General and Comparative Endocrinology 206 (2014) 96–110

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Identification of the first neuropeptides from the Amphipoda (Arthropoda, Crustacea) Andrew E. Christie ⇑ Békésy Laboratory of Neurobiology, Pacific Biosciences Research Center, University of Hawaii at Manoa, 1993 East-West Road, Honolulu, HI 96822, USA

a r t i c l e

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Article history: Received 10 May 2014 Revised 11 July 2014 Accepted 14 July 2014 Available online 21 July 2014 Keywords: Echinogammarus veneris Hyalella azteca Melita plumulosa Bioinformatics Transcriptome shotgun assembly

a b s t r a c t Despite being used as models in the field of ecotoxicology, including use in studies of endocrine disruption, little is known about the hormonal systems of amphipods, particularly their peptidergic signaling systems. Here, transcriptome shotgun assembly (TSA) sequences were used to predict the structures of the first neuropeptides from members of this crustacean order. Using a well-established workflow, BLAST searches of the extant amphipod TSA data were conducted for putative peptide-encoding transcripts. The pre/preprohormones deduced from the identified TSA sequences were then used to predict the mature structures of amphipod neuropeptides. In total, 43 putative peptide-encoding transcripts were identified from three amphipods, Echinogammarus veneris, Hyalella azteca and Melita plumulosa. Collectively, 139 distinct mature peptides (110 from E. veneris alone) were predicted from these TSA sequences. The identified peptides included members of the adipokinetic hormone/red pigment concentrating hormone, allatostatin A, allatostatin B, allatostatin C, bursicon a, bursicon b, crustacean hyperglycemic hormone, diuretic hormone 31, FLRFamide, molt-inhibiting hormone, myosuppressin, neuroparsin, neuropeptide F, orcokinin, pigment dispersing hormone (PDH), proctolin, RYamide, SIFamide, sulfakinin and tachykinin-related peptide families. Of particular note were the identifications of orcokinins possessing SFDEIDR– rather than the typical NFDEIDR– amino-termini, e.g. SFDEINRSNFGFN, a carboxyl-terminally amidated orcokinin, i.e. SFDEINRSNFGFSamide, PDHs longer than the stereotypical 18 amino acids, e.g. NSELLNTLLGSKSLAALRAAamide, and a 13 rather than 12 amino acid long SIFamide, i.e. GPYRKPPFNGSIFamide. These data not only provide the first descriptions of native amphipod neuropeptides, but also represent a new resource for initiating investigations of peptidergic signaling in the Amphipoda. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction The Amphipoda is a crustacean order within the Malacostraca that is comprised of approximately 10,000 living species (Horton et al., 2013). Amphipods can be found in essentially all marine and freshwater habitats, with a few species being primarily terrestrial (Horton et al., 2013). For many aquatic ecosystems, members of the Amphipoda constitute a considerable portion of the biomass, and serve both as major herbivores/detritivores and as significant food sources for higher-level consumers (Horton et al., 2013). Given their abundance and trophic position in aquatic ecosystems, and their sensitivity to many environmental toxicants, amphipods are routinely used for ecotoxicological studies and/or as environmental indicator species. For example, the freshwater amphipod Hyalella azteca is one of the most commonly used invertebrates for laboratory tests of aquatic chemical toxicity and ⇑ Fax: +1 808 956 6984. E-mail address: [email protected] http://dx.doi.org/10.1016/j.ygcen.2014.07.010 0016-6480/Ó 2014 Elsevier Inc. All rights reserved.

sediment bioaccumulation (e.g. Anderson et al., 2014; de Perre et al., 2014; Gott et al., 2014; Jensen-Fontaine et al., 2014; Kunz et al., 2013; Li et al., 2014; Pedersen et al., 2013; Poynton et al., 2013; Vangheluwe et al., 2013; Willming et al., 2013). Despite their use as models in the field of ecotoxicology, and specifically for their growing use in studies of endocrine disruption and disruption of neural control (e.g. Bossus et al., 2014; Hyne, 2011; Nyman et al., 2013), very little is currently known about the native hormonal systems of amphipods. This is particularly true for their neuropeptides, which, as in all crustaceans, undoubtedly constitute the largest and most diverse class of chemicals used for hormonal communication (for recent reviews of crustacean peptidergic signaling see: Christie (2011), Christie and McCoole (2012), and Christie et al. (2010a)). In fact, to the best of this author’s knowledge, no peptide hormones have thus far been described from any member of Amphipoda. With the advent of new technologies for high-throughput sequencing, and ever decreasing costs for such endeavors, large transcriptomic datasets have been generated and publicly

A.E. Christie / General and Comparative Endocrinology 206 (2014) 96–110

deposited for many crustacean species (e.g. Lenz et al., 2014). When available, these data have proven to be rich resources for peptide discovery, with large peptidomes recently predicted via in silico transcriptome mining for a variety of crustaceans (Christie, 2014a–e; Christie et al., 2013; Gard et al., 2009; Toullec et al., 2013; Yan et al., 2012). For example, the first neuropeptides from any member of the Remipedia were recently predicted from the transcriptome of Speleonectes cf. tulumensis (Christie, 2014a); the first neuropeptides from a member of the Branchiura were also recently deduced via transcriptome mining (Christie, 2014c). The public deposition of transcriptome shotgun assembly (TSA) datasets for several amphipods (e.g. Cattonaro, 2014 unpublished direct submission to GenBank; Hook, Twine, Simpson, Spadaro, Moncuquet and Wilkins, 2013 unpublished direct submission to GenBank; Weston et al., 2013) now provides a resource for the first peptide discovery in the Amphipoda, and here, the results of such an undertaking are presented. As the data that follow will show, 43 putative neuropeptideencoding transcripts were identified from the extant TSA data for the Amphipoda. The identified sequences included ones from three amphipod species, Echinogammarus veneris, Hyalella azteca and Melita plumulosa, though those from E. veneris accounted for the majority of the positive BLAST hits. Via a well-established workflow, 139 mature peptides were predicted from these three species, 110 from E. veneris, 23 from H. azteca and six from M. plumulosa. The predicted amphipod peptides included members of the adipokinetic hormone (AKH)/red pigment concentrating hormone (RPCH), allatostatin A (AST-A), allatostatin B (AST-B), allatostatin C (AST-C), bursicon a, bursicon b, crustacean hyperglycemic hormone (CHH), diuretic hormone 31 (DH31), FLRFamide, molt-inhibiting hormone (MIH), myosuppressin, neuroparsin, neuropeptide F (NPF), orcokinin, pigment dispersing hormone (PDH), proctolin, RYamide, SIFamide, sulfakinin and tachykinin-related peptide (TRP) families, as well as many linker/precursor-related peptides that do not fit into any commonly categorized family. These peptides are the only ones thus fare described from the Amphipoda, and as such, provide a new resource for investigating peptidergic signaling in members of this ecologically important crustacean order. 2. Materials and methods 2.1. Database searches Database searches were conducted (on or before May 1, 2014) using methods modified from a well-vetted protocol (e.g. Christie, 2008a,b, 2014a–e; Christie et al., 2008, 2010b, 2011a,b; Gard et al., 2009; Ma et al., 2009, 2010). Specifically, the database of the online program tblastn (National Center for Biotechnology Information, Bethesda, MD; http://blast.ncbi.nlm.nih.gov/Blast.cgi) was set to ‘‘Transcriptome Shotgun Assembly (TSA)’’ and restricted to sequence data from the ‘‘Amphipoda (taxid:6821)’’. Known crustacean and/or insect peptide precursors were input into tblastn as the query sequences, and all hits returned by a given search were fully translated using the ‘‘Translate’’ tool of ExPASy (http:// web.expasy.org/translate/) and then checked manually for homology to the target query. The complete list of peptides families search for in this study, as well as the specific queries used, is provide in Table 1; this table also provides the BLAST-generated maximum score and E-value for each of the transcripts identified as encoding a putative peptide precursor. It should be noted that a reciprocal BLAST search was conducted for each of the amphipod precursor proteins deduced here. These searched were done to identify missing amino (N)- and/or carboxyl (C)-termini for partial pre/preprohormones, and to identify additional transcripts (and ultimately proteins) that were unidentified

