Elsevier

Journal of Proteomics

Volume 135, 1 March 2016, Pages 51-61
Journal of Proteomics

Proteogenomic insights into the core-proteome of female reproductive tissues from crustacean amphipods

https://doi.org/10.1016/j.jprot.2015.06.017Get rights and content

Highlights

  • We conducted a comparative proteomic analysis of ovaries from five Senticaudata amphipods.

  • Mass spectrometry data were interpreted with several databases for taking into account molecular biodiversity.

  • The conserved proteins are among the most abundant, thus protein concentrations are under strong evolutionary pressure.

  • We noted an important polymorphism of large lipid transfer proteins in the analyzed animals.

Abstract

As a result of the poor genome sequence coverage of crustacean amphipods, characterization of their evolutionary biology relies mostly on phenotypic traits. Here, we analyzed the proteome of ovaries from five amphipods, all from the Senticaudata suborder, with the objective to obtain insights into the core-proteome of female reproductive systems. These amphipods were from either the Gammarida infraorder: Gammarus fossarum, Gammarus pulex, Gammarus roeseli, or the Talitrida infraorder: Parhyale hawaiensis and Hyalella azteca. Ovaries from animals sampled at the end of their reproductive cycle were dissected. Their whole protein contents were extracted and their proteomes were recorded by high-throughput nanoLC–MS/MS with a high-resolution mass spectrometer. We interpreted tandem mass spectrometry data with the protein sequence resource from G. fossarum and P. hawaiensis, both recently established by RNA sequencing. The large molecular biodiversity within amphipods was assessed by the ratio of MS/MS spectra assigned for each sample, which tends to diverge rapidly along the taxonomic level considered. The core-proteome was defined as the proteins conserved along all samples, thus detectable by the homology-based proteomic assignment procedure. This specific subproteome may be further enriched in the future with the analysis of new species and update of the protein sequence resource.

Introduction

Arthropods, including insects, arachnids and crustaceans, show an astonishing variety of adaptations and are considered to be the largest animal phylum in terms of species diversity. Among crustaceans, the Amphipoda order is one of the most diverse, with more than 9600 species currently identified and principally included in the Senticaudata suborder. These “shrimp-like” organisms inhabit a wide range of aquatic environments, where they constitute an important biomass and occupy key roles in ecosystem processes, being both an important food resource for aquatic communities and, themselves, detritivores [1], [2].

Because of their ecological relevance, amphipod organisms are popular biological models in environmental sciences. Due to their sensitivity to many pollutants, they are employed worldwide as test species in hazard assessment of environmental contaminations, both in the laboratory and in field experiments: Melita plumulosa for testing estuarine sediment toxicity in Australia [3], [4], the marine Echinogammarus marinus for assessing endocrine disruption and reproductive impairments in the UK [5], [6], and in freshwater ecotoxicology using Hyalella azteca in North America [7], [8] or Gammarus pulex and Gammarus fossarum in Europe reviewed by [9]. Amphipods are also studied in developmental and evolutionary biology. Parhyale hawaiensis, distributed in circumtropical marine habitats, is an emerging model for studying crustacean embryonic development. Several molecular techniques, such as stable transgenesis and gene knockdown, have been developed for this purpose [10], [11]. Amphipod organisms occur in cryptic species complexes, i.e. species with similar morphological traits but genetic divergence. Several studies investigating the underlying mechanisms leading to cryptic phenomena have been carried out within the H. azteca complex [12], the G. fossarum/pulex complex [13] and the M. plumulosa complex [14]. Finally, amphipods are also biological models in parasitology. Indeed, it has been shown that in Gammarus roeseli [15] and Gammarus duebeni [16], the reproductive organs can be infected by vertically transmitted parasites. When the testes are infected, these microsporidian parasites actively convert males into females in order to enhance their transmission to the offspring, leading to a feminized phenotype.

Among the different fields employing amphipod organisms as biological models, knowledge of their reproductive biology is of utmost importance. For example, in ecotoxicology, due to the critical role of reproduction in population viability, measurement of reproductive success in response to pollutants is a highly relevant endpoint [17]. In females, the molting, embryonic and oogenic cycles are coordinated. Whereas the physiological parameters involved in the reproductive process have been well described, the associated molecular mechanisms have been poorly characterized, due to the lack of comprehensive genomic resources. To date, the only crustacean genome available is that of the water flea, Daphnia pulex (Branchiopoda Cladocera) [18]. Due to the large phylogenetic distances between crustaceans, conservation of genes and associated function remains to be established. This is especially true when investigating proteins and genes involved in reproductive function, these biomolecules being highly species specific as a result of sexual selection in evolution [19]. To date, genome sequencing projects have been announced for two amphipod species: H. azteca and P. hawaiensis [20]. Meanwhile, transcriptomes sequenced by next-generation technology have been released for M. plumulosa whole organism [4], and P. hawaiensis [10] and E. marinus [6] reproductive organs.

