General and Comparative Endocrinology 201 (2014) 74–86

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Prediction of the first neuropeptides from a member of the Remipedia (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

i n f o

Article history: Received 26 October 2013 Revised 10 January 2014 Accepted 28 January 2014 Available online 11 February 2014 Keywords: Speleonectes cf. tulumensis BLAST Bioinformatics Neurohormone Neuropeptide Transcriptome shotgun assembly

a b s t r a c t The Remipedia is a small, recently described crustacean class that inhabits submerged marine/anchialine cave systems. Phylogenetic and morphological investigations support a sister group relationship between these animals and the hexapods. The recent deposition of numerous (>100,000) transcriptome shotgun assembly sequences for Speleonectes cf. tulumensis provides a unique resource to identify proteins of interest from a member of the Remipedia. Here, this dataset was mined for sequences encoding putative neuropeptide pre/preprohormones, with the mature peptides predicted from the deduced precursors using an established workflow. The structures of 40 mature peptides were obtained via this strategy, including members of 11 well-known arthropod peptide families (adipokinetic hormone/corazonin-like peptide [ACP], allatostatin A, allatostatin C, diuretic hormone 31, eclosion hormone, ion transport peptide/crustacean hyperglycemic hormone, neuropeptide F, proctolin, SIFamide, sulfakinin and tachykinin-related peptide); these are the only peptides thus far described from any member of the Remipedia. Comparison of the Speleonectes isoforms with those from other crustaceans and hexapods revealed the peptidome of this species to have characteristics of both subphyla (e.g. it possesses the stereotypical decapod crustacean SIFamide and tachykinin-related peptide isoforms, while simultaneously being the only crustacean with an insect AKC). Moreover, BLAST searches in which the deduced Speleonectes precursors were compared to the pancrustacean protein database most frequently returned insect homologs as the closest matches. The peptidomic analyses presented here are consistent with the hypothesized phylogenetic position of the Remipedia within the Pancrustacea, and serve as a foundation from which to launch future investigations of peptidergic signaling in remipedes. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction In 1981, a new species of crustacean, Speleonectes lucayensis, was described from four individuals collected by divers from an anchialine cave (Lucayan Cavern) located on Grand Bahama Island, Commonwealth of the Bahamas (Yager, 1981). The morphology of S. lucayensis, including its large number of trunk sections, each containing laterally-directed, biramous swimming appendages, precluded its inclusion in any extant crustacean class. Thus, a new taxon of crustaceans was proposed, the Remipedia, named for the oar-like shape of the swimming legs of S. lucayensis (Yager, 1981). Since this initial description, a number of other remipedes have been identified, all from neotropical marine or anchialine cave systems, with the class now comprised of approximately 30 living species (e.g. Hoenemann et al., 2013). Arthropod phylogeny has been an area of ongoing debate for some time. While the myriapods and hexapods were initially ⇑ Fax: +1 808 956 6984. E-mail address: [email protected] http://dx.doi.org/10.1016/j.ygcen.2014.01.017 0016-6480/Ó 2014 Elsevier Inc. All rights reserved.

grouped as sister taxa, and the crustaceans more distantly related (e.g. Heymons, 1901; Pocock, 1893), this grouping, the Atelocerata, has fallen from favor, and today there is a general consensus that the Crustacea and Hexapoda are more closely related, forming a monophyletic clade known as the Tetraconata or Pancrustacea (e.g. Dunn et al., 2008; Friedrich and Tautz, 1995; Richter, 2002; Roeding et al., 2007, 2009). Despite strong support for the pancrustacean lineage, which crustacean taxon is the sister group to the hexapods has been controversial, with many taxa proposed as occupying this position (e.g. Jenner, 2010). Recently there has been growing molecular and morphological data suggesting that it is the Remipedia that is the sister group to the hexapods (e.g. Ertas et al., 2009; Fanenbruck et al., 2004; Harzsch, 2006; Regier et al., 2010; Stemme et al., 2012, 2013; von Reumont et al., 2012), which is somewhat surprising given its initial basal placement as the sister group to the entire pancrustacean line (e.g. Schram, 1986). If the sister group status of the remipedes and hexapods is correct, then the Remipedia is a key player for any complete understanding of evolution within the Pancrustacea.

A.E. Christie / General and Comparative Endocrinology 201 (2014) 74–86

Recently, a large transcriptome shotgun assembly (TSA) sequence dataset for Speleonectes cf. tulumensis (recently proposed as a member of the newly erected genus Xibalbanus (Hoenemann et al., 2013), a remipede from the Yucatan Peninsula of Mexico, was deposited in GenBank; these data are the result of a sequencing project designed to assess pancrustacean phylogeny, including the placement of the Remipedia (von Reumont et al., 2012). This TSA dataset, which consists of over 100,000 sequences, is the only major, publicly accessible transcriptomic resource for this enigmatic group of crustaceans, and thus provides a unique platform for protein discovery in remipedes. The arthropods, particularly members of the Crustacea and Hexapoda, have long served as models for the field of peptide biology, specifically physiological/behavioral control by peptide hormones (for recent reviews see: Altstein and Nässel, 2010; Audsley et al., 2008; Audsley and Weaver, 2009; Bendena, 2010; Christie, 2011; Christie et al., 2010a; Clynen et al., 2010; Nässel and Winther, 2010; Scherkenbeck and Zdobinsky, 2009; Taghert and Nitabach, 2012). Included among the extant studies are many that have focused on characterizing the peptidomes of crustaceans and insects, both via mass spectrometry and/or via genome/transcriptome mining (e.g. Christie, 2008a; Christie et al., 2008, 2010b, 2011a, 2013; Clynen et al., 2006; Dircksen et al., 2011; Fu et al., 2005; Gard et al., 2009; Hauser et al., 2010; Hewes and Taghert, 2001; Hui et al., 2013; Hummon et al., 2006; Huybrechts et al., 2003, 2005, 2010; Li et al., 2003; Ma et al., 2008, 2009a, 2010; Stemmler et al., 2007; Verleyen et al., 2004; Weaver and Audsley, 2008). These studies provide useful references for comparisons of individual peptide isoforms across the Pancrustacea, and have provided support for phylogenetic inferences made via other methodologies (e.g. that the peptidome of the cladoceran crustacean Daphnia pulex appears quite insect-like (Christie et al., 2011a; Dircksen et al., 2011; Gard et al., 2009), which matches well with data gleaned from several molecular phylogenies that support a close branchiopod/hexapod association (e.g. Cook et al., 2005; Dunn et al., 2008; Mallatt and Giribet, 2006; Meusemann et al., 2010; Regier et al., 2005a,b; Strausfeld and Andrew, 2011). Given the proposed sister group status of the Remipedia to the Hexapoda (e.g. von Reumont et al., 2012), the study presented here asks two questions: 1) ‘‘Can a putative neuropeptidome be predicted from S. cf. tulumensis TSA data?’’ and 2) If a peptidome can be predicted, are the mature remipede isoforms (and precursor proteins) more similar to those currently known from crustaceans or insects?’’. The data that follow show that the answer to the first question is ‘‘Yes’’, albeit a somewhat limited one, with the answer to the second being ‘‘A crustacean-insect intermediate’’. The answer to the latter question is consistent with the hypothesized position of the Remipedia within the Pancrustacea, i.e. the sister group to the Hexapoda, while the predicted peptides may encourage molecular, anatomical and physiological investigations of peptidergic signaling in members of this crustacean taxon.

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part, previously identified D. pulex or Drosophila melanogaster neuropeptide precursors (see Table 1 for accession numbers) were used as the query sequences for these BLAST searches (Table 1); these species were chosen as each has a large number peptide precursors that are publicly accessible in GenBank. All hits returned by tblastn were fully translated using the ‘‘Translate’’ tool of ExPASy (http://www.web.expasy.org/translate/) and then manually checked for homology to the target query. For each of the putative neuropeptide-encoding transcripts identified, the BLAST-generated maximum score and E-value for significant alignment to each query sequence are provided in Table 2.