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in the original BLAST analyses. Notation of the transcripts identified via these searches is also provided in Table 1. 2.2. Peptide prediction The structures of mature peptides were predicted using a wellestablished workflow (e.g. Christie 2008a,b, 2014a–e; Christie et al., 2008, 2010b, 2011a–c, 2013; Gard et al., 2009; Ma et al., 2009, 2010). Specifically, each of the deduced precursor proteins was assessed for the presence of a signal peptide using the online program SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/; Petersen et al., 2011); the D-cutoff values for SignalP were set to ‘‘Sensitive’’. Prohormone cleavage sites were identified based on the information presented in Veenstra (2000) and/or by homology to known pre/preprohormone processing schemes. When present, prediction of the sulfation state of tyrosine residues was done using the online program Sulfinator (http://www.expasy.org/ tools/sulfinator/; Monigatti et al., 2002). Disulfide bonding between cysteine residues was predicted by homology to known peptide isoforms and/or by using the online program DiANNA (http://www.clavius.bc.edu/~clotelab/DiANNA/; Ferrè and Clote, 2005). Other post-translational modifications, e.g. cyclization of N-terminal glutamine/glutamic acid residues and C-terminal amidation at glycine residues, were predicted by homology to known arthropod peptide isoforms. Figure 1 shows three examples of mature peptide structural prediction using this workflow; the mature structures of all peptides predicted in this study are provided in Tables 2–4. All protein/peptide alignments were done using the online program MAFFT version 7 (http://mafft.cbrc.jp/ alignment/software/; Katoh and Standley, 2013). 3. Results Thirty-six distinct peptide families were searched for within the extant amphipod TSA dataset (Table 1). In the interest of space, only those searches that resulted in the identification of putative precursor-encoding transcripts are described here (Table 1), with the data presented in alphabetical order base on family name. Proteins described as ‘‘full-length’’ exhibit a functional signal sequence (including a ‘‘start’’ methionine) and are flanked on their C-terminal end by a stop codon; proteins described as ‘‘partial’’ lacked a start methionine (referred to here as C-terminal partial proteins), a stop codon (referred to here as N-terminal partial proteins), or both of these features (referred to here as internal protein fragments). 3.1. Adipokinetic hormone/red pigment concentrating hormone Two transcripts, both from E. veneris, were identified as encoding putative AKH/RPCH precursors (Table 1). Translation of these sequences revealed one to encode a 50 amino acid N-terminal partial preprohormone, and the other a 79 amino acid C-terminal partial protein (Table 1). As the two partial sequences possess a common region of overlap, a 113 amino acid full-length precursor (Echve-prepro-RPCH; Fig. 1A) was obtained by combining the two partial proteins. Four distinct peptides were predicted from Echveprepro-RPCH (Table 2), including one, pQLNFSPGWamide, whose structure is identical to that of authentic RPCH (for a recent review of this peptide family in crustaceans see Christie et al. (2010a)). 3.2. Allatostatin A Three transcripts, all from E. veneris, were identified as encoding putative AST-A precursors (Table 1). Translation of these sequences revealed each to encode a distinct partial protein, with no regions

Peptide Family

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Table 1 Putative amphipod peptide-encoding transcripts identified via in silico transcriptome shotgun assembly (TSA) mining. Amphipod TSA transcript/protein identifications

tblasn search statistics *

 



Transcript accession number

Transcript length

Deduced protein length

Deduced protein type

BLAST score

E-value

E. veneris

GARO01016992 GARO01016991

400 288

79 50

C N

31.2 29.6

0.11 0.25

ACP AST-A

E. veneris

AST-B AST-C

E. veneris E. veneris

H. azteca M. plumulosa

GARO01001929 GARO01001928 GARO01028151 GARO01007400 GARO01000094 GARO01008747 GARO01003800 GAJQ01008793 GAKD01008343

320 4265 234 337 1902 2063 1474 493 283

63 204 77 96 106 41 83 109 84

C C I C F C C F C

55.8 47.4 43.1 34.7 82.4 26.2 25.8 26.9 78.2

8e-10 7e-06 2e-05 0.023 5e-19 5.3 7.1 2.6 2e-19

E. veneris E. veneris

GARO01008548 GARO01012567

1914 1768

149 142

F F

196 125

2e-59 5e-34

E. veneris

GARO01004012

2127

99

C

99.8

2e-24

E. veneris

GARO01002199 GARO01002200 GAKD01021450

3418 272 392

126 32 115

C N N

73.9 35.4 72.4

1e-15 8e-04 1e-16

E. veneris

GARO01007248 GARO01020007 GARO01023024 GARO01020006

1037 491 349 426

154 158 39 20

C C C C

28.5 86.7 30.4 29.6

8.5 1e-21 0.15 0.34

E. veneris H. azteca

GARO01012047 GAJP01003058 GAJQ01003936 GAJQ01005755 GAJP01002079 GAKD01020548 GARO01009877 GAJQ01004351 GARO01004968 GARO01014944 GARO01000399 GAJQ01005023 GARO01010272 GARO01014169 GARO01003692 GAJQ01001266

