Characterization of the Carcinus maenas neuropeptidome by mass spectrometry and functional genomics

Abstract

Carcinus maenas, commonly known as the European green crab, is one of the best-known and most successful marine invasive species. While a variety of natural and anthropogenic mechanisms are responsible for the geographic spread of this crab, its ability to adapt physiologically to a broad range of salinities, temperatures and other environmental factors has enabled successful establishment in these new habitats. To extend our understanding of hormonal control in C. maenas, including factors that allow for its extreme adaptability, we have undertaken a mass spectral/functional genomics investigation of the neuropeptides used by this organism. Via a strategy combining MALDI-based high resolution mass profiling, biochemical derivatization, and nanoscale separation coupled to tandem mass spectrometric sequencing, 122 peptide paracrines/hormones were identified from the C. maenas central nervous system and neuroendocrine organs. These peptides include 31 previously described Carcinus neuropeptides (e.g. NSELINSILGLPKVMNDAamide [β-pigment dispersing hormone] and PFCNAFTGCamide [crustacean cardioactive peptide]), 49 peptides only described in species other than the green crab (e.g. pQTFQYSRGWTNamide [Arg 7 -corazonin]), and 42 new peptides de novo sequenced here for the first time (e.g. the pyrokinins TSFAFSPRLamide and DTGFAFSPRLamide). Of particular note are a collection of 25 FMRFamide-like peptides (including 9 new isoforms sequenced de novo) and a collection of 25 A-type allatostatin peptides (including 10 new sequences reported for the first time) in this study. Both peptide families are among the most diverse families, each containing a large number of isoforms in arthropod species. Also of interest was the identification of two SIFamide isoforms, GYRKPPFNGSIFamide and VYRKPPFNGSIFamide, the latter peptide known previously only from members of the astacidean genus Homarus. Using transcriptome analyses, 15 additional peptides were characterized, including an isoform of bursicon β and a neuroparsin-like peptide. Collectively, the data presented in this study * Correspondence to: Dr. Lingjun Li, School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, Wisconsin 53705-2222 USA; Phone: 608-265-8491; Fax: 608-262-5345; lli@pharmacy.wisc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. not only greatly expand the number of identified C. maenas neuropeptides, but also provide a framework for future investigations of the physiological roles played by these molecules in this highly adaptable species. NIH Public Access KeywordsCarcinus maenas; European green cr...

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.…”