2.1.2. Peptide prediction Peptides were predicted using an established workflow (e.g. Christie, 2008a,b; Christie et al., 2008, 2010b, 2011a,b,c, 2013; Gard et al., 2009; Ma et al., 2009a, 2010). Specifically, each deduced protein 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 the program were set to ‘‘Sensitive’’. Prohormone cleavage sites were identified based on the information presented in Veenstra (2000) and/or 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), while disulfide bonding between cysteine residues was predicted using the online program DISULFIND (http://disulfind.dsi.unifi.it/; Ceroni et al., 2006). Other post-translational modifications, e.g. cyclization of amino (N)-terminal glutamine/glutamic acid residues and carboxyl (C)-terminal amidation at glycine residues, were predicted by homology to known arthropod peptide isoforms. Examples of this peptide prediction workflow are shown in Fig. 1. All protein/ peptide alignments were done using the online program MAFFT version 7 (http://www.mafft.cbrc.jp/alignment/software/; Katoh and Standley, 2013).

2.2. Reciprocal blastp analyses To identify the known pancrustacean proteins most similar to each of the S. cf. tulumensis preprohormones identified in this study, each of the deduced Speleonectes proteins was used as the query sequence in a blastp search of the non-redundant, combined crustacean and hexapod protein databases curated at NCBI (all searches were conducted on or before October 18, 2013). For these analyses, the database of blastp was set to ‘‘Non-redundant protein sequences (nr)’’ and limited to the combined set of crustacean and hexapod sequences (taxid:6657 + taxid:6960).

3. Results 2. Materials and methods

3.1. In silico prediction of S. cf. tulumensis neuropeptides

2.1. In silico peptide discovery

In an attempt to identify S. cf. tulumensis transcripts encoding neuropeptide precursors, known D. pulex (a cladoceran crustacean) and D. melanogaster (a member of the Hexapoda) proteins were used as query sequences for searches of the remipede TSA database (Table 1); in several case other insect or crustacean pre/preprohormones were used as the query (Table 1). In total, the Speleonectes dataset was probed for the presence of 34 different peptide families/subfamilies (Table 1), with transcripts putatively encoding members of 11 of these targets identified. In the interest of space, only the searches that resulted in transcript identifications are described below.

2.1.1. Database searches Database searches were conducted (on or before October 18, 2013) using methods modified from a well-vetted protocol (e.g. Christie, 2008a,b; Christie et al., 2008, 2010b, 2011b,c; Gard et al., 2009; Ma et al., 2009a, 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 Remipedia (taxid:84343). For the most

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Table 1 Putative Speleonectes cf. tulumensis peptide precursor-encoding transcripts identified using known hexapod and/or crustacean protein queries. Pre/preprohormone query (subfamily)

Adipokinetic hormone (AKH)/red pigment concentrating hormone (RPCH) Adipokinetic hormone-corazonin-like peptide (ACP) Allatostatin A (AST-A) Allatostatin B (AST-B) Allatostatin C (AST-C) Allatotropin (ATR) Bursicon a Bursicon b CAPA/periviscerokinin/pyrokinin CCHamide Corazonin (COR) Crustacean cardioactive peptide (CCAP) DENamide Diuretic hormone 31 (DH31) Diuretic hormone 44 (DH44) Ecdysis-triggering hormone (ETH) Eclosion hormone (EH) FMRFamide-like peptide (myosuppressin) FMRFamide-like peptide (neuropeptide F) FMRFamide-like peptide (short neuropeptide F) FMRFamide-like peptide (sulfakinin) FMRFamide-like peptide (other) Insulin-like peptide (ILP) Intocin Ion transport peptide (ITP)/crustacean hyperglycemic hormone (CHH) Leucokinin Neuroparsin Orcokinin Pigment dispersing factor (PDF)/pigment dispersing hormone (PDH) Proctolin RYamide SIFamide Tachykinin Tachykinin-related peptide (TRP)

Speleonectes transcripts identified Hexapoda

Crustaceanb

0 11 1 0 1 02 0 0 0 0 0 0 NA 3 0 0 1 0 1 0 3 0 0 03 3 0 06 0 0 1 NA 7 07 1

0 NA 1 0 2 0 0 0 0 0 0 0 0 3 0 0 1 0 1 0 3 0 0 0 34 05 0 0 0 1 0 7 NA 1

Drosophila queries: AKH/RPHC, AAB00099; AST-A, AAF56331; AST-B, AAF49354; AST-C, AAF53062 and AAF53063; bursicona, CAH74223; bursiconb, CAH74224; CAPA, AAF56969; CCHamide, AAF55014 and AAF54942; COR, AAF55046; CCAP, AAF56045; DH31, AAF52685; DH44, AAF54421; ETH, AAF47275; EH, AAF55423; FMRFamide, AAA28536; ILP, AAF50205, AAF50204, AAF50203, AAF50202, AAS65048, AAF45773, AAF45885 and AAF49378; ITP, ABI31114; leucokinin, AAF49731; myosuppressin, AAF56283; NPF, AAF55339; PDF, AAF56593; proctolin, AAF52572; sNPF, AAN11060; SIFamide, AAX52685; sulfakinin, AAF52173; TRP, AAF54735. Daphnia queries: AKH/RPCH, EFX68649; AST-A, EFX87432; AST-B, EFX77444; AST-C, EFX85706; ATR, EFX71302; bursicona, EFX87546; bursiconb, EFX87749; CAPA, Dircksen et al., 2011; CCHamide, EFX80320; COR, EFX86608; CCAP, EFX70015; DENamide, Dircksen et al., 2011; DH31, EFX90445; DH44, Dircksen et al., 2011; ETH, Dircksen et al., 2011; EH, Dircksen et al., 2011; FMRFamide, EFX67846; ILP, EFX76240, EFX70023 and EFX82116; intocin, EFX71881; myosuppressin, Dircksen et al., 2011; neuroparsin, EFX67953; NPF, Dircksen et al., 2011; orcokinin, EFX70781; PDH, EFX87718; proctolin, EFX69425; RYamide, Dircksen et al., 2011; sNPF, EFX90018; SIFamide, EFX67946; sulfakinin, EFX80896; TRP, EFX86778. Other hexapod queries: 1Tribolium castaneum prepro-ACP, NP_001159497; 2Manduca sexta prepro-ATR, AAB08759; 3T. castaneum prepro-intocin, ABX52000; 6Bombyx mori pre-neuroparsin, BAG50366; 7Aedes aegypti prepro-sialokinin I, AAD16886. Other crustacean queries: 4Daphnia magna prepro-ITP, ABO43963; 5Calanus finmarchicus prepro-leucokinin (partial), Christie et al., 2013. a Drosophila melanogaster sequences used unless otherwise noted (see numerical superscripts). b Daphnia pulex sequences used unless otherwise noted (see numerical superscripts).

3.1.1. Adipokinetic hormone-corazonin-like peptide (ACP) A single transcript encoding a putative ACP precursor was identified via a tblastn search of the S. cf. tulumensis TSA dataset using the sequence of Tribolium castaneum prepro-ACP (Accession No. ADF28807; Hansen et al., 2010) as the protein query (Table 2). This transcript is predicted to encode an 88 amino acid, N-terminal partial preprohormone (Fig. 1), with the 50 but not 30 end of the putative open reading frame (ORF) of the transcript bounded by a stop codon (data not shown). Two peptides were predicted from the extant portion of the deduced precursor (Fig. 1), one of which has the structure pQVTFSRDWNAamide (Table 3), which is identical to an isoform of ACP previously reported from the mosquitoes, Anopheles gambiae, Aedes aegypti, and Culex pipiens, and the assassin bug Rhodnius prolixus (Hansen et al., 2010). The other peptide, likely missing a portion of its C-terminus, appears to be a novel sequence (Table 3). 3.1.2. Allatostatin A (AST-A) A 117-amino acid, putative C-terminal partial prepro-AST-A (Fig. 2A) was deduced from a S. cf. tulumensis transcript identified by tblastn analysis using either the D. pulex (Accession No.