818 792 852 645 1174 423 1087 774 3199 762 1104 710 689 763 1184 298

104 108 108 115 112 83 95 93 144 115 91 81 76 76 83 75

C F F F F C F F C F F F F F F F

53.5 58.2 58.2 65.9 63.2 61.6 92.4 77.8 58.5 37.7 25.4 78.6 75.1 74.7 59.7 24.3

5e-09 1e-10 1e-10 4e-14 2e-12 1e-12 2e-23 2e-18 3e-10 0.002 6.6 1e-18 9e-18 2e-17 1e-11 9.3

Allatotropin Bursicon a Bursicon b CAPA CCHamide Corazonin CCAP CHH DENamide DH31

M. plumulosa DH44 DXXRLamide ETH EH FLRFamide

FXGGXamide GSEFLamide ILP Intocin Leucokinin MIHa

Myosuppressin Neuroparsin NPF Orcokinin PDH

Proctolin

H. azteca H. azteca M. plumulosa E. veneris H. azteca E. veneris E. veneris H. azteca E. veneris H. azteca

A.E. Christie / General and Comparative Endocrinology 206 (2014) 96–110

Species AKH/RPCH

Table 1 (continued) Peptide Family

Amphipod TSA transcript/protein identifications Species

RYamide sNPF SIFamide Sulfakinin TRP

E. veneris E. veneris E. veneris

tblasn search statistics

Transcript accession number

Transcript length*

Deduced protein length 

Deduced protein type–

BLAST score

E-value

GAJP01004752 GAJP01009440 GAJQ01008636

577 333 497

75 72 72

F F F

153 53.9 53.1

7e-48 2e-10 5e-10

GARO01003670 GARO01023039 GARO01002294

1740 628 3760

76 91 124

F C C

44.6 38.5 33.1

6e-07 0.002 0.23

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Peptide family abbreviations: AKH/RPCH, adipokinetic hormone/red pigment concentrating hormone; ACP, adipokinetic hormone-corazonin-like peptide; AST-A, allatostatin A; AST-B, allatostatin B; AST-C, allatostatin C; CCAP, crustacean cardioactive peptide; CHH, crustacean hyperglycemic hormone; DH31, diuretic hormone 31; DH44, diuretic hormone 44; ETH, ecdysis-triggering hormone; EH, eclosion hormone; ILP, insulin-like peptide; MIH, moltinhibiting hormone; NPF, neuropeptide F; PDH, pigment dispersing hormone; sNPF, short neuropeptide F; TRP, tachykinin-related peptide. Species abbreviations: E. veneris, Echinogammarus veneris; H. azteca, Hyalella azteca; M. plumulosa, Melita plumulosa. Query proteins: AKH/RPCH, Carcinus maenas prepro-RPCH (AAB28133 (Linck et al., 1993)); ACP, Speleonectes cf. tulumensis prepro-ACP (Christie, 2014a); AST-A, Daphnia pulex prepro-AST-A (EFX87432 (Colbourne et al., 2011)); ASTB, D. pulex prepro-AST-B (EFX77444 (Colbourne et al., 2011)); AST-C, D. pulex prepro-AST-C (EFX85706 (Colbourne et al., 2011)); allatotropin, D. pulex prepro-allatotropin (EFX71302 (Colbourne et al., 2011)); bursicon a, D. pulex pre-bursicon a (EFX87546 (Colbourne et al., 2011)); bursicon b, D. pulex pre-bursicon b (EFX87749 (Colbourne et al., 2011)); CAPA, D. pulex prepro-periviscerokinin (Dircksen et al., 2011); CCHamide, D. pulex prepro-CCHamide (EFX80320 (Colbourne et al., 2011)); corazonin, D. pulex prepro-corazonin (EFX86608 (Colbourne et al., 2011)); CCAP, D. pulex prepro-CCAP (EFX70015 (Colbourne et al., 2011)); CHH, Cancer productus prepro-CHH I (ABQ41269 (Hsu et al., 2008)); DENamide, D. pulex prepro-DENamide (Dircksen et al., 2011); DH31, D. pulex prepro-DH31 (EFX90445 (Colbourne et al., 2011)); DH44, Drosophila melanogaster prepro-DH44 A (AAF54421 (Adams et al., 2000)); DXXRLamide, Tigriopus californicus prepro-DXXRLamide Ia (Christie, 2014b); ETH, D. pulex prepro-ETH (Dircksen et al., 2011); EH, D. pulex pre-EH (Dircksen et al., 2011); FLRFamide, Procambarus clarkii prepro-FLRFamide A (BAE06262 [Yasuda-Kamatani and Yasuda, 2006)); FXGGXamide, T. californicus prepro-FXGGXamide Ia (Christie, 2014b); GSEFLamide, T. californicus prepro-GSEFLamide Ia (Christie, 2014b); ILP, D. pulex-prepro-ILP 2 (EFX70023 (Colbourne et al., 2011)); intocin, D. pulex prepro-intocin (EFX71881 (Colbourne et al., 2011)); leucokinin, T. californicus prepro-leucokinin (Christie, 2014b); myosuppressin, Homarus americanus prepro-myosuppressin (ACX46385 (Stevens et al., 2009)); neuroparsin, T. californicus pre-neuroparsin I (Christie, 2014b); NPF, Litopenaeus vannamei prepro-NPF (AEC12204 (Christie et al., 2011d)); orcokinin, D. pulex prepro-orcokinin (EFX70781 (Colbourne et al., 2011); PDH, D. pulex prepro-PDH (EFX87718 (Colbourne et al., 2011)); proctolin, D. pulex prepro-proctolin (EFX69425 (Colbourne et al., 2011)); RYamide, L. vannamei prepro-RYamide (Christie, 2014e); sNPF, D. pulex prepro-sNPF (EFX90018 (Colbourne et al., 2011)); SIFamide, D. pulex prepro-SIFamide (EFX67946 (Colbourne et al., 2011)); sulfakinin, D. pulex prepro-sulfakinin (EFX80896 (Colbourne et al., 2011)); TRP, D. pulex prepro-TRP (EFX86778 (Colbourne et al., 2011)). Transcripts/proteins shown in red font were identified via reciprocal BLASTs using amphipod protein queries. * Length in nucleotides.   Length in amino acids. – Deduced protein type: F, full-length; N, amino-terminal partial; I, internal protein fragment; C, carboxyl-terminal partial. a Identified during the search for crustacean hyperglycemic hormone precursors (see query list below).

