposes to develop new generation of endocrine disruptor (ED) biomarkers, making possible their use for field survey. The first objective of the proposal is to develop and propose relevant ED biomarkers for the crustacean G. fossarum in order to improve hazard assessment of pollutants in aquatic systems. The second objective aims to propose transferable (universal) analytical methods for measuring proposed ED biomarkers on Gammarus species, in order to investigate the vulnerability of gammarid populations to ED and the relevance of G. fossarum as a surrogate species of gammarid diversity. In parallel, to quantify these biomarkers, a new approach based on the Luminex xMAP® technology will be implemented. This methodology allows developing multiplex immunoassays to quantify simultaneously some biomarkers in a single analysis, through the identification and quantification of proteotypic peptides for each biomarker. Based on large transcriptome dataset available on our reference population of G. fossarum, specific proteins of the male and female reproductive tract will be identified, and among them, candidates for endocrine disruptor biomarkers will be proposed according to (i) describing their pattern during the reproductive cycle via proteomic shotgun analysis and and (ii) assessing their sensitivity towards models compounds and during field experiments. To address the representativity of G. fossarum as a surrogate species toward gammarid diversity in regard to the impacts of ED, a proteogenomic approach will be performed on five species mostly observed in France and Europe, i.e. G. fossarum, G. pulex, G roeseli, G. tigrinus and Dikerogammarus villosus. Pan-proteomes will be recorded on ovary and testis of animals from the five species and interpreted based on a pan-RNAseq derived database. This comparative high-throughput proteomic approach will allow deciphering a set of proteotypic peptides that could be specific of proteins identified as ED biomarker in G. fossarum, but conserved amongst the different species, thus “universal” ED biomarkers for European Gammarus species. ProteoGam will thus (i) improve knowledge on ecophysiology of G. fossarum, especially proteins playing a key role in reproduction, (ii) exemplify the relevance of new omic approaches for biomarkers development, and (iii) propose an original validated multiplex assay as a diagnostic tool for field survey of ED impacts.