EFX87432; Colbourne et al., 2011) or the D. melanogaster (Accession No. AAF56331; Adams et al., 2000) AST-A precursor as the initial query sequence (Table 2); the putative partial ORF for this protein is bounded on its 30 but not 50 end by a stop codon (data not shown). The structures of six novel peptides were predicted from the deduced partial preprohormone (Table 3). Three of the predicted peptide, DGNKFSFGLamide, SMSYGFGLamide and SPAYGFGLamide, possess the C-terminal motif – YXFGLamide (where X represents a variable amino acid), which is the hallmark of the AST-A family (e.g. Christie et al., 2010a). 3.1.3. Allatostatin C (AST-C) A 93 amino acid, putative full-length prepro-AST-C (Fig. 1B) was deduced from two S. cf. tulumensis transcripts identified within the TSA dataset using the sequence of a D. pulex AST-C precursor (Accession No. EFX85706; Colbourne et al., 2011) as the query protein (Table 2). One of the two Speleonectes transcripts contains the full ORF for the preprohormone, with a stop codon preceding and following it (Table 2). The other S. cf. tulumensis sequence encodes an 84 amino acid, N-terminal partial protein (Table 2) that is

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A.E. Christie / General and Comparative Endocrinology 201 (2014) 74–86 Table 2 Putative Speleonectes cf. tulumensis neuropeptide precursor protein-encoding transcripts identified via in silico transcriptome shotgun assembly (TSA) mining. Target precursor

ACP AST-A AST-C DH31

EH NPF Sulfakinin

ITP/CHH

Proctolin SIFamide

TRP

Speleonectes cf. tulumensis TSA transcript/protein identifications c

Tblastn search statistics d

TSA Accession No.

Transcript length

Deduced protein length

JL122470 JL205783 JL134697 JL151960 JL136212 JL100892 JL114474 JL143836 JL205812 JL176613 JL143916 JL157951 JL178755 JL124400 JL136094 JL181547 JL186572 JL173353 JL108619 JL199871 JL126285 JL107015 JL105825 JL122362

314 384 485 356 527 646 372 827 383 713 558 651 519 436 439 413 313 445 346 393 460 462 701 329

88e 117f 93g 84e 112g 90f 71e 81g 90g 119g 82f 119g 111g 88e 75e 83g 79e 72g 72g 72g 72g 72g 72g 87e

Hexapod querya

Crustacean queryb

BLAST score

E-value

BLAST score

E-value

38.5 30.8 29.3 NA 87.4 87.0 38.5 63.2 42.7 51.6 50.8 49.7 69.3 62.8 45.8 29.6 36.6 37.0 35.8 35.8 35.8 35.8 35.8 33.5

6e05 0.21 1.9 NA 6e22 1e21 2e04 6e13 3e06 1e08 2e08 7e08 3e15 4e13 4e07 0.54 0.001 0.002 0.003 0.004 0.006 0.006 0.008 0.041

NA 34.7 102 82.8 99.0 91.7 50.4 63.2 48.5 62.4 60.8 60.1 89.4 82.8 50.8 29.3 43.9 40.8 43.9 43.9 43.9 43.9 41.2 33.9

NA 0.023 3e28 6e21 2e26 3e23 8e09 8e13 3e08 6e12 1e11 3e11 2e22 3e20 8e09 0.14 6e07 8e06 6e07 6e07 8e07 8e07 2e05 0.030

Abbreviations: ACP, adipokinetic hormone-corazonin-like peptide; AST-A, allatostatin A; AST-C, allatostatin C; DH31, diuretic hormone 31; EH, eclosion hormone; NPF, neuropeptide F; ITP/CHH, ion transport peptide/crustacean hyperglycemic hormone; TRP, tachykinin-related peptide. a Hexapod searches were primarily done using Drosophila melanogaster sequences as the query proteins (see Table 1 for exceptions and accession numbers). b Crustacean searches were primarily done using Daphnia pulex sequences as the query proteins (see Table 1 for exceptions and accession numbers). c Length in base pairs. d Length in amino acids. e Amino (N)-terminal partial protein. f Carboxy (C)-terminal partial protein. g Full-length protein.

identical in sequence to the corresponding portion of full-length preprohormone (data not shown). A search of the Speleonectes TSA sequences using the D. melanogaster allatostatin double C precursor (Accession No. AAF53063; Adams et al., 2000) also identified the transcript containing the complete ORF as encoding a putative prepro-AST-C (Table 2); the transcript encoding the N-terminal partial protein was not returned as a hit using the Drosophila precursor as the query sequence. Two peptides, including SYWKQCAFNAVSCFamide (a disulfide bond predicted between the position 6 and 13 cysteine residues), were predicted from the deduced S. cf. tulumensis prepro-AST-C (Table 3). SYWKQCAFNAVSCFamide, with its disulfide bridging, is identical in structure to an AST-C originally identified from the bee Apis mellifera genome (Hummon et al., 2006); this peptide has subsequently been shown to be broadly conserved in both insects and crustaceans (e.g. Dickinson et al., 2009; Ma et al., 2009b; Veenstra, 2009). 3.1.4. Diuretic hormone 31 (DH31) A 112 amino acid, putative full-length DH31 preprohormone (Fig. 2B) was deduced from three S. cf. tulumensis transcripts identified within the TSA dataset using the sequence of either D. pulex (Accession No. EFX90445; Colbourne et al., 2011) or D. melanogaster prepro-DH31 (Accession No. AAF52685; Adams et al., 2000) as the query protein (Table 2). One of the Speleonectes transcripts contains the full ORF for the precursor protein (Table 2), with the other two encoding partial proteins (one a 71 amino acid, N-terminal partial sequence and the other a 90 amino acid, C-terminal partial protein; Table 2). The two partial preprohormones are identical in sequence to the corresponding portions of full-length precursor (data not shown). Three novel peptides were predicted from the deduced precursor protein (Table 3), including one with the

putative mature structure GLDLGLGRGFSGSLAAKHLMGLAAANFA GGPamide, a peptide that differs in sequence from an isoform of DH31 previously identified from both the beetle T. castaneum (Li et al., 2008) and the lobster Homarus americanus (Christie et al., 2010c) at just one position (i.e. a leucine for glutamine substitution at position 14).

3.1.5. Eclosion hormone (EH) An 81 amino acid, putative full-length EH precursor (Fig. 2C) was deduced from a S. cf. tulumensis transcript identified by tblastn analysis using either the D. pulex (Dircksen et al., 2011) or the D. melanogaster (Accession No. AAF55423; Adams et al., 2000) EH precursor as the initial query sequence (Table 2). Two novel peptides were predicted from the deduced prepro-hormone (Table 3), including SYVGSCIRNCGQCMYGDYFHGQACAVSCIETSGMTVPDCNNPSTINRFL (disulfide bonds predicted between the position 6 and 39, 10 and 28, and 13 and 24 cysteine residues), which exhibits structural similarity (including disulfide bridging patterns) to members of the EH family (Christie et al., 2010a).

