Proteogenomics of Gammarus fossarum to document the reproductive system of amphipods

Abstract

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. We identified and annotated the most relevant proteins detected through a shotgun tandem mass spectrometry analysis carried out on the proteomes from three major tissues involved in the organism's reproductive function: the male and female reproductive systems, and the cephalon, where different neuroendocrine glands are present. The 1,873 mass-spectrometry-certified proteins represent the largest crustacean proteomic resource to date, with 218 proteins being lineage specific. Comparative proteomics between the male and female reproductive systems indicated key proteins with strong sexual dimorphism. Protein expression profiles during spermatogenesis at seven different stages highlighted the major gammarid proteins involved in the different facets of reproduction.

Fig. 1.

Images of G. fossarum in amplexus and sampled tissues. A, sexually mature organisms in precopulatory mate guarding. B, the cephalon and the four attached hepatopancreatic caeca. C, a female oocyte. D, transversal view of a male gammarid with attached reproductive tissue. M, male reproductive tissue (orange); F, female reproductive tissue (black); C, cephalon; H, hepatopancreatic caeca; ovd., oviduct.

Fig. 2.

General proteogenomic strategy for discovering key proteins from G. fossarum.

Fig. 3.

Analysis of the 1,873 MS/MS-identified proteins. A, distribution of identified proteins by class of 150 proteins according to their cumulative normalized spectral abundance factor (NSAF) and respective contribution of homolog and orphan proteins. B, number of proteins identified in, respectively, four, three, two, and one biological tissues; male reproductive tissue is represented twice in the global analysis (Testis 1, protein catalog; Testis 2, spermatogenesis experiment).

Fig. 4.

Proteome dynamics of the male reproductive tissue from G. fossarum. Five replicates were sampled at seven different spermatogenesis stages. The specific tissue was excised from individual organisms and analyzed as indicated.

Fig. 5.

Comparative analysis of male and female reproductive tissue. A, total number of proteins identified in male and female reproductive tissues. B, distribution of the proteins among the different categories obtained after data analysis using the Tfold module of the PatternLab software program: Blue class (fold change > 2 and p value < 0.05), Green class (fold change > 2 and p value > 0.05), and Red class (fold change > 2 and/or p value > 0.05). C, number of proteins with significant sexual dimorphism (fold change > 2 and p value < 0.05) according to the Tfold analysis. D, distribution of proteins with significant sexual dimorphism according to their fold change value.

Fig. 6.

Clustered temporal abundance variations of proteins during spermatogenesis, before and after copulation. The different trends were obtained from clustering the spectral count using the TrendQuest module of the PatternLab program and are displayed in A–E. Cluster health was set at 0.9, and the minimum number of items per cluster was three.