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Fig. 1. Three examples of the in silico workflow used for the prediction of putative mature amphipod peptide structures. (A) Predicted processing scheme for Echinogammarus veneris (Echve)-prepro-red pigment concentrating hormone (RPCH). The structure of the putative mature RPCH isoform is shown in red, with those of putative mature linker/ precursor-related peptides shown in blue. (B) Predicted processing scheme for E. veneris prepro-allatostatin C (AST-C) I. The structure of the putative mature AST-C isoform is shown in red, with those of two putative mature linker/precursor-related peptides shown in blue. In this schematic, the disulfide bond predicted between the cysteine residues in the putative mature AST-C is indicated by the inverted red bracket. (C) Predicted processing scheme for E. veneris-prepro-sulfakinin (a carboxyl [C]-terminal partial protein). The structures of the putative mature sulfakinins are shown in red, with those of putative mature linker/precursor-related peptides shown in blue. In this panel, the presence of sulfated tyrosine residues is indicated by ‘‘Y(SO3H)’’, with pyroglutamic acid indicated by ‘‘pQ’’. The ‘‘+’’ symbol at the amino-terminus of one of the linker/precursor-related peptides indicates the presence of unknown additional amino acids. A detailed description of the workflow used for peptide prediction is provided in Section 2.2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

of overlap present between any of them. Two of the proteins are Cterminal partial precursors, one being 63 amino acids long (Echveprepro-AST-A I; Fig. 2A1), and the other 204 amino acids in overall length (Echve-prepro-AST-A II; Fig. 2A2); the third protein is a 77 amino acid internal precursor fragment (Echve-prepro-AST-A III; Fig. 2A3). The structures of six distinct peptides were predicted from Echve-prepro-AST-A I, with 15 and five distinct peptides each predicted from Echve-prepro-AST-A II and III, respectively (Table 2). Of the peptides collectively predicted from the three partial precursors, 18 (17 full-length and one partial) possess –

YXFGLamide C-termini (or a close approximation thereof), which is the hallmark of the AST-A family (Christie et al., 2010a); 16 of the 18 AST-As are distinct in structure (Table 2), with one isoform, VQDYAFGLamide (Table 2), shared by both Echve-prepro-AST-A I and III (Fig. 2A1 and A3). 3.3. Allatostatin B A single E. veneris transcript was identified as encoding a putative AST-B precursor (Table 1). Translation of this sequence

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Table 2 Predicted peptidome of Echinogammarus veneris.

(continued on next page)

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⁄ Identified from partial FMRFamide-like peptide precursors. Abbreviations: a, amidated C-terminus; +, likely partial peptide with additional unknown amino acids; pQ/pE, pyroglutamic acid; C, disulfide bonded cysteine (see text for descriptions of bonding partners); Y(SO3H), sulfated tyrosine. Prediction of sulfation state of tyrosine residues and disulfide bridging between cysteines was done only for full-length peptides. A detailed description of the workflow used for peptide prediction is provided in Section 2.2.

Table 3 Predicted peptidome of Hyalella azteca.

Abbreviations: a, amidated C-terminus; pQ, pyroglutamic acid; C, disulfide bonded cysteine (see text for descriptions of bonding partners). A detailed description of the workflow used for peptide prediction is provided in Section 2.2.

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Table 4 Predicted peptidome of Melita plumulosa.

Abbreviations: a, amidated C-terminus; +, likely partial peptide with additional unknown amino acids; C, disulfide bonded cysteine (see text for descriptions of bonding partners); Y(SO3H), sulfated tyrosine. Prediction of sulfation state of tyrosine residues and disulfide bridging between cysteines was done only for full-length peptides. A detailed description of the workflow used for peptide prediction is provided in Section 2.2.

revealed it to encode a 96 amino acid C-terminal partial protein (Echve-prepro-AST-B; Fig. 2B). Seven peptides, including four possessing –WX6Wamide C-termini, the hallmark of the AST-B family (Christie et al., 2010a), were predicted from Echve-prepro-AST-B. Of the AST-Bs, two are encoded as single copies within the extant portion of the precursor, with two copies present of one isoform, i.e. DPENNWGNFRGSWamide (Fig. 2B and Table 2). All of the linker/precursor-related peptides possess unique structures (Table 2). 3.4. Allatostatin C Stereotypical members of the AST-C family are characterized by a pyroglutamine blocked N-terminus, the unamidated C-terminal motif – PISCF, and a disulfide bridge between the cysteine residues located at positions 7 and 14, though variations in these features has been noted, e.g. the peptide SYWKQCAFNAVSCFamide (a disulfide bridge predicted between the positions 6 and 13 cysteine residues), a broadly conserved arthropod AST-C variant (see Christie et al. (2010a) for review). Five transcripts were identified within the amphipod TSA dataset as encoding putative AST-C precursors, three from E. veneris and one each from H. azteca and M. plumulosa (Table 1). Translation of the E. veneris sequences revealed one to encode a 106 amino acid full-length preprohormone (Echve-prepro-AST-C I; Fig. 1B), with the other two encoding C-terminal partial proteins of 41 (Echve-prepro-AST-C II; Fig. 2C1) and 83 (Echveprepro-AST-C III; Fig. 2C2) amino acids, respectively. Three distinct peptides were predicted from Echve-prepro-AST-C I (Table 2), including SYWKQCAFNAVSCFamide (a disulfide bridge predicted between the positions 6 and 13 cysteine residues), which is identical in structure to the broadly conserved arthropod AST-C variant noted above. Three and four distinct peptides were predicted from Echve-prepro-AST-C II and III, respectively (Table 2). In each of these precursors, one of the predicted peptides, GQPDGRLYWRCYFNAVSCF (disulfide bridging between the cysteines at positions 11 and 18; Table 2) in Echve-prepro-AST-C II and pQIRYHQCYFNPISCF (a disulfide bridge predicted between the positions 7 and 14 cysteines; Table 2) in Echve-prepro-AST-C III, exhibits structural characteristics of the AST-C family. Translation of the H. azteca transcript revealed a 109 amino acid full-length AST-C precursor (Hyaaz-prepro-AST-C; Fig. 3A). Four distinct peptides were predicted from this preprohormone (Table 3), including pQVRYHQCYFNPISCF (disulfide bridging between the positions 7 and 14 cysteine residues), which possesses all of the structural hallmarks of a stereotypical isoform of AST-C. Translation of the M. plumulosa TSA sequence revealed an 84 amino acid C-terminal partial protein (Melpl-prepro-AST-C; Fig. 4A). Two distinct peptides were predicted from the extant portion of this precursor (Table 4), including SYWKQCAFNAVSCFa-