3.1.6. FMRFamide-like peptide – neuropeptide F (NPF) A 90 amino acid, putative full-length prepro-NPF (Fig. 2D) was deduced from a S. cf. tulumensis transcript identified by tblastn analysis using either the D. pulex NPF 1 precursor (Dircksen et al., 2011) or a NPF preprohormone from D. melanogaster (Accession No. AAF55339; Adams et al., 2000) as the initial query sequence (Table 2). Two novel peptides were predicted from the deduced preprohormone (Table 3), including KPDPGQLAAMADALKYLQELDKYYSQVARPRFamide, which exhibits the hallmarks of members of the NPF family, i.e. an overall length of >30 amino acids, an – RPRFamide C-terminus,

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Fig. 1. Three examples of the in silico workflow used for predicting the structures of putative mature peptides from deduced Speleonectes cf. tulumensis preprohormone sequences. (A) Predicted processing of the partial adipokinetic hormone-corazonin-like peptide (ACP) precursor. The structure of the putative mature ACP isoform is shown in red, with that of a partial linker/precursor-related peptide shown in blue. In this processing scheme, the presence of an amino (N)-terminal pyroglutamic acid is indicated by ‘‘pQ’’, while the ‘‘+’’ indicates the potential of additional amino acid residues at the carboxyl (C)-terminus of the precursor. (B) Predicted processing of the allatostatin C (ASTC) precursor. The structure of the putative mature AST-C isoform is shown in red, with that of a putative mature linker/precursor-related peptide shown in blue. The disulfide bond predicted between the two cysteine residues in the putative mature AST-C is indicated by the inverted red bracket. (C) Predicted processing of the sulfakinin precursor. The structures of two putative mature sulfakinin isoforms are shown in red, with those of two putative mature linker/precursor-related peptides shown in blue. In this schematic, tyrosine residues that are predicted to be sulfated are shown as ‘‘Y(SO3H)’’. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

and tyrosine residues at positions 10 and 17 from the C-terminus (Christie et al., 2010a). 3.1.7. FMRFamide-like peptide – sulfakinin A 119 amino acid, putative full-length prepro-sulfakinin (Fig. 1C) was deduced from three S. cf. tulumensis transcripts identified within the TSA dataset using the sequence of either the D. pulex (Accession No. EFX80896; Colbourne et al., 2011) or the

D. melanogaster (Accession No. AAF52173; Adams et al., 2000) sulfakinin precursor as the query protein (Table 2). Two of the Speleonectes transcripts encode full-length precursor proteins that are identical in sequence save an uncalled residue in one sequence, with the third encoding an 82 amino acid, C-terminal partial protein (Table 2). The partial preprohormone is identical in sequence to the corresponding portions of full-length precursor (data not shown). Four novel peptides were predicted from the deduced

Table 3 Predicted Speleonectes cf. tulumensis peptides. Peptide (subfamily)

Predicted structure

Adipokinetic hormone-corazonin-like peptide Adipokinetic hormone-corazonin-like peptide linker/precursor-related peptide Allatostatin-A

Allatostatin-A linker/precursor-related peptide

Allatostatin-C

Diuretic hormone 31 Diuretic hormone 31 linker/precursor-related peptide Eclosion hormone Eclosion hormone linker/precursor-related peptide FMRFamide-like peptide (neuropeptide F) Neuropeptide F linker/precursor-related peptide FMRFamide-like peptide (sulfakinin) Sulfakinin linker/precursor-related peptide Ion transport peptide/crustacean hyperglycemic hormone Ion transport peptide/crustacean hyperglycemic hormone linker/precursor-related peptide

DGNKFSFGLa SMSYGFGLa SPAYGFGLa EVDPTELDDMLMRSDDEEE SASDSDTEKDNFSENNEHLIPKE QKHKKAYDVSVGETIKPYNSKIEKSERKTGSVGQ SYWKQCAFNAVSCFa KSLSQVEKERFGNELDLVDDDGSMENALLNYLFAKQMVSRLRSNMDVSDLQ GLDLGLGRGFSGSLAAKHLMGLAAANFAGGPa APLSSHLAETEDPDY(SO3H)VLDLLSKLGQSIIRADELENS SAGAKEGKPSDSL SYVGSCIRNCGQCMYGDYFHGQACAVSCIETSGMTVPDCNNPSTINRFL Li KPDPGQLAAMADALKYLQELDKYYSQVARPRFa MDSRPLGSVENVIDGNEKLWSHL pQFDDY(SO3H)GHMRFa DFDDY(SO3H)GHMRFa SHPSRKMDLSRLTNFIVPYLEAHSKPDLPPVKQSMRTPVAPAPVDEGNDFEDPDMMKFHGAE SAVEE SFLDIECKGVYDKSIFTKLNMVCEGCYHLYRDPELHTLCKQDCWSTKYFEGCLNALVRGA EKEEIKDILNDLHaSFLDIECKGVYDKSIFTKLNMVCEGCYISTGIQNSTRF+ MIVPRTIS

Proctolin Proctolin linker/precursor-related peptide

RYLPT SDDNRVE IRELLKDLIESEVE DYD FVF EVPAPVHQMAPVEGLMDH

SIFamide SIFamide linker/precursor-related peptide

A.E. Christie / General and Comparative Endocrinology 201 (2014) 74–86

Allatostatin-C linker/precursor-related peptide

pQVTFSRDWNAa SGAAALYPDCAMPERALLSEVSKLIHNEAQRMVSCQAWTVLRG+

GYRKPPFNGSIFa GSTAEY(SO3H)EGAGKSLYAMCEIAVEACSAWFPTADN GSTAEY(SO3H)EGAGKSLYAMCEIAVEACSAWFSTADN GSTAEY(SO3H)EGAGKSLYAMCEIAVEACSAWFP TTEEQTGLN+

Tachykinin-related peptide Tachykinin-related peptide linker/precursor-related peptide

APSGFLGMRa QDTD DAVDEYSGNVD DSDDVASSDDLEAD APS+

Abbreviations: a, amidated C-terminus; +, likely partial peptide with additional unknown amino acids following the symbol; pQ, pyroglutamic acid; C, disulfide bonded cysteine; Y(SO3H), sulfated tyrosine.

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Fig. 2. Putative Speleonectes cf. tulumensis preprohormones deduced from transcriptome shotgun assembly sequence data. (A) The carboxyl (C)-terminal portion of preproallatostatin A. (B) Prepro-diuretic hormone 31. (C) Prepro-eclosion hormone. (D) Prepro-neuropeptide F. (E) Prepro-proctolin. (F) The amino (N)-terminal portion of preprotachykinin-related peptide. In all panels, signal peptides are shown in gray, while all mono/dibasic cleavage loci are shown in black. Similarly, for each sequence, the isoform(s) of the peptide for which the precursor is named is/are shown in red, with all other linker/precursor related peptides shown in blue. The ‘‘+’’ in panels A and F indicate the presence of additional, unknown, amino acid residues at the N- or C-terminus, respectively. In panel C a putative dibasic cleavage locus that is likely not cleaved (based on homology to known eclosion hormone isoforms) is highlighted in yellow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

