Neuropeptide discovery in Proasellus cavaticus: Prediction of the first large-scale peptidome for a member of the Isopoda using a publicly accessible transcriptome

Highlights

• The first peptidome for a member of the Isopoda is described.

• 171 putative Proasellus cavaticus neuropeptides were predicted.

• The P. cavaticus peptidome includes members of 28 different peptide families.

• Many P. cavaticus neuropeptides are identical to known decapod/amphipod isoforms.

• Conserved structures suggest conserved functions for peptides in malacostracans.

Abstract

In silico transcriptome mining is one of the most effective methods for neuropeptide discovery in crustaceans, particularly for species that are small, rare or from geographically inaccessible habitats that make obtaining the large pools of tissue needed for other peptide discovery platforms impractical. Via this approach, large peptidomes have recently been described for members of many of the higher crustacean taxa, one notable exception being the Isopoda; no peptidome has been predicted for any member of this malacostracan order. Using a publicly accessible transcriptome for the isopod Proasellus cavaticus, a subcentimeter subterranean ground water dweller, the first in silico-predicted peptidome for a member of the Isopoda is presented here. BLAST searches employing known arthropod neuropeptide pre/preprohormone queries identified 49 transcripts as encoding putative homologs within the P. cavaticus transcriptome. The proteins deduced from these transcripts allowed for the prediction of 171 distinct mature neuropeptides. The P. cavaticus peptidome includes members of the adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin B, allatostatin C, allatotropin, bursicon α, bursicon β, CCHamide, crustacean cardioactive peptide, crustacean hyperglycemic hormone/molt-inhibiting hormone, diuretic hormone 31, eclosion hormone, elevenin, FMRFamide-like peptide, glycoprotein hormone α2, leucokinin, myosuppressin, neuroparsin, neuropeptide F, pigment dispersing hormone, pyrokinin, red pigment concentrating hormone, RYamide, short neuropeptide F, sulfakinin, tachykinin-related peptide and trissin families, as well as many linker/precursor-related sequences that may or may not represent additional bioactive molecules. Interestingly, many of the predicted P. cavaticus neuropeptides possess structures identical (or nearly so) to those previously described from members of several other malacostracan orders, i.e., the Decapoda, Amphipoda and Euphausiacea, a finding that suggests broad phylogenetic conservation of bioactive peptide structures, and possibly functions, may exist within the Malacostraca.

Introduction

Peptides represent the largest single class of compounds used by animals for paracrine/endocrine signaling, particularly within and from the nervous system (e.g., [1]). For more than half a century, crustaceans have been used to elucidate the basic principles underlying neuropeptidergic signaling, and much work has focused on the identification and characterization of native neuropeptides in these animals using a variety of techniques (e.g., [2], [3]). Most early studies employed biochemical isolation and Edman degradation to purify and characterize individual neuropeptides of interest (e.g., [4], [5], [6], [7], [8], [9], [10]), or used targeted molecular cloning for neuropeptide discovery (e.g., [11], [12], [13], [14]), with a peptidome for a species built over time by adding new discoveries in a one-by-one fashion. More recently, mass spectrometry, in particular accurate mass matching and de novo tandem mass spectrometric sequencing, has become a common means for neuropeptide identification (e.g., [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25]); mass spectral approaches have the capacity for rapid peptidome elucidation when sufficient starting material is available for analysis (e.g., [19], [26], [27], [28], [29], [30], [31], [32], [33]). However, for species that are small, rare and/or geographically inaccessible, it is often not practical to obtain the large pools of tissue needed for biochemical- and/or mass spectral-based neuropeptide identification. For these animals, in silico genome/transcriptome mining with subsequent bioinformatics peptide structural prediction provides an alternative means for neuropeptide discovery, as genomes and transcriptomes can often be produced using tissue from a single individual as the starting material.

Over the past several years, genome/transcriptome mining has been used extensively to predict peptidomes for a wide variety of crustacean species (e.g., [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51], [52], [53], [54]), including many for which biochemical and/or mass spectral approaches would be highly impractical due to their geographic inaccessibility and/or small size, e.g., Speleonectes c.f. tulumensis, an anchialine cave dwelling remipede [35], Euphausia crystallorophias, a species of Antarctic krill [51], and multiple planktonic/parasitic members of the Copepoda [36], [38], [41]. In fact, via this approach, large peptidomes have now been described for at least one member of most of the major taxa that comprise the Crustacea, one notable exception being isopods.

The Isopoda is a crustacean order within the class Malacostraca and superorder Peracarida; also included in the Malacostraca, among other taxa, are the Amphipoda, another order within the Peracarida, and the Decapoda and Euphausiacea, two orders within the superorder Eucarida (e.g., [55]). Over 10,000 species of isopods, many endemic, are currently known, with members of this taxon occurring globally and existing in essentially all possible aquatic and terrestrial habitats (e.g., [56], [57], [58]). The reproductive strategy seen in isopods, which typically involves internal fertilization, brooding of the young in a ventral pouch, direct development, small brood size, and often a limited number of lifetime broods, is likely a major contributor to the high degree of endemism and habitat specialization seen in this group of crustaceans (e.g., [58]). Interestingly, and despite the variations in physiology/behavior that must be used by members of the Isopoda that have colonized and succeeded in radically different habitats, little work has focused on identifying the native neuropeptides present in members of crustacean order (e.g., [46], [59], [60], [61], [62], [63], [64]), which undoubtedly are key players in modulating the physiological and behavioral control systems of these animals.

Here, using a publicly deposited transcriptome for Proasellus cavaticus (BioProject No. PRJEB14193; T. Lefebure, direct GenBank submission), a subcentemeter, blind, pigmentless, subterranean ground water dwelling member of the Isopoda [65], the first peptidome for an isopod has been deduced via in silico transcriptome mining and bioinformatics peptide prediction. Specifically BLAST searches of the P. cavaticus transcriptome were conducted using known arthropod neuropeptide precursors as the query sequences. Via this strategy, 49 putative peptide-encoding transcripts were identified, allowing for the prediction of the mature structures of a 171-sequence peptidome. The P. cavaticus peptidome contains members of 28 known arthropod neuropeptide families/subfamilies, as well as a large number of linker/precursor-related sequences whose structures do not place them into any generally recognized peptide group, and which may or may not be bioactive. Comparisons of the peptide isoforms predicted for P. cavaticus with those identified previously from members of the Amphipoda, Decapoda and Euphausiacea show that the structures of many peptide isoforms are broadly conserved in malacostracans, a finding that suggests that many peptides may serve conserved physiological roles within this crustacean class.