mide (bridging predicted between the positions 6 and 13 cysteines), which is the same isoform of AST-C derived from Echve-prepro-AST-C I (see above). 3.5. Bursicon a A single E. veneris TSA sequence was identified as encoding a putative bursicon a precursor (Table 1). Translation of this transcript revealed it to encode a 149 amino acid prehormone (Echve-pre-bursicon a; Fig. 2D). A single 120 amino acid peptide was predicted from Echve-pre-bursicon a (Table 2). Analysis of this peptide by DiANNA predicts disulfide bridges between the cysteine residues located at positions 3 and 27, 17 and 89, 31 and 49, 52 and 87, and 66 and 92 (Table 2). This pattern of bridging, as well as the amino acid sequence of the peptide, strongly supports it being a member of the bursicon a family (Christie et al., 2010a). 3.6. Bursicon b One E. veneris transcript was identified as encoding a putative bursicon b precursor (Table 1). Translation of this sequence revealed a 142 amino acid prehormone (Echve-pre-bursicon b; Fig. 2E). A single 113 amino acid peptide was predicted from Echve-pre-bursicon b; analysis of this peptide by DiANNA suggests disulfide bridging between its position 6 and 61, 30 and 64, 39 and 106, 43 and 104, and 79 and 109 cysteines (Table 2). The amino acid sequence of the peptide derived from Echve-pre-bursicon b, as well as its pattern of disulfide bridges, strongly supports it being a member of the bursicon b family (Christie et al., 2010a). 3.7. Crustacean hyperglycemic hormone A single E. veneris TSA sequence was identified as encoding a putative CHH precursor (Table 1). Translation of this transcript revealed a 99 amino acid C-terminal partial protein (Echve-prepro-CHH; Fig. 2F). Two peptides, one a 72 amino acid full-length peptide and the other a partial sequence, were predicted from Echve-prepro-CHH (Table 2). The full-length peptide, with disulfide bridges predicted between the first and fifth, second and fourth, and third and sixth cysteines and an amidated C-terminus (Table 2), shows sequence homology and a pattern of disulfide bridging similar to known crustacean members of the CHH superfamily (e.g. Böcking et al., 2002). 3.8. Diuretic hormone 31 Three transcripts, two from E. veneris and one from M. plumulosa, were identified as encoding putative DH31 precursors

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Fig. 2. Putative Echinogammarus veneris (Echve) pre/preprohormones deduced from transcriptome shotgun assembly sequence data. (A1) The carboxyl (C)-terminal portion of Echve-prepro-allatostatin A I. (A2) The C-terminal portion of Echve-prepro-allatostatin A II. (A3) An internal fragment of Echve-prepro-allatostatin A III. (B) The C-terminal portion of Echve-prepro-allatostatin B. (C1) The C-terminal portion of Echve-prepro-allatostatin C II. (C2) The C-terminal portion of Echve-prepro-allatostatin C III. (D) Echvepre-bursicon a. (E) Echve-pre-bursicon b. (F) The C-terminal portion of Echve-prepro-crustacean hyperglycemic hormone. (G) Echve-prepro-diuretic hormone 31. (H1) The Cterminal portion of Echve-prepro-FLRFamide I. (H2) The C-terminal portion of Echve-prepro-FLRFamide IIa. (H3) The C-terminal portion of Echve-prepro-FLRFamide IIb. (I) The C-terminal portion of Echve-pre-molt-inhibiting hormone. (J) Echve-prepro-neuropeptide F. (K) The C-terminal portion of Echve-prepro-orcokinin. (L1) Echve-prepropigment dispersing hormone I. (L2) Echve-prepro-pigment dispersing hormone II. (M1) Echve-prepro-proctolin Ia. (M2) Echve-prepro-proctolin Ib. (M3) Echve-preproproctolin II. (N) Echve-prepro-SIFamide. (O) The C-terminal portion of Echve-prepro-tachykinin-related peptide. In this figure, signal peptides are shown in gray, while all mono/dibasic cleavage loci are shown in black. For each sequence, the isoform(s) of the peptide for which the precursor is named is/are shown in red, with all linker/ precursor-related peptides shown in blue. The ‘‘+’’ symbol indicate the presence of additional, unknown amino acid residues. In panels H2 and H3, an isoform of RYamide is shown in green. A detailed description of the workflow used for peptide prediction is provided in Section 2.2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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3 Fig. 3. Putative Hyalella azteca (Hyaaz) pre/preprohormones deduced from transcriptome shotgun assembly sequence data. (A) Hyaaz-prepro-allatostatin C. (B) Hyaaz-premolt-inhibiting hormone. (C) Hyaaz-prepro-myosuppressin. (D) Hyaaz-pre-neuroparsin. (E) Hyaaz-prepro-neuropeptide F. (F) Hyaaz-prepro-pigment dispersing hormone. (G1) Hyaaz-prepro-proctolin I. (G2) Hyaaz-prepro-proctolin IIa. (G3) Hyaaz-prepro-proctolin IIb. In this figure, signal peptides are shown in gray, while all mono/dibasic cleavage loci are shown in black. For each sequence, the isoform of the peptide for which the precursor is named is shown in red, with all linker/precursor related peptides shown in blue. A detailed description of the workflow used for peptide prediction is provided in Section 2.2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. Putative Melita plumulosa (Melpl) pre/preprohormones deduced from transcriptome shotgun assembly sequence data. (A) The carboxyl (C)-terminal portion of Melplprepro-allatostatin C. (B) The amino (N)-terminal portion of Melpl-prepro-diuretic hormone 31. (C) The C-terminal portion of Melpl-pre-neuroparsin. In this figure, signal peptides are shown in gray, while all mono/dibasic cleavage loci are shown in black. For each sequence, the isoform of the peptide for which the precursor is named is shown in red, with all linker/precursor related peptides shown in blue. The ‘‘+’’ symbol indicate the presence of additional, unknown amino acid residues. A detailed description of the workflow used for peptide prediction is provided in Section 2.2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(Table 1). Translation of the two E. veneris sequences revealed one to encode a 126 amino acid C-terminal partial preprohormone and the other a 32 amino acid N-terminal partial protein. The two sequences share a 19 amino acid region of overlap with variation at just one residue. Combining the two partial proteins allowed for the deduction of a 139 amino acid full-length protein (Echveprepro-DH31; Fig. 2G), with the variable reside being position 14 (a serine in the C-terminal partial protein (shown in Fig. 2G) vs. an alanine in the N-terminal partial sequence), which is located in the signal peptide. Three distinct peptides were predicted from Echve-prepro-DH31, one of which, GLDLGLGRGFSGSQAAKHLMGL SAANFAGGPamide, possesses the structural hallmarks of the DH31 family, i.e. an overall length of 31 amino acids, an amidated C-terminus, and 13 highly conserved residues, i.e.