precursor protein (Table 3). Two of these peptides, pQFDDY(SO3H)GHMRFamide and DFDDY(SO3H)GHMRFamide, exhibit the C-terminal motif – Y(SO3H)GHM/LRFamide, which is considered the hallmark of the sulfakinins (Christie et al., 2010a). 3.1.8. Ion transport peptide (ITP)/crustacean hyperglycemic hormone (CHH) Three S. cf. tulumensis transcripts were identified as encoding putative ITP/CHH precursors using the sequence of either Daphnia magna prepro-ITP (Accession No. ABO43963; Montagne and Toullec, unpublished direct submission) or a prepro-ITP from D. melanogaster (Accession No. ABI31114; Adams et al., 2000) as the query protein (Table 2). One of the three transcripts encode a 111 amino acid, putative full-length precursor, with the other two encoding N-terminal partial proteins of 88 and 75 amino acids, respectively (Fig. 3A). The 88 amino acid partial preprohormone is identical in sequence to the corresponding portion of the full-length protein, while the 75 amino acid sequences shows a serine for phenylalanine substitution at position 17, as well as a novel C-terminal sequence (likely partial) starting at position 65 (Fig. 3A). Three peptides are predicted from the collective set of deduced precursors (Table 3), including one full-length and one partial isoform of ITP/CHH, i.e. SFLDIECKGVYDKSIFTKLNMVCEGCYHLYRDPELHTLCKQDCWSTKYF EGCLNALVRGAEKEEIKDILNDLHamide and SFLDIECKGVYDKSIFTKL NMVCEGCYISTGIQNSTRF+. In the full-length ITP/CHH isoform, disulfide bonds are predicted between the cysteine residues at positions 7 and 26, 23 and 43, and 39 and 52 (Table 3); no cysteine bridging is predicted in the partial ITP/CHH (Table 3). All of the peptides predicted from the deduced S. cf. tulumensis prepro-ITP/CHHs are novel. 3.1.9. Proctolin An 83 amino acid, putative full-length proctolin precursor (Fig. 2E) was deduced from a S. cf. tulumensis transcript identified by tblastn analysis using either D. pulex (Accession No. EFX69425; Colbourne et al., 2011) or D. melanogaster (Accession No. AAF52572; Adams et al., 2000) prepro-proctolin as the query sequence (Table 2). Six peptides were predicted from the deduced precursor protein (Table 3). The structure of one of these peptides, RYLPT, is identical to that of proctolin, a broadly conserved pancrus-

tacean pentapeptide (Christie et al., 2010a; Isaac et al., 2004); the remaining five peptides are predicted to possess novel structures. 3.1.10. SIFamide Seven S. cf. tulumensis transcripts were identified as encoding putative SIFamide precursors using the sequence of either D. pulex (Accession No. EFX67946; Colbourne et al., 2011) or D. melanogaster (Accession No. AAX52685; Adams et al., 2000) prepro-SIF as the query protein (Table 2). Six of the seven transcripts encode 72 amino acid, putative full-length precursors, five of which are identical in sequence, with the sixth possessing a serine for proline substitution at position 68 (Fig. 3B). The protein deduced from the remaining transcript is a 79 amino acid, putative N-terminal partial preprohormone, that is identical in sequence through position 68 to that of the major 72 amino acid isoform, but possessing a novel 11 amino acid extension C-terminal to this conserved region (Fig. 3B). Four peptides were predicted from the collective set of Speleonectes precursors (Table 3), including GYRKPPFNGSIFamide, a highly conserved decapod isoform of SIFamide (e.g. Christie et al., 2010a); GYRKPPFNGSIFamide is also native to a number of mosquito species (e.g. Verleyen et al., 2009). 3.1.11. Tachykinin-related peptide (TRP) An 87 amino acid, putative N-terminal partial TRP precursor (Fig. 2F) was deduced from a S. cf. tulumensis transcript identified by tblastn analysis using either the D. pulex (Accession No. EFX86778; Colbourne et al., 2011) or a D. melanogaster (Accession No. AAF54735; Adams et al., 2000) TRP preprohormone as the query sequence (Table 2). Six peptides were predicted from the partial protein (Table 3), including two copies of APSGFLGMRamide, a broadly conserved decapod crustacean TRP (Christie et al., 2010a); the remaining four peptides (including one partial sequence) are novel. 3.2. Comparison of S. cf. tulumensis neuropeptide precursors with those of insects and other crustaceans As an assessment of the relatedness of the deduced S. cf. tulumensis peptide precursors to those from other crustaceans and

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Fig. 3. Alignments of the deduced amino acid sequences of multiple Speleonectes cf. tulumensis ion transport peptide (ITP)/crustacean hyperglycemic hormone (CHH) and SIFamide precursors. (A) Alignment of three full-length or partial ITP/CHH precursor proteins. (B) Alignment of seven full-length or partial SIFamide prepro-hormones. In both panels, signal peptides are shown in gray, with dibasic cleavage loci shown in black. In A, isoforms of ITP/CHH are shown in red. In B, SIFamide sequences are colored red. In both A and B, all linker/precursor related peptides are shown in blue. In this figure, the ‘‘+’’ at the carboxyl-terminus of several of the sequences indicates the presence of additional, unknown, amino acid residues. In the line immediately below each sequence grouping, ‘‘⁄’’ indicates amino acids that are identical in all of the aligned proteins, while ‘‘.’’ and ‘‘:’’ denote amino acids that are similar in structure between all sequences. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

insects, each was used as the query sequence in a blastp search of the combined hexapod and crustacean non-redundant protein databases curated at NCBI. The top 10 hits returned for each search are provided in Table 4. As can be seen from this table, the vast majority of hits returned were hexapod sequences (95 of combined 110 total sequences). With respect to the top hit for each search, the Hexapoda also predominated, with eight of 11 the searches returning a hexapod sequence as most similar to the Speleonectes query, e.g. the SIFamide and TRP searches, and this despite the fact that the S. cf. tulumensis precursors are predicted to give rise to wellknown crustacean isoforms of these respective peptide families. Interestingly, when the top hit was a crustacean sequence, it was always from a derived line, i.e. the Decapoda (e.g. the top hits for the Speleonectes DH31 and sulfakinin precursors were proteins from the lobster H. americanus). In fact, of the 15 crustacean hits in the collective set, 10 sequences were from members of the Malacostraca (all decapods); of the remaining five crustacean hits, four were from members of the Branchiopoda (all Daphnia sequences) and one was from a member of the Maxillopoda (a barnacle species). Thus, it appears that the peptide precursor proteins of S. cf. tulumensis are more hexapod-like than they are crustacean-like, and within the latter subphylum, they are more similar to the derived decapods than they are to members of taxa generally considered to be basal.

S. cf. tulumensis is one of only a handful of crustaceans for which large datasets (>100,000 sequences) are publicly available (von Reumont et al., 2012). Thus, these Speleonectes sequences are a significant resource for protein discovery in the Crustacea, and are the only extant resource currently available for such work on any member of the Remipedia. The transcriptome sequencing of S. cf. tulumensis was originally undertaken in order to resolve phylogenetic relationships between crustaceans and hexapods (von Reumont et al., 2012), and data gleaned from its transcriptome has helped advance the hypothesis that the Remipedia is the crustacean sister group to the Hexapoda (e.g. Ertas et al., 2009; Fanenbruck et al., 2004; Harzsch, 2006; Regier et al., 2010; Stemme et al., 2012, 2013; von Reumont et al., 2012). Given the proposed phylogenetic position of remipedes within the Pancrustacea, this taxon is fundamentally important to any understanding of evolution within the Arthropoda, including the evolution of neurochemical signaling systems, in particular those utilizing peptides. In the study presented here the S. cf. tulumensis TSA dataset was used to mine for transcripts encoding putative neuropeptide precursor proteins, with the ultimate goal of determining if the peptidome of this species is also consistent with the proposed sister group status of the Remipedia and Hexapoda.

