Combining in silico transcriptome mining and biological mass spectrometry for neuropeptide discovery in the Pacific white shrimp Litopenaeus vannamei

Abstract

The shrimp Litopenaeus vannamei is arguably the most important aquacultured crustacean, being the subject of a multi-billion dollar industry worldwide. To extend our knowledge of peptidergic control in this species, we conducted an investigation combining transcriptomics and mass spectrometry to identify its neuropeptides. Specifically, in silico searches of the L. vannamei EST database were conducted to identify putative prepro-hormone-encoding transcripts, with the mature peptides contained within the deduced precursors predicted via online software programs and homology to known isoforms. MALDI-FT mass spectrometry was used to screen tissue fragments and extracts via accurate mass measurements for the predicted peptides, as well as for known ones from other species. ESI-Q-TOF tandem mass spectrometry was used to de novo sequence peptides from tissue extracts. In total 120 peptides were characterized using this combined approach, including 5 identified both by transcriptomics and by mass spectrometry (e.g. pQTFQYSRGWTNamide, Arg 7 -corazonin, and pQDLDHVFLRFamide, a myosuppressin), 49 predicted via transcriptomics only (e.g. pQIRYHQCYFNPISCF and pQIRYHQCYFIPVSCF, two C-type allatostatins, and RYLPT, authentic proctolin), and 66 identified solely by mass spectrometry (e.g. the orcokinin NFDEIDRAGMGFA). While some of the characterized peptides were known L. vannamei isoforms (e.g. the pyrokinins DFAFSPRLamide and ADFAFNPRLamide), most were novel, either for this

Distinct or shared actions of peptide family isoforms: II. Multiple pyrokinins exert similar effects in the lobster stomatogastric nervous system

“…This contrasts sharply with the case in most other crustaceans, as well as most insects. To date, pyrokinins have been identified in six decapod crustacean species, including a shrimp (L. vannamei), three species of crab (C. borealis, Carcinus maenas and C. sapidus), the crayfish Procambarus clarkii and the lobster H. americanus (Christie, 2014a;Hui et al, 2012;Ma et al, 2009Ma et al, , 2010Ma et al, , 2008Saideman et al, 2007;Torfs et al, 2001;Christie and Chi, 2015); in all of these except H. americanus, pyrokinins with extended C-termini have been identified. In contrast to the case in insects, where many functions have been ascribed to pyrokinins (e.g.…”

“…This suggests that there are multiple pyrokinins in H. americanus, including in the STNS. In most crustaceans thus far examined, this is the case (Christie, 2014a,b;Christie et al, 2013;Hui et al, 2012;Ma et al, 2009Ma et al, , 2010Ma et al, , 2008Saideman et al, 2007;Torfs et al, 2001;Christie and Chi, 2015), with at least one species (the shrimp L. vannamei) possessing 11 distinct PBAN/diapause hormone/pyrokinin/ periviscerokinin superfamily members (Christie, 2014a;Ma et al, 2010;Torfs et al, 2001). The differences in responses of the cardiac and stomatogastric ganglia to different pyrokinins would also be explained if there were at least two pyrokinin receptors, one of which was highly specific, while the other(s) was much more promiscuous in its binding properties -a situation that exists in at least some insects (e.g.…”

“…Recent studies of the neuropeptidomes of several decapod crustaceans, for example, have shown that these species often contain upwards of 100 or more distinct peptides (e.g. Fu et al, 2005;Hui et al, 2013Hui et al, , 2012Ma et al, 2009Ma et al, , 2010Ma et al, , 2008. Many of these peptides display high levels of amino acid similarity, suggesting that they evolved from a single common precursor (e.g.…”

“…Originally identified in insects , members of the pyrokinin peptide family have recently been found in several crustacean species (Christie, 2014a,b;Christie et al, 2013;Hui et al, 2012;Ma et al, 2009Ma et al, , 2010Ma et al, , 2008Saideman et al, 2007;Torfs et al, 2001;Christie and Chi, 2015). The pyrokinins are members of the pheromone biosynthesis activating neuropeptide (PBAN)/diapause hormone/pyrokinin/periviscerokinin superfamily of peptides, which typically possess the carboxy (C)-terminal sequence -FXPRLamide, or a close approximation thereof (e.g.…”

“…Baggerman et al, 2002;Hewes and Taghert, 2001;Kean et al, 2002;Meng et al, 2002;Vanden Broeck, 2001). Twenty different pyrokinins have been identified in six different decapod crustaceans (Table 1; Christie, 2014a;Hui et al, 2012;Ma et al, 2009Ma et al, , 2010Ma et al, , 2008Saideman et al, 2007;Torfs et al, 2001;Christie and Chi, 2015), with each species having from one [the lobster Homarus americanus (Ma et al, 2008) and the crab Callinectes sapidus (Hui et al, 2012)] to 11 [the penaeid shrimp Litopenaeus vannamei (Christie, 2014a;Ma et al, 2010;Torfs et al, 2001)] different isoforms. However, only two studies to date (Saideman et al, 2007; and our accompanying paper, Dickinson et al, 2015) have examined the biological activity of these peptides in the crustaceans; both studies found that the pyrokinins are active neuromodulators.…”