XXDXGLXRGXSGXXXAKXXXXXXXANXXXGPamide (Christie et al., 2010a). Translation of the M. plumulosa transcript revealed it to encode a 115 amino acid N-terminal partial protein (Melpl-prepro-DH31; Fig. 4B). Three peptides were predicted from the extant portion of this precursor (Table 4), including GIDLGMGRGFSGSQAAKHLMGLSAATF ANGPamide, which possesses many of the structural features common to members of the DH31 family. 3.9. FLRFamide Four transcripts, all from E. veneris, were identified as encoding putative FLRFamide-like peptide precursors (Table 1). Three distinct, but similar, N-terminal partial proteins were deduced

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from this collection of TSA sequences. One of the partial proteins, named here Echve-prepro-FLRFamide I (Fig. 2H1), is 154 amino acids in length and is predicted to liberate six distinct peptides (Table 2), including three isoforms of FL/IRFamide, i.e. AFPRSFLRFamide, pEPSDLHFIRFamide and ARNNKNFLRFamide. The second partial precursor, Echve-prepro-FLRFamide IIa (Fig. 2H2), is 158 amino acids long and is predicted to produce eight distinct peptides (Table 2), including the FLRFamides AFPVNFSRFamide and pEPSDLNFIRFamide. The final peptide predicted from Echve-prepro-FLRFamide IIa possesses structural similarity to the RYamide family of peptides (see Section 3.17). A partial protein encoded by two transcripts is highly similar to the C-terminus of Echveprepro-FLRFamide IIa, and was named here Echve-prepro-FLRFamide IIb (Fig. 2H3). It too contains an isoform of RYamide (see Section 3.17), which is distinct from that present in Echve-preproFLRFamide IIa (Table 2), as well as a partial linker/precursorrelated peptide that is identical to the corresponding portion of one present in Echve-prepro-FLRFamide IIa (Table 2). 3.10. Molt-inhibiting hormone Three amphipod transcripts, one from E. veneris and two from H. azteca, were identified as encoding putative MIH precursors (Table 1). Translation of the E. veneris sequence revealed a 104 amino acid C-terminal partial prehormone (Echve-pre-MIH; Fig. 2I). The MIH predicted from Echve-pre-MIH is 89 amino acids long, possesses an amidated C-terminus, and is predicted to possess disulfide bridges between its first and third, second and fourth, and fifth and sixth cysteine residues (Table 2), which is a variant on the bridging pattern commonly exhibited by members of the CHH superfamily, i.e. the first and fifth, second and fourth, and third and sixth cysteines are typically bridged (e.g. Böcking et al., 2002). Translation of the two H. azteca transcripts revealed each to encode an identical 108 amino acid full-length prehormone (Hyaaz-pre-MIH; Fig. 3B). As for Echve-pre-MIH, an 89 amino acid, Cterminally amidated MIH isoform (with disulfide bridging between the first and third, second and fourth, and fifth and sixth cysteines) was predicted from Hyaaz-pre-MIH (Table 3). 3.11. Myosuppressin A single H. azteca transcript was identified as encoding a putative myosuppressin precursor (Table 1). Translation of this sequence revealed it to encode a 115 amino acid full-length preprohormone (Hyaaz-prepro-myosuppressin; Fig. 3C). Three peptides were predicted from this precursor (Table 3), including pQDLDHVFLRFamide, a known and broadly conserved decapod isoform of myosuppressin (Christie et al., 2010a). 3.12. Neuroparsin Two amphipod TSA sequences, one from H. azteca and the other from M. plumulosa, were identified as encoding putative neuroparsin precursors (Table 1). Translation of the H. azteca transcript revealed it to encode a 112 amino acid full-length prehormone (Hyaaz-pre-neuroparsin; Fig. 3D). A single 76 amino acid peptide was predicted from Hyaaz-pre-neuroparsin, and analysis of this peptide by DiANNA suggests disulfide bridging is present between its position 4 and 50, 18 and 27, 32 and 61, 40 and 70, 56 and 76, and 58 and 64 cysteines (Table 3). This pattern of disulfide bridges, as well as the amino acid sequence, suggests that this peptide is a member of the neuroparsin family (e.g. Badisco et al., 2007). Translation of the M. plumulosa transcript revealed it to encode an 83 amino acid C-terminal partial protein (Melpl-pre-neuroparsin; Fig. 4C). A single 77 amino acid peptide was predicted from

Melpl-pre-neuroparsin (the signal peptide cleavage locus in this protein was identified by homology to that of Hyaaz-pre-neuroparsin rather that via SignalP analysis), with DiANNA suggesting disulfide bridges between its positions 4 and 51, 19 and 33, 28 and 59, 41 and 77, 57 and 71, and 62 and 65 cysteines (Table 4). As with the peptide predicted from Hyaaz-pre-neuroparsin, this peptide’s sequence and disulfide bridging pattern suggest it is a member of the neuroparsin family. 3.13. Neuropeptide F Members of the NPF family are typified by an overall length of 30 or more amino acids, the C-terminal motif –GRPRFamide, and tyrosine residues at positions 10 and 17 from the C-terminus (Christie et al., 2010a). Two transcripts, one from E. veneris and the other from H. azteca, were identified as encoding putative NPF precursors (Table 1). Translation of the E. veneris sequence revealed it to encode a 95 amino acid full-length preprohormone (Echve-prepro-NPF; Fig. 2J). Two peptides were predicted from Echve-prepro-NPF (Table 2), including RPEPAQLVAMADALKYLH QLDKYYSQVARPRFamide, which shows considerable structural similarity to members of the NPF family. Translation of the H. azteca transcript revealed it to encode a 93 amino acid full-length precursor (Hyaaz-prepro-NPF; Fig. 3E). As with the E. veneris preprohormone, two peptides were predicted from Hyaaz-prepro-NPF (Table 3), including RPEPAQLVAMADAL KYLHQLDKYYSQVARPRFamide, which is identical in structure to the isoform of NPF predicted from Echve-prepro-NPF. 3.14. Orcokinin Stereotypical members of the orcokinin family possess the Nterminal motif NFDEIDR–, an overall length of 13 amino acids, and are non-amidated at their C-termini (Christie et al., 2010a). A single E. veneris transcript was identified as encoding a putative orcokinin precursor (Table 1). Translation of this sequence revealed it to encode a 144 amino acid C-terminal partial protein (Echveprepro-orcokinin; Fig. 2K). Ten peptides were predicted from the extant portion of Echve-prepro-orcokinin (nine full-length and one partial; Table 2), including seven variant orcokinin family members (i.e. slight modifications of the N-terminal consensus motif, length, and/or C-terminal amidation state). Of these orcokinins, five are present within Echve-prepro-orcokinin as single copies, with two copies of NFDEINRSAFGFN contained within the extant portion of the preprohormone. 3.15. Pigment dispersing hormone Among the first invertebrate neuropeptides fully characterized were members of the PDH family, i.e. NSGMINSILGIPRVMTEAamide (a-PDH; Fernlund, 1976) and NSELINSILGLPKVMNDAamide (b-PDH; Rao et al., 1985), so named for their ability to disperse pigment granules in chromatophores. Since their initial discovery, other structurally related peptides have been identified, with these isoforms also being 18 amino acids in overall length and C-terminally amidated (Christie et al., 2010a). Three amphipod transcripts, two from E. veneris and one from H. azteca, were identified as encoding putative PDH precursors (Table 1). Translation of one E. veneris sequence revealed a 115 amino acid full-length preprohormone (Echve-prepro-PDH I; Fig. 2L1). Two peptides were predicted from Echve-prepro-PDH I (Table 2), including NSELINSLLGLPKIL RGPLGLPKGSDKNSAN, which appears to be a C-terminally extended, non-amidated PDH variant. Translation of the second E. veneris transcript revealed a 91 amino acid full-length preprohormone (Echve-prepro-PDH II; Fig. 2L2). Three peptides were pre-