4.2. In silico neuropeptide discovery in S. cf. tulumensis 4. Discussion 4.1. Molecular resources for the Remipedia Despite methodological advances and decreased costs for highthroughput sequencing, there are still relatively few crustaceans that have been the subjects of large-scale genome/transcriptome projects (e.g. Christie et al., 2013; Colbourne et al., 2011; Li et al., 2012, 2013; Ma et al., 2012; Jung et al., 2011). The remipede

Over the past several decades peptide discovery in crustaceans has moved from one-by-one identifications to the elucidation of complete peptidomes, the full complement of peptides present in a species (e.g. Christie et al., 2010a). While many techniques have been used for peptidomic analyses, biological mass spectrometry, in particular high-resolution mass profiling and tandem mass spectrometric sequencing, has been the primary approach used for this type of investigation (e.g. Fu et al., 2005; Huybrechts et al., 2003; Li et al., 2003; Ma et al., 2008, 2009a,b, 2010; Stemmler et al., 2007). While

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Table 4 blastp analyses of putative Speleonectes cf. tulumensis peptide precursor vs. all NCBI non-redundant hexapod-crustacean protein sequences. Preprohormone

Top 10 blastp hits Accession No.

Protein name

Species

Class

ACP

ABB58739 ABY81279 NP_001124365 XP_001661197 AGH25545 CAY77167 XP_001843277 CAY77162 EFA12888 NP_001159497

AKH-I preprohormone Type I adipokinetic prohormone precursor Adipokinetic hormone 2 precursor Hypothetical protein AaeL_AAEL010950 Adipokinetic 2 precursor Adipokinetic hormone 2 preprohormone Conserved hypothetical protein Adipokinetic hormone 2 preprohormone AKH-like peptide precursor AKH/corazonin-related peptide preproprotein

Tribolium castaneum Bombyx mori Bombyx mori Aedes aegypti Helicoverpa armigera Culex pipiens Culex quinquefasciatus Aedes aegypti Tribolium castaneum Tribolium castaneum

H H H H H H H H H H

BLAST score 48.9 47.4 45.8 54.1 42.2 42.7 42.4 42.0 41.2 41.2

E-value 2e07 6e07 3e06 6e06 4e05 4e05 6e05 7e05 1e04 1e04

AST-A

CAA62500 ACN42938 O44314 CAD87569 P12764 AAC72895 CAD48593 CAC83078 AAC72893 BAF64528

Allatostatin precursor Allatostatin precursor Helicostatin peptide precursor Statin precursor Allatostatin precursor Allatostatin neuropeptide precursor Allatostatin A precursor Allatostatin A prohormone precursor Allatostatin neuropeptide precursor Allatostatin precursor protein

Periplaneta americana Reticulitermes flavipes Helicoverpa armigera Spodoptera frugiperda Diploptera punctata Blatta orientalis Spodoptera frugiperda Gryllus bimaculatus Blaberus craniifer Panulirus interruptus

H H H H H H H H H C

53.5 52.4 50.4 49.7 48.5 48.1 47.0 47.4 47.4 46.2

9e08 3e07 5e07 1e06 4e06 6e06 6e06 9e06 1e05 3e05

AST-C

XP_003699263 BAO00971 P85798 XP_003399378 XP_003488230 EFN70796 XP_001121443 EFN89652 XP_003693246 EFX85706

Prohormone-1-like precursor Allatostatin C precursor Prohormone-1 precursor Prohormone-1-like precursor Prohormone-1-like precursor Prohormone-1 precursor Prohormone-1 precursor Prohormone-1 precursor Prohormone-1-like precursor Allatostatin C preprohormone

Megachile rotundata Nilaparvata lugens Apis mellifera Bombus terrestris Bombus impatiens Camponotus floridanus Apis mellifera Harpegnathos saltator Apis florea Daphnia pulex

H H H H H H H H H C

119 118 117 117 116 110 109 106 108 102

3e34 6e34 2e33 2e33 6e33 1e30 2e30 3e29 4e29 1e27

DH31

ACX46386 BAO00939 XP_003696343 XP_003395619 AEA51302 XP_001945901 ACX47068 EEZ99367 XP_003395620 EFZ22024

Prepro-calcitonin-like diuretic hormone Diuretic hormone 31 precursor Diuretic hormone class 2-like Diuretic hormone class 2-like isoform 1 Calcitonin-like diuretic hormone prepropeptide Diuretic hormone class 2-like precursor Calcitonin-like diuretic hormone prepropeptide Diuretic hormone 31-like protein precursor Diuretic hormone class 2-like isoform 2 Hypothetical protein SINV_00307

Homarus americanus Nilaparvata lugens Apis florea Bombus terrestris Rhodnius prolixus Acyrthosiphon pisum Rhodnius prolixus Tribolium castaneum Bombus terrestris Solenopsis invicta

C H H H H H H H H H

118 117 110 108 108 108 105 104 104 99.0

7e33 1e32 1e29 1e29 2e29 3e29 2e28 3e28 4e28 8e26

EH

BAO00951 AFK81936 ENN80798 XP_003395267 XP_003487278 EEZ99174 XP_002432310 XP_969164 BAO00950 XP_003246438

Eclosion hormone 2 precursor Eclosion hormone precursor Hypothetical protein YQE_02807 Eclosion hormone-like protein precursor Eclosion hormone-like protein precursor Eclosion hormone-like protein precursor Eclosion hormone precursor Eclosion hormone precursor Eclosion hormone 1 precursor Eclosion hormone-like protein precursor

Nilaparvata lugens Amphibalanus amphitrite Dendroctonus ponderosae Bombus terrestris Bombus impatiens Tribolium castaneum Pediculus humanus corporis Tribolium castaneum Nilaparvata lugens Acyrthosiphon pisum

H C H H H H H H H H

86.7 77.4 73.9 72.0 72.0 70.9 69.7 68.6 67.4 67.4

8e22 1e17 7e17 5e16 8e16 9e16 5e15 1e14 2e14 3e14

NPF

AEC12206 AGM32387 AEC12204 ACV86032 P86442 AFD50506 AEC12207 AEC12205 XP_002054350 XP_004523106

Preproneuropeptide F I Neuropeptide F precursor Preproneuropeptide F I Neuropeptide F precursor Neuropeptide F precursor Neuropeptide F prepropeptide Preproneuropeptide F II precursor Preproneuropeptide F II precursor GJ24394 Neuropeptide F-like precursor

Melicertus marginatus Coptotermes formosanus Litopenaeus vannamei Reticulitermes flavipes Locusta migratoria Schistocerca gregaria Melicertus marginatus Litopenaeus vannamei Drosophila virilis Ceratitis capitata

C H C H H H C C H H

83.2 82.8 79.0 67.4 66.2 66.2 65.9 61.6 52.8 52.4

6e24 7e24 3e22 6e18 2e17 2e17 4e17 2e15 2e12 2e12

Sulfakinin

ABQ95346 EFX80896 BAO00978 BAH11170 CAL48349 XP_004529342 XP_003249150 XP_001846221 XP_002019319 XP_001359636

Preprosulfakinin Sulfakinin-like peptide precursor Sulfakinin precursor Preprosulfakinin Preprosulfakinin Callisulfakinin-like precursor Hypothetical protein LOC100579011 Sulfakinin precursor GL12302 Drosulfakinin precursor

Homarus americanus Daphnia pulex Nilaparvata lugens Psacothea hilaris hilaris Gryllus bimaculatus Ceratitis capitata Apis mellifera Culex quinquefasciatus Drosophila persimilis Drosophila pseudoobscura pseudoobscura

C C H H H H H H H H

80.9 63.5 60.8 58.5 58.2 56.6 58.2 55.8 54.3 54.3

6e22 3e15 2e14 1e13 2e13 6e13 1e12 1e12 4e12 4e12

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A.E. Christie / General and Comparative Endocrinology 201 (2014) 74–86 Table 4 (continued) Preprohormone

Top 10 blastp hits Accession No.

