The power of next-generation sequencing as illustrated by the neuropeptidome of the crayfish Procambarus clarkii
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.
References (58)
- et al.
Neuroparsins, a family of conserved arthropod neuropeptides
Gen. Comp. Endocrinol.
(2007) - et al.
Identification of a steroidogenic neurohormone in female mosquitoes
J. Biol. Chem.
(1998) - et al.
Isoform-specific expression of the neuropeptide orcokinin in Drosophila melanogaster
Peptides
(2015) Prediction of the first neuropeptides from a member of the Remipedia (Arthropoda, Crustacea)
Gen. Comp. Endocrinol.
(2014)Identification of the first neuropeptides from the Amphipoda (Arthropoda, Crustacea)
Gen. Comp. Endocrinol.
(2014)Expansion of the Litopenaeus vannamei and Penaeus monodon peptidomes using transcriptome shotgun assembly sequence data
Gen. Comp. Endocrinol.
(2014)- et al.
Identification of putative crustacean neuropeptides using in silico analyses of publicly accessible expressed sequence tags
Gen. Comp. Endocrinol.
(2008) - et al.
Bioinformatic analyses of the publicly accessible crustacean expressed sequence tags (ESTs) reveal numerous novel neuropeptide-encoding precursor proteins, including ones from members of several little studied taxa
Gen. Comp. Endocrinol.
(2010) An insulin-like growth factor found in hepatopancreas implicates carbohydrate metabolism of the blue crab Callinectes sapidus
Gen. Comp. Endocrinol.
(2014)- et al.
Cloning of an insulin-like androgenic gland factor (IAG) from the blue crab, Callinectes sapidus: implications for eyestalk regulation of IAG expression
Gen. Comp. Endocrinol.
(2011)
Identification of the endogenous cysteine-rich peptide trissin, a ligand for an orphan G protein-coupled receptor in Drosophila
Biochem. Biophys. Res. Commun.
Identification of a novel insect neuropeptide, CNMa and its receptor
FEBS Lett.
The eyestalk transcriptome of red swamp crayfish Procambarus clarkii
Gene
Signalling through pigment dispersing hormone-like peptides in invertebrates
Prog. Neurobiol.
Identification and characterization of receptors for ion transport peptide (ITP) and ITP-like (ITPL) in the silkworm Bombyx mori
J. Biol. Chem.
Molecular cloning and biological activity of ecdysis-triggering hormones in Drosophila melanogaster
FEBS Lett.
Best practices in insect genome sequencing: what works and what doesn’t
Curr. Opin. Insect Sci.
Ecdysis triggering hormone signaling in arthropods
Peptides
Control of lipid metabolism by tachykinin in Drosophila
Cell Rep.
OKB, a novel family of brain-gut neuropeptides from insects
Insect Biochem. Mol. Biol.
Allatostatin C and its paralog allatostatin double C: the Arthropod somatostatins
Insect Biochem. Mol. Biol.
Neurohormones and neuropeptides encoded by the genome of Lottia gigantea, with reference to other mollusks and insects
Gen. Comp. Endocrinol.
What the loss of the hormone neuroparsin in the melanogaster subgroup of Drosophila can tell us about its function
Insect Biochem. Mol. Biol.
In silico cloning of genes encoding neuropeptides, neurohormones and their putative G-protein coupled receptors in a spider mite
Insect Biochem. Mol. Biol.
Characterization of the shrimp neuroparsin (MeNPLP): RNAi silencing resulted in inhibition of vitellogenesis
FEBS Open Bio
Characterization of crustacean hyperglycemic hormone from the crayfish (Procambarus clarkii): multiplicity of molecular forms by stereoinversion and diverse functions
Gen. Comp. Endocrinol.
Identification of GYRKPPFNGSIFamide (crustacean-SIFamide) in the crayfish Procambarus clarkii by topological mass spectrometry analysis
Gen. Comp. Endocrinol.
Identification of orcokinin gene-related peptides in the brain of the crayfish Procambarus clarkii by the combination of MALDI-TOF and on-line capillary HPLC/Q-Tof mass spectrometries and molecular cloning
Gen. Comp. Endocrinol.
A new family of receptor tyrosine kinases with a venus flytrap binding domain in insects and other invertebrates activated by aminoacids
PLoS One
Cited by (85)
Activation and characterization of G protein-coupled receptors for CHHs in the mud crab, Scylla paramamosain
2024, Comparative Biochemistry and Physiology -Part A : Molecular and Integrative PhysiologyDual roles of crustacean female sex hormone during juvenile stage in the kuruma prawn Marsupenaeus japonicus
2023, General and Comparative EndocrinologyRobust strategy for disease resistance and increasing production breeding in red swamp crayfish (Procambarus clarkii)
2022, Fish and Shellfish ImmunologyImmunomodulatory role of crustacean cardioactive peptide in the mud crab Scylla paramamosain
2022, Fish and Shellfish Immunology