Because of their ecological importance, amphipod crustacea are employed worldwide as test species in environmental risk assessment. Although proteomics allows new insights into the molecular mechanisms related to the stress response, such investigations are rare for these organisms because of the lack of comprehensive protein sequence databases. Here, we propose a proteogenomic approach for identifying specific proteins of the freshwater amphipod Gammarus fossarum, a keystone species in European freshwater ecosystems. After deep RNA sequencing, we created a comprehensive ORF database. Next-generation proteomics, relying on ultra-rapid and subparts-per-million mass spectrometry analyzers, is able to offer in-depth insights into the molecular players sustaining the physiology of complex organisms. Identifying and quantitating thousands of proteins has become, over the past decade, a routine task for most proteomic platforms with the development of high-throughput shotgun proteomics. The interpretation of large-scale MS/MS data is only possible if a highquality database of nucleic acid sequences is available. Homology-driven proteomics using cross-species matching is a first alternative if the genome of interest is unknown, and de novo sequencing (i.e. interpretation of MS/MS data to establish the exact sequence of each peptide from scratch) is another possibility. However, major drawbacks of these two approaches lead to a scarcity of results as soon as a non-model organism, distantly related to a sequenced organism, is analyzed. Indeed, only highly conserved and ubiquitous proteins will be identified and carefully annotated with such approaches.
Because of their ecological representativeness, invertebrates are commonly employed as test organisms in ecotoxicological assessment; however, to date, biomarkers employed for these species were the result of a direct transposition from vertebrates, despite deep evolutionary divergence. To gain efficiency in the diagnostics of ecosystem health, specific biomarkers must be developed. In this sense, next-generation proteomics enables the specific identification of proteins involved in key physiological functions or defense mechanisms, which are responsive to ecotoxicological challenges. However, the analytical investment required restricts use in biomarker discovery. Routine biomarker validation and assays rely on more conventional mass spectrometers. Here, we describe how proteomics remains a challenge for ecotoxicological test organisms because of the lack of appropriate protein sequences databases, thus restricting the analysis on conserved and ubiquitous proteins. These limits and some strategies used to overcome them are discussed. These new tools, such as proteogenomics and targeted proteomics, should result in new biomarkers specific to relevant environmental organisms and applicable to routine ecotoxicological assessment.
A 12-month active biomonitoring study was performed in 2008-2009 on the Vesle river basin (Champagne-Ardenne, France) using the freshwater mussel Dreissena polymorpha as a sentinel species; allochthonous mussels originating from a reference site (Commercy) were exposed at four sites (Bouy, Sept-Saulx, Fismes, Ardre) within the Vesle river basin. Selected core biomarkers (acetylcholinesterase (AChE) activity, glutathione-S transferase (GST) activity, metallothionein concentration), along with digestive enzyme activities (amylase, endocellulase) and energy reserve concentrations (glycogen, lipids), were monitored throughout the study in exposed mussels. At the Fismes and Ardre sites (downstream basin), metallic and organic contamination levels were low but still high enough to elicit AChE and GST activity induction in exposed mussels (chemical stress); besides, chemical pollutants had no apparent deleterious effects on mussel condition. At the Bouy and Sept-Saulx sites (upstream basin), mussels obviously suffered from adverse food conditions which seriously impaired individual physiological state and survival (nutritional stress); food scarcity had however no apparent effects on core biomarker responses. Digestive enzyme activities responded to both chemical and nutritional stresses, the increase in energy outputs (general adaptation syndrome-downstream sites) or the decrease in energy inputs (food scarcity-upstream sites) leading to mid- or long-term induction of digestive carbohydrase activities in exposed mussels (energy optimizing strategy). Complex regulation patterns of these activities require nevertheless the use of a multi-marker approach to allow data interpretation. Besides, their sensitivity to natural confounding environmental factors remains to be precised.
While the decrease in human sperm count in response to pollutants is a worldwide concern, little attention is being devoted to its causes and occurrence in the biodiversity of the animal kingdom. Arthropoda is the most species-rich phyla, inhabiting all aquatic and terrestrial ecosystems. During evolution, key molecular players of the arthropod endocrine system have diverged from the vertebrate counterparts. Consequently, arthropods may have different sensitivities toward endocrine disrupting chemicals (EDCs). Here alteration of sperm quality in a crustacean, Gammarus fossarum, a popular organism in freshwater risk assessment, was investigated after laboratory exposure to various concentrations of three different xenobiotics: cadmium, methoxyfenozide, and pyriproxyfen. The integrity of the reproductive process was assessed by means of sperm-quality markers. For each substance, semiquantitative/relative proteomics based on spectral counting procedure was carried out on male gonads to observe the biological impact. The changes in a total of 871 proteins were monitored in response to toxic pressure. A drastic effect was observed on spermatozoon production, with a dose-response relationship. While exposure to EDCs leads to strong modulations of male-specific proteins in testis, no induction of female-specific proteins was noted. Also, a significant portion of orphans proved to be sensitive to toxic stress.
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