Protein name

Species

Class

BLAST score

E-value

a

ACZ96371 XP_002427293 EFN85846 ERL89918 ABO43964 Q26492 DAA35082 ABN79657 NP_001076808 XP_001604056

Ion transport protein isoform B CHH XO-type, variant 4 precursor Ion transport peptide-like precursor Hypothetical protein D910_07277 Putative ion transport peptide-like precursor Ion transport peptide-like precursor Ion transport peptide-like protein Ion transport peptide isoform A precursor Ion transport peptide precursor Ion transport peptide-like isoform 1 precursor

Rhodnius prolixus Pediculus humanus corporis Harpegnathos saltator Dendroctonus ponderosae Daphnia magna Schistocerca gregaria Atta cephalotes Tribolium castaneum Tribolium castaneum Nasonia vitripennis

H H H H C H H H H H

109 106 105 104 104 103 103 103 103 105

1e29 9e29 1e27 1e27 1e27 2e27 2e27 2e27 3e27 3e27

Proctolin

EFA05689 BAO00972 XP_002432200 AEX08669 XP_001949738 XP_002064642 XP_004521909 EFX88050 XP_002087991 NP_609158

Proctolin precursor Proctolin precursor Hypothetical protein Phum_PHUM576440 Proctolin prepropeptide Hypothetical protein LOC100163518 isoform 1 GK23968 Uncharacterized protein LOC101462442 M4-proctolin precursor GE18327 Proctolin precursor

Tribolium castaneum Nilaparvata lugens Pediculus humanus corporis Rhodnius prolixus Acyrthosiphon pisum Drosophila willistoni Ceratitis capitata Daphnia pulex Drosophila yakuba Drosophila melanogaster

H H H H H H H C H H

63.5 57.8 55.5 50.8 43.9 33.5 33.5 32.0 32.0 31.6

6e13 1e10 1e09 4e08 1e05 0.100 0.15 0.23 0.53 0.65

SIFamideb

XP_001814498 ACT35307 BAO00977 ERL92324 XP_003245357 XP_001654051 NP_001124359 XP_001846474 AGH25569 XP_308708

Similar to conserved hypothetical protein SIFamide precursor SIFamide precursor Hypothetical protein D910_09641 FMRFamide-related neuropeptides-like precursor hypothetical protein AaeL_AAEL009858 IMFamide precursor Conserved hypothetical protein SIFamide precursor AGAP007056-PA

Tribolium castaneum Rhodnius prolixus Nilaparvata lugens Dendroctonus ponderosae Acyrthosiphon pisum Aedes aegypti Bombyx mori Culex quinquefasciatus Helicoverpa armigera Anopheles gambiae

H H H H H H H H H H

87.4 86.7 85.5 82.0 79.3 75.9 73.9 73.9 73.2 71.6

3e22 5e22 2e21 4e20 3e19 9e18 5e17 5e17 7e17 3e16

TRP

AAX11211 EFA09176 XP_975364 XP_001652135 NP_001124364 AAX11212 AGH25571 BAC82426 BAD06363 ACB41786

Preprotachykinin Tachykinin precursor Similar to preprotachykinin Hypothetical protein AaeL_AAEL006644 Tachykinin precursor Preprotachykinin Tachykinin precursor Preprotachykinin Preprotachykinin B Preprotachykinin

Rhyparobia maderae Tribolium castaneum Tribolium castaneum Aedes aegypti Bombyx mori Periplaneta americana Helicoverpa armigera Procambarus clarkii Panulirus interruptus Homarus americanus

H H H H H H H C C C

71.6 68.9 70.5 64.3 62.0 62.4 60.8 60.5 59.7 58.9

2e14 5e14 7e14 2e12 2e11 4e11 4e11 4e11 8e11 2e10

ITP/CHH

C, Crustacea; H, Hexapoda. a Full-Length precursor derived from JL178755 used as the query sequence. b Full-Length precursor derived from JL108619 used as the query sequence.

useful for work on many species, peptide discovery via mass spectrometry often requires a relatively large amount of starting material, and thus for microscopic, rare and/or geographically inaccessible animals, it is often not practical. In contrast, in silico mining of large genome/transcriptome datasets provides an alternative strategy for elucidating peptidomes in small and/or difficult to obtain species; here more limited starting material is need to generate the genomic/transcriptomic data, and through the use of well-vetted bioinformatics workflows, the structures of mature peptide isoforms can be predicted from the identified genes/transcripts. Genome/transcriptome mining has been successfully used for peptidome discovery in a number of planktonic crustaceans (Christie et al., 2011a, 2013; Dircksen et al., 2011; Gard et al., 2009), and here it was employed for peptide discovery in a member of the Remipedia, S. cf. tulumensis, which, while not planktonic, is certainly a difficult species to collect given the environment it inhabits (i.e. submerged marine/anchialine cave systems in the Yucatan Peninsula of Mexico). Using publicly accessible TSA data for S. cf. tulumensis, 24 putative peptide precursor-encoding transcripts were identified. These sequences encompassed 11 different peptide families/subfamilies (ACP, AST-A, AST-C, DH31, EH, ITP/CHH, NPF, proctolin, SIFamide, sulfakinin and TRP), and allowed for the prediction of 40 mature S. cf. tulumensis peptides (36 full-length and 4 partial ones). Prior to

this study, no peptide hormones were known from any member of the Remipedia. 4.3. The peptidome of S. cf. tulumensis – a crustacean-insect intermediate? One goal of this study was to determine if a peptidome could be predicted for S. cf. tulumensis using the publicly accessible TSA dataset available for it. The answer to this question is ‘‘Yes’’, as 40 peptides encompassing 11 families/subfamilies were deduced. However, not all of the peptide families/subfamilies queried for had transcripts identified (i.e. no sequences encoding homologs of 23 of the 34 targeted peptide families were found). This is not surprising as mining of other transcriptome datasets using the same workflow have, on average, produced two peptide precursor-encoding transcript identifications per 10,000 sequences in the database being searched (e.g. Christie 2008a,b; Christie et al., 2008, 2010b, 2011b, 2013; Gard et al., 2009; Ma et al., 2009a, 2010). Given the 100,000 TSA sequences available for Speleonectes, 20 peptide-encoding transcripts are predicted to exist in the dataset, and 24 were found. Whether or not S. cf. tulumensis possesses isoforms of the unidentified peptide groups remains an open question, one that