Next-generation tandem mass spectrometry allows large proteome surveys and also comparative analysis, where quantities of hundreds or thousands of proteins are compared between various conditions, but to date has been used in a very limited number of studies regarding only one amphipod species [21], [22]. While the lack of a genomic resource for these animals, and their wide diversity, are the major reasons for restricting proteomic applications, we recently reported a large proteogenomic survey to decipher the proteins involved in the reproductive function of G. fossarum [23]. For this, a protein sequence database was created based on RNA-seq data recorded specifically for this purpose and translated into the six possible reading frames. Proteogenomics, which was initially proposed to better annotate genomes with proteomic information, turns out to be powerful not only for handling draft genomes, but also unannotated genomes or transcriptomes [24]. For G. fossarum, we reported the existence of 1873 mass-spectrometry-certified contigs, a dataset that represents the largest crustacean proteomic resource to date. In addition, comparative proteomics of the testis at seven different stages during spermatogenesis revealed some major proteins involved in male reproduction. We also conducted a proteomic analysis of G. fossarum subjected to three different xenobiotics in order to propose specific biomarkers for toxicity assays [25]. In the same vein as what is done in comparative genomics, comparative proteomics is possible as soon as several proteomes of the same branch of the tree of life can be recorded, with the advantage from a functional point of view of highlighting the most crucial items by means of their abundance. However, comparative proteomics, i.e. the analysis and comparison of various species proteomes, is still rather in its infancy and is restricted to the analysis of bacteria due to the experimental cost. Christie-Oleza et al. [26] reported different adaptive strategies after comparing the proteomes of twelve marine bacteria. Toueille et al. [27] and Bouthier de la Tour et al. [28] identified the most important proteins involved in the structure of the Deinococcus nucleoid after comparing two different species. By similarity to comparative genomics, the concepts of core-proteome, i.e. the proteins which are conserved in all the species of a given life branch and produced for a given condition, and pan-proteome, i.e. all the proteins which are present in a given condition for all the species of a given life branch, can be proposed.

With the objective to obtain insights into the core-proteome of female reproductive systems for amphipods, we investigated here the proteomes of ovaries sampled from five different species, all from the Senticaudata suborder: G. fossarum, G. pulex, G. roeseli, P. hawaiensis and H. azteca. Their proteomes were recorded by high-throughput nanoLC–MS/MS with the high-resolution mass spectrometer, merging the results from five animals per species. First, we analyzed the effects of database search when using either a generalist protein sequence resource such as NCBInr or genome data from a distantly related animal, or more specific RNA-seq-derived protein databases. We then delineated the core-proteome for this specific reproductive tissue, highlighting which of the most abundant proteins assessed by a label-free approach were conserved.

Section snippets

Sampling of animals and preparation of biological samples

The amphipods from the Gammarida infraorder were sampled from rivers in mid-eastern France. They were collected by kick sampling, as previously described [23]. The organisms were determined by phenotypic criteria [29]. G. pulex organisms were collected in the Tanche River (latitude, 47°052′815″; longitude: 5°639′305″) while G. fossarum and G. roeseli organisms were collected in the Bourbre River (45°569′442″; 5°459′115″ and 45°716′018″, 5°159′666″, respectively). The organisms from the Talitrida

Protein database search strategy for interspecies comparative proteomics

Fig. 1 shows the strategy in terms of animal sampling, proteomic analysis and MS/MS assignments. For studying the reproductive proteome, the female ovaries were chosen because i) this organ is crucial for the development of the new generation by accumulating yolk reserves along the oogenesis process, and ii) because of the availability of ovary transcriptomes for P. hawaiensis and G. fossarum. Ovaries at the end of the oogenesis are fully developed, containing all the materials required for

Conclusion

The proteome data measured on five different amphipods from the Senticaudata suborder shows the importance of a comprehensive protein sequence database for interpretation of MS/MS spectra. The best interpretation could be obtained for G. fossarum organisms because of the specific RNA-seq protein database previously established [23]. Indeed, several reports highlighted how RNA-seq is useful to inform proteomics carried out on either non-model organisms or natural biomaterials [21], [42], [43],

Acknowledgments

We thank the Institut National de Recherche en Sciences et Technologies pour l'Environnement et l'Agriculture (France), the Commissariat à l'Energie Atomique et aux Energies Alternatives (France) through the transversal toxicology program (PPTOX), the Agence Nationale de la Recherche program “ProteoGam” (ANR-14-CE21-0006-02) and the Agence Nationale de la Recherche CESA program “GAMMA” 021 02 “Variability–adaptation–diversity and Ecotoxicology in gammarids” (2012–2015) for their financial

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