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dicted from this precursor (Table 2), including NSELLNTLLGSK SLAALRAAamide, a novel 20 amino acid isoform of PDH. Translation of the H. azteca transcript revealed an 81 amino acid full-length preprohormone (Hyaaz-prepro-PDH; Fig. 3F). Three peptides were predicted from Hyaaz-prepro-PDH (Table 3), including NSELLNTLLGSKNLVALRAAamide, another novel 20 amino acid PDH variant.

from this protein (Table 2), including GPYRKPPFNGSIFamide, which is identical to the highly conserved decapod SIFamide isoform GYRKPPFNGSIFamide, save the addition of a proline residue at position 2. The linker/precursor-related peptide predicted from Echve-prepro-SIFamide, i.e. SGISNFVEEDASRMAAVCRVALDTCS TWFPEADQD, is predicted by DiANNA to possess a disulfide bridge between its positions 18 and 25 cysteines (Table 2).

3.16. Proctolin

3.19. Sulfakinin

The pentapeptide RYLPT, commonly referred to as proctolin, is a broadly conserved arthropod neuropeptide (Christie et al., 2010a). Seven amphipod transcripts, three from E. veneris and four from H. azteca, were identified as encoding putative proctolin precursors (Table 1). Translation of the E. veneris sequences revealed each to encode a full-length precursor, with two proteins being 76 amino acids long, and the third, 83 amino acids in overall length. The two 76 amino acid precursors differ from one another at two residues (one in the signal peptide and one in a linker/precursorsrelated peptide) and have been named here Echve-prepro-proctolin Ia (Fig. 2M1) and Ib (Fig. 2M2). Five peptides were predicted from each of these preprohormones (Table 2), including RYLPT (authentic proctolin); Echve-prepro-proctolin Ia and Ib share in common three of their four linker/precursor-related peptides (Fig. 2M1 and M2). The 83 amino acid precursor (named here Echve-prepro-proctolin II; Fig. 2M3) is also predicted to give rise to five peptides (Table 2), one of which is authentic proctolin; the four linker/precursor-related peptides derived from Echve-preproproctolin II are distinct from those predicted from Echve-preproproctolin Ia and Ib. Translation of the four H. azteca transcripts also revealed each to encode a full-length preprohormone, with two encoding 75 amino acid proteins and the others, 72 amino acid precursors. The two 75 amino acid proteins are identical in amino acid sequence (Hyaaz-prepro-proctolin I; Fig. 3G1). Five distinct peptides were predicted from Hyaaz-prepro-proctolin I (Table 3), including RYLPT. The two 72 amino acid precursors differ from one another at a single residue (located in a linker/precursor-related peptide) and have been named here Hyaaz-prepro-proctolin IIa (Fig. 3G2) and IIb (Fig. 3G3). Four peptides each were predicted from these preprohormones (Table 3), including authentic proctolin; all but one of the linker/precursor-related peptides is shared by these precursors, though all are distinct from those derived from Hyaaz-prepro-proctolin I.

The C-terminal motif –Y(SO3H)GHM/LRFamide is the defining character of members of the sulfakinin family (Christie et al., 2010a). A single E. veneris transcript was identified as encoding a putative sulfakinin precursor (Table 1). Translation of this sequence revealed a 91 amino acid C-terminal partial protein (Echve-prepro-sulfakinin; Fig. 1C). Five peptides (Table 2), four full-length and one partial, were predicted from Echve-preprosulfakinin, including two, pQYADY(SO3H)GHLRFamide and AEFGDY(SO3H) GHLRFamide, that possess the structural hallmark of the sulfakinin family.

3.17. RYamide While no amphipod RYamide preprohormone-encoding transcripts similar to that recently identified from the shrimp Litopenaeus vannamei (Christie, 2014e) were discovered via BLAST searches of the extant TSA data, two E. veneris peptides possessing –RYamide C-termini, NLLRYamide and NFLRYamide (Table 2), were predicted from partial FLRFamide-like peptide precursors (see Section 3.9). 3.18. SIFamide Stereotypical members of the SIFamide family possess the structure XYRKPPFNGSIFamide, where X in most decapod crustaceans is glycine (Christie et al., 2010a), though quite variant sequences have been discovered, e.g. TRKLPFNGSIFamide from cladoceran Daphnia pulex (Verleyen et al., 2009). Within the amphipod TSA dataset, a single E. veneris transcript was identified as encoding a putative SIFamide precursor (Table 1). Translation of this sequence revealed a 76 amino acid full-length preprohormone (Echve-prepro-SIFamide; Fig. 2N). Two peptides were predicted