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can only be addressed with additional sequence information. What can be said is that the peptides that were deduced include a number of novel isoforms, as well as a mixture of known crustacean and insect variants. For example, the theorized isoforms of Speleonectes AST-A, DH31, EH, NPF, sulfakinin and ITP/CHH all possess novel structures, though many are very close in sequence to known arthropod isoforms. For example, the native DH31 of S. cf. tulumensis differs from an isoform previously reported from both the beetle T. castaneum (Li et al., 2008) and the lobster H. americanus (Christie et al., 2010c) at just one of its 31 total residues, while the native NPF differs at just 4 of 32 residues from an isoform found in three penaeid shrimp (Christie et al., 2008, 2011d). With respect to known peptides, the native S. cf. tulumensis AST-C and proctolin are identical in structure to previously reported and broadly conserved isoforms present in many crustaceans and insects (e.g. Christie et al., 2010a; Dickinson et al., 2009; Isaac et al., 2004; Ma et al., 2009b; Veenstra, 2009), while the native SIFamide is a well-known decapod crustacean variant that has also been described from a number of mosquito species (e.g. Christie et al., 2010a; Verleyen et al., 2009). Likewise, the TRP predicted from the S. cf. tulumensis TSA dataset is a known and highly conserved decapod peptide (Christie et al., 2010a). Of particular note was the identification of pQVTFSRDWNAamide, whose theorized structure is identical to that of a peptide that is structurally an adipokinetic hormone-corazonin intermediate: adipokinetic hormonecorazonin-like peptide or ACP (Hansen et al., 2010). The ACP family was originally proposed as hexapod-specific, as isoforms of it, as well as its receptor, are broadly conserved in insects, but absent in the genome of the cladoceran crustacean D. pulex (Hansen et al., 2010), a member of the Branchiopoda, which, at the time the ACPs were first described, was proposed as the direct ancestor of the Hexapoda (Glenner et al., 2006). The particular isoform of ACP found in Speleonectes is the same as that found in the mosquitoes, A. gambiae, A. aegypti, and C. pipiens, as well as in the assassin bug R. prolixus, and differs at just one residue from that present in the beetle T. castaneum (Hansen et al., 2010). Taken collectively, the native peptides of S. cf. tulumensis include both unique and known crustacean/hexapod isoforms, including one previously believed to be a member of an insect-specific peptide family. Thus, while still limited in scope, the peptidome of this species appears to be a crustacean-hexapod intermediate, supporting the proposed phylogenetic position of the Remipedia within the Pancrustacea. As additional peptide sequences are added to the collection presented here, it will be interesting to see if this trend continues to hold true. 4.4. BLAST analyses reveal many remipede neuropeptide precursors to be insect-like Publicly accessible protein sequences for members of the Crustacea are currently much more limited than those available for hexapods. A scan of the proteins accessible through NCBI for members of each taxon (conducted on October 22, 2013) revealed 130,000 crustacean sequences to have been publicly deposited vs. over 1.7 million sequences accessible for members of the Hexapoda. While there is a 10-fold discrepancy in sequence abundance between the two datasets, for all of the precursor protein families identified from S. cf. tulumensis, there are at least some publicly deposited homologs from members of each taxon. Despite the presence of crustacean precursor protein sequences in the NCBI database, few were returned as top 10 hits when Speleonectes sequences were used to query the combined crustacean and hexapod protein dataset using blastp searches. Moreover, for only three searches, DH31, NPF and sulfakinin, were crustacean sequences returned as the top hit, and here, each from a decapod. Again, while care must be taken in interpreting these findings

due to the differences in sequence richness of the datasets, it appears that the precursor proteins of S. cf. tulumensis are more insect-like in terms of their sequence structure and organization than they are crustacean-like, further supporting a close phylogenetic relationship between the remipedes and hexapods. Moreover, these data also suggest that the peptide precursors of this member of the Remipedia are more similar to those of the derived Decapoda than they are to those of crustaceans from more basal taxa (e.g. the Branchiopoda), though, again, this hypothesis is based on rather limited sequence comparisons. 4.5. Conclusions and future directions The data presented here represent the first identifications of putative peptide-encoding transcripts, peptide precursors and mature peptide isoforms from a member of the Remipedia, the crustacean taxon hypothesized to be the sister group to the Hexapoda. Fourteen distinct preprohormones representing 11 well-known arthropod peptide families were discovered, and the structures of 40 mature peptides were predicted. The deduced peptidome of Speleonectes, while limited in peptide richness, contains isoforms characteristic of both crustaceans and hexapods. BLAST comparisons of the Speleonectes preprohormones from which the peptides were derived with those from other crustaceans and hexapods revealed insect homologs as the closest matches. Thus, these data are consistent with the hypothesized phylogenetic position of the Remipedia within the Pancrustacea, and provide a starting point for future studies of the evolution of peptidergic and other neurochemical signaling systems in this and other remipedes. Acknowledgments The author thanks the Cades Foundation (Honolulu, Hawaii) for funding this project. References Adams, M.D., Celniker, S.E., Holt, R.A., Evans, C.A., Gocayne, J.D., Amanatides, P.G., Scherer, S.E., Li, P.W., Hoskins, R.A., Galle, R.F., George, R.A., Lewis, S.E., Richards, S., Ashburner, M., Henderson, S.N., Sutton, G.G., Wortman, J.R., Yandell, M.D., Zhang, Q., Chen, L.X., Brandon, R.C., Rogers, Y.H., Blazej, R.G., Champe, M., Pfeiffer, B.D., Wan, K.H., Doyle, C., Baxter, E.G., Helt, G., Nelson, C.R., Gabor, G.L., Abril, J.F., Agbayani, A., An, H.J., Andrews-Pfannkoch, C., Baldwin, D., Ballew, R.M., Basu, A., Baxendale, J., Bayraktaroglu, L., Beasley, E.M., Beeson, K.Y., Benos, P.V., Berman, B.P., Bhandari, D., Bolshakov, S., Borkova, D., Botchan, M.R., Bouck, J., Brokstein, P., Brottier, P., Burtis, K.C., Busam, D.A., Butler, H., Cadieu, E., Center, A., Chandra, I., Cherry, J.M., Cawley, S., Dahlke, C., Davenport, L.B., Davies, P., de Pablos, B., Delcher, A., Deng, Z., Mays, A.D., Dew, I., Dietz, S.M., Dodson, K., Doup, L.E., Downes, M., Dugan-Rocha, S., Dunkov, B.C., Dunn, P., Durbin, K.J., Evangelista, C.C., Ferraz, C., Ferriera, S., Fleischmann, W., Fosler, C., Gabrielian, A.E., Garg, N.S., Gelbart, W.M., Glasser, K., Glodek, A., Gong, F., Gorrell, J.H., Gu, Z., Guan, P., Harris, M., Harris, N.L., Harvey, D., Heiman, T.J., Hernandez, J.R., Houck, J., Hostin, D., Houston, K.A., Howland, T.J., Wei, M.H., Ibegwam, C., Jalali, M., Kalush, F., Karpen, G.H., Ke, Z., Kennison, J.A., Ketchum, K.A., Kimmel, B.E., Kodira, C.D., Kraft, C., Kravitz, S., Kulp, D., Lai, Z., Lasko, P., Lei, Y., Levitsky, A.A., Li, J., Li, Z., Liang, Y., Lin, X., Liu, X., Mattei, B., McIntosh, T.C., McLeod, M.P., McPherson, D., Merkulov, G., Milshina, N.V., Mobarry, C., Morris, J., Moshrefi, A., Mount, S.M., Moy, M., Murphy, B., Murphy, L., Muzny, D.M., Nelson, D.L., Nelson, D.R., Nelson, K.A., Nixon, K., Nusskern, D.R., Pacleb, J.M., Palazzolo, M., Pittman, G.S., Pan, S., Pollard, J., Puri, V., Reese, M.G., Reinert, K., Remington, K., Saunders, R.D., Scheeler, F., Shen, H., Shue, B.C., Sidén-Kiamos, I., Simpson, M., Skupski, M.P., Smith, T., Spier, E., Spradling, A.C., Stapleton, M., Strong, R., Sun, E., Svirskas, R., Tector, C., Turner, R., Venter, E., Wang, A.H., Wang, X., Wang, Z.Y., Wassarman, D.A., Weinstock, G.M., Weissenbach, J., Williams, S.M., Woodage, T., Worley, K.C., Wu, D., Yang, S., Yao, Q.A., Ye, J., Yeh, R.F., Zaveri, J.S., Zhan, M., Zhang, G., Zhao, Q., Zheng, L., Zheng, X.H., Zhong, F.N., Zhong, W., Zhou, X., Zhu, S., Zhu, X., Smith, H.O., Gibbs, R.A., Myers, E.W., Rubin, G.M., Venter, J.C., 2000. The genome sequence of Drosophila melanogaster. Science 287, 2185–2195. Altstein, M., Nässel, D.R., 2010. Neuropeptide signaling in insects. Adv. Exp. Med. Biol. 692, 155–165. Audsley, N., Weaver, R.J., 2009. Neuropeptides associated with the regulation of feeding in insects. Gen. Comp. Endocrinol. 162, 93–104. Audsley, N., Matthews, H.J., Price, N.R., Weaver, R.J., 2008. Allatoregulatory peptides in Lepidoptera, structures, distribution and functions. J. Insect Physiol. 54, 969– 980.

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