The power of next-generation sequencing as illustrated by the neuropeptidome of the crayfish Procambarus clarkii

https://doi.org/10.1016/j.ygcen.2015.06.013Get rights and content

Highlights

  • Fifty-eight neuropeptide precursors are present in the Procambarus transcriptome.

  • It has three neuroparsins and two neuroparsin receptors that are widely expressed.

  • It has a crustacean female sex hormone ortholog that is expressed in the ovary.

  • RNAseq data suggests significant expression of pigment dispersing hormones in ovaries.

  • Putative hyperglycemic hormone receptors are expressed in the hepatopancreas.

Abstract

Transcriptomes of the crayfish Procambarus clarkii were analyzed for the presence of transcripts encoding neurohormones, neuropeptides and their receptors. A total of 58 different transcripts were found to encode such ligands and another 82 for their receptors. A very large number of the neuropeptide transcripts appeared to be complete and for those that were not only small parts seemed to be lacking. Transcripts for the neuropeptide GPCRs as well as for the putative receptors for insulin, neuroparsin and eclosion hormone were often also complete or almost so. Of particular interest is the presence of three different neuroparsin genes and two putative neuroparsin receptors. There are also three pigment dispersing hormones as well three likely receptors for these neuropeptides. CNMamide, calcitonin, CCRFamide, natalisin, trissin and relaxin appear to be new crustacean neuropeptides. The recently identified crustacean female sex hormone was also found and in the crayfish appears to be not only expressed in the eyestalk, but in the ovary as well (though not in the testis). Interestingly, there are two other proteins in the crayfish with a structure similar to crustacean female sex hormone, that could be precursors of neurohormones, but these are not expressed by the ovary. The ovary also appears to contain significant numbers of transcripts encoding pigment dispersing hormones, CNMamide as well as glycoprotein B5, but not glycoprotein A2.

Introduction

Recombinant DNA technology has revolutionized biology. Whereas manual sequencing allowed the determination of relatively small recombinant DNA sequences, fluorescent labeling and automation made it possible to determine whole genome sequences by the year 2000. The first sequenced genomes were relatively small but a draft for the entire human sequenced followed very quickly. Next-generation sequencing (NGS) has once again moved the limits of feasibility and can be used to sequence entire genomes. However, this remains a challenge when the size of the genome is large and when the species to be sequenced is small and/or when no homozygous individuals are available (see e.g. Richards and Murali, 2015).

The largest genome sequenced to date is that of the migratory locust Locusta migratoria and this sequence represents a major milestone (Wang et al., 2014). Although current improvements in NGS technology will no doubt make such large genomes easier to sequence, in many cases one is more interested in the coding and non-coding RNAs generated by a genome than in the genomic sequences per se. The NGS technologies have been phenomenal in determining which RNAs are expressed by the genome, whether in specific tissues or in entire individuals. Not surprisingly, it is extensively used for a large number of different projects. Many of these concern the expression of every gene under different experimental conditions in a particular tissue, while the objective of other studies is to determine protein sequences from different species and use those for the construction of phylogenetic trees, such as those that were used to generate such a tree for Arthropods (Misof et al., 2014). As a result there is a enormous amount of data that is often only very partially exploited. For the comparative endocrinologist this allows the exploration of unprecedented amounts of DNA sequences, which has allowed us to document the presence of numerous neuropeptides in species that have never been used before in endocrinological research (e.g. Christie et al., 2008, Christie et al., 2010, Christie, 2014a, Christie, 2014b). In some cases such data may lead to interesting experiments on those species, but in many cases this will probably not be the case. Although such data is very interesting and valuable, it is of haphazard nature in that it does not supply the entire neuropeptidome of a single species such as a complete genome sequence may provide. It is more interesting to deduce neuropeptidomes from species that have a large genome and are used in biological research. Studies on several crustaceans have yielded significant numbers of neuropeptide transcripts (e.g. Christie, 2014c), but many of these transcripts are incomplete and one suspects that these neuropeptidomes also remain incomplete. We here describe the neuropeptidome of the crayfish Procambarus clarkii. The genome of this species has been estimated as 6.2 pg (Bachmann and Rheinsmith, 1973), which would correspond to about 6064 Mbp and this crustacean is commonly used as a research model for neuropeptide research (e.g. Yasuda et al., 1994, Yasuda et al., 2004, Nagasawa et al., 1996, Yasuda-Kamatani and Yasuda, 2000, Yasuda-Kamatani and Yasuda, 2004, Yasuda-Kamatani and Yasuda, 2006). Much to our surprise, even though there are only seven short read archives (SRAs) and two transcriptome shotgun assemblies (TSAs) at NCBI for this species (Tom et al., 2014, Shen et al., 2014, Manfrin et al., 2015), they contain sufficient information to get a very complete picture of the neurohormones, neuropeptides and their receptors encoded by this very large genome.

Section snippets

Materials and methods

Two P. clarkii TSAs (GBEV00000000.1 and GARH00000000.1) and seven SRAs (SRR870673, SRR1144630, SRR1144631, SRR1265966, SRR1509456, SRR1509457 and SRR1509458) were downloaded from NCBI, while an additional TSA file was graciously made available by Dr. Huaishun Shen. Dr Shen also supplied the crude RNAseq data from the hepatopancreas described by him and his colleagues (Shen et al., 2014). The fasta sequences were extracted from the SRA’s with the SRA toolkit (//www.ncbi.nlm.nih.gov/Traces/sra/?view=software

Results and discussion

A total of 58 putative neuropeptide precursors were identified (Fig. 1), including a number of recently identified arthropod peptides such as trissin (Ida et al., 2011), EFLamide (Veenstra et al., 2012), natalisin (Jiang et al., 2013), CNMamide (Jung et al., 2014), calcitonin (Veenstra, 2014) and CCRFamide (Conzelmann et al., 2013) as well as the crustacean female sex hormone (Zmora and Chung, 2014). In the absence of a genome it is impossible to know how complete this list is, but it is

Conclusion

The DNA sequences tentatively deduced here for the various crayfish neuropeptides and their receptors should allow analysis of their expression by RT-PCR. Though SNPs may from time to time frustrate such attempts, there is so much sequence information that they should not become a major problem. The information should also be useful for the design of effective RNAi species for the knockdown of specific gene products. In a similar vein, one should be able to do physiology using peptide sequence

Acknowledgments

The author gratefully acknowledges all those responsible for the publicly-accessible sequences that made this work possible with a special mention to Dr. Huaishun Shen of the Chinese Academy of Fishery Sciences. I also thank the constructive comments by a reviewer that allowed me to improve the manuscript. My work is funded by institutional funds from the CNRS.

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