3.20. Tachykinin-related peptide A single E. veneris transcript was identified as encoding a putative TRP precursor (Table 1). Translation of this sequence revealed it to encode a 124 amino acid C-terminal partial preprohormone (Echve-prepro-TRP; Fig. 2O). Nine peptides, eight full-length and one partial, were predicted from Echve-prepro-TRP (Table 2), including APYGFVGMRamide (2 copies), APSGFVGMRamide, APMGFFGMRamide, and APMGFMGMRamide, peptides that possess the C-terminal motif –FXGXRamide, the structural hallmark of the TRP family (Christie et al., 2010a). 4. Discussion 4.1. Transcriptome mining continues to be a rapid and powerful new method for crustacean peptide discovery In the late 1960’s/early 1970’s, Fernlund and Josefsson isolated and characterized the first neuropeptide from any invertebrate, i.e. RPCH from the eyestalk ganglia of the shrimp Pandalus borealis (Fernlund and Josefsson 1968, 1972). The discovery of RPCH by these authors was achieved using a bioassay-directed purification process involving chromatographic/biochemical isolation of the peptide from large pool of starting tissue (Fernlund and Josefsson 1968), followed by structural analysis using a combination of proteolytic cleavage, Edman analysis, and mass spectrometry (Fernlund and Josefsson 1972). For the next quarter-century this strategy was a standard method for crustacean peptide discovery, with the isolation and characterization of first crustacean members of the PDH, FLRFamide and TRP families, among others, achieved using this methodology (Christie et al., 1997; Fernlund, 1976; Trimmer et al., 1987). With the development and implementation of mass spectral techniques for identifying and characterizing peptide structures, a shift in focus occurred in the early 2000’s from the biochemical isolation/purification and structural elucidation of individual crustacean neuropeptides to the elucidation of peptidomes, the full complement of neuropeptides present in a species (e.g. Huybrechts et al., 2003; Li et al., 2003). While mass spectrometry continues to be a major contributor to peptidome discovery in crustaceans (e.g. Hui et al., 2012, 2013), the public deposition of large genomic/transcriptomic data sets for members of the Crustacea has provided an alternative strategy for neuropeptide discovery, namely in silico genome/transcriptome mining. For small, rare, and/or geographically inaccessible species, genomics/

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transcriptomics is often the only tractable means for peptidomic analyses, as the collection of the large tissue pools needed for standard biochemical and/or mass spectral peptide discovery is not feasible. Via in silico means, the first peptidomes for members of the Cladocera (Christie et al., 2011c; Dircksen et al., 2011; Gard et al., 2009), the Cirripedia (Yan et al., 2012), the Copepoda (Christie, 2014b,d; Christie et al., 2013), the Euphausiacea (Toullec et al., 2013), the Remipedia (Christie, 2014a) and the Branchiura (Christie, 2014c) have recently been deduced. Here, using the in silico mining of TSA sequence datasets, the first neuropeptides from members of the Amphipoda have been predicted. Clearly, in silico transcriptome mining has become a major contributor to peptide discovery in the Crustacea, and with ongoing efforts to sequence more crustacean transcriptomes, likely will continue to do so for the foreseeable future. 4.2. Identification of the first neuropeptides from members of the Amphipoda The public deposition of TSA sequences for members of the Amphipoda has allowed for the prediction of the first neuropeptides from this crustacean order. Using a well-vetted protocol, putative peptide-encoding transcripts from three species, E. veneris (29 transcripts), H. azteca (11 transcripts) and M. plumulosa (three transcripts), were identified via tblastn searches using known crustacean or insect precursor proteins as queries. Translation of the identified transcripts allowed for the determination of pre/ preprohormone sequences, which were subjected to a peptide prediction workflow to determine the structures of putative mature amphipod neuropeptides. In total, 139 peptides were predicted from the three species, 110 from E. veneris, 23 from H. azteca, and six from M. plumulosa. The predicted E. veneris peptides included isoforms of AKH/RPCH, AST-A, AST-B, AST-C, bursicon a, bursicon b, CHH, DH31, FLRFamide, MIH, NPF, orcokinin, PDH, proctolin, RYamide, SIFamide, sulfakinin and TRP, while those deduced for H. azteca included members of the AST-C, MIH, neuroparsin, NPF, PDH and proctolin families. M. plumulosa isoforms of AST-C, DH31 and neuroparsin were also discovered. With a few exceptions, e.g. pQLNFSPGWamide (authentic RPCH) SYWKQCAFNAVSCFa and pQIRYHQCYFNPISCF (two AST-Cs), pQDLDHVFLRFamide (myosuppressin) and RYLPT (authentic proctolin), which are isoforms of their respective families shared with and broadly conserved in members of the Decapoda, the peptide structures predicted for these amphipods are novel, being described here for the first time. Among the noteworthy amphipod peptide discoveries were the identifications of orcokinins possessing SFDEIDR– rather than the typical NFDEIDR– N-termini, e.g. SFDEINRSNFGFN, and a C-terminally amidated isoform of this peptide family, i.e. SFDEINRSNFGFSamide. The PDHs identified from the Amphipoda were also atypical, being longer than the standard 18 amino acids seen in most other arthropods, e.g. NSELLNTLLGSKSLAALRAAamide, with one family member being significantly extended (31 amino acids long) and unamidated, i.e. NSELINSLLGLPKILR GPLGLPKGSDKNSAN. Similarly the SIFamide discovered here is a 13 rather than 12 amino acid long variant, i.e. GPYRKPPFNGSIFamide. Again, the peptides predicted in this study are the only ones thus far described from the Amphipoda, and as such, they provide a foundation for initiating investigations of peptidergic control of physiology and behavior in members of this ecologically important crustacean order. 4.3. Amphipod neuropeptides – potential targets for studies of endocrine disruption As outlined in the Introduction, amphipods are routinely used as model species for studies of aquatic toxicity. In amphipods,

changes in food intake, growth and reproduction are three aspects of physiology and behavior commonly used to assess the effects of chemical toxicants (Agostinho et al., 2012; Charron et al., 2013; Dedourge-Geffard et al., 2009; Dutra et al., 2009; Jacobson and Sundelin, 2006; Mann et al., 2011; Pedersen et al., 2013; Tong et al., 2010). Since each of these processes has been shown to be under hormonal control in other crustaceans, it is highly likely that they will ultimately be shown to play similar roles in amphipods (for review of the functional roles played by crustacean neuropeptides see Christie et al. (2010a)). For example, members of the NPF family have been shown to be powerful modulators of food intake in shrimp (Christie et al., 2011d), while members of the CHH superfamily have been demonstrated to play key roles in reproductive control in a variety of crustacean species (e.g. Zmora et al., 2009). Similarly, members of the CHH and bursicon families have been shown to be major contributors to the control of ecdysis in crustaceans, a process critical for growth (e.g. Webster et al., 2013). Given that each of these peptide families has been identified from at least one member of the Amphipoda, these, and the other peptide groups described in this study, provide, for the first time, a set of discrete targets for assessing the effects of chemical toxicants on peptide hormone expression in this group of animals. Acknowledgment The Cades Foundation of Honolulu, Hawaii provided funding for this study. 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Identification of the first neuropeptides from the Amphipoda (Arthropoda, Crustacea).

Despite being used as models in the field of ecotoxicology, including use in studies of endocrine disruption, little is known about the hormonal syste...
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