Similarities between decapod and insect neuropeptidomes

DNA sequences

The following short read archives (SRAs) were downloaded from NCBI using the prefetch command from the SRA Toolkit (http://www.ncbi.nlm.nih.gov/books/NBK158900/): for C. maenas: SRR1564428, SRR1572181, SRR1586326, SRR1589617, SRR1612556, SRR1632279, SRR1632285, SRR1632289, SRR1632290, SRR1632291, SRR1632292 and SRR1632293 (Verbruggen et al., 2015); for P. clarkii: SRR1144630, SRR1144631, SRR1265966, SRR1509456, SRR1509457, SRR1509458 and SRR870673 (Jiang et al., 2014; Tom et al., 2014; Shen et al., 2014; Manfrin et al., 2015); for M. rosenbergii: DRR023219, SRR1559288, SRR345608, SRR572725, DRR023253, SRR1653452, SRR345609, SRR896637, SRR1138560, SRR1653453, SRR345610, SRR896638, SRR1138561, SRR1653454, SRR345611, SRR896645, SRR1138562, SRR567391, SRR896646, SRR1138563, SRR572719, SRR896647, SRR1138564, SRR572720, SRR896649, SRR1138565, SRR572721, SRR896650, SRR1138572, SRR2082768, SRR572722, SRR896651, SRR1138573, SRR2082769, SRR572723, SRR1559287, SRR2082770, SRR572724 (Jung et al., 2011; Ventura et al., 2013; Suwansa-Ard et al., 2015); for S. paramamosain: SRR1310332, SRR1310333, SRR1205999, SRR3086589, SRR834579, SRR1206015, SRR3086590, SRR834580, SRR1310331 and SRR3086592 (Gao et al., 2014; Ma et al., 2014; Bao et al., 2015); for L. vanamei: SRR1037362, SRR1407789, SRR1460505, SRR1952625, SRR2103853, SRR2103860, SRR2895158, SRR1037363, SRR1104812, SRR1407790, SRR1609917, SRR2060962, SRR2103854, SRR2103861, SRR346404, SRR1037364, SRR1105791, SRR1407791, SRR1618514, SRR2060963, SRR2103855, SRR2103862, SRR554363, SRR1037365, SRR1104084, SRR1104085, SRR1460493, SRR1951370, SRR2060964, SRR2103856, SRR2103863, SRR554364, SRR1037366, SRR1184416, SRR1460494, SRR1951371, SRR2060965, SRR2103857, SRR2103864, SRR554365, SRR1039534, SRR1407787, SRR1460495, SRR1951372, SRR2103851, SRR2103858, SRR2103865, SRR556131, SRR1104083, SRR1104080, SRR1104086, SRR1104087, SRR1407788, SRR1460504, SRR1951373, SRR2103852, SRR2103859 and SRR2103866 (Li et al., 2012; Chen et al., 2013; Wei et al., 2014; Gao et al., 2015; Peng et al., 2015); for E. sinensis: ERR336998, SRR1555734, SRR2170964, SRR579530, SRR1199039, SRR1576649, SRR2170970, SRR579531, SRR1199053, SRR1735503, SRR2180019, SRR579532, SRR1199058, SRR1735536, SRR2180020, SRR769751, SRR1199228, SRR1735537, SRR546086, SRR770582, SRR1205971, SRR2073826 and SRR579529 (He et al., 2012; Hui et al., 2014; Li et al., 2014; Sun et al., 2014; Liu et al., 2015; Xu et al., 2015; Cui et al., 2015; Song et al., 2015; Wang et al., 2016); and for H. americanus: SRR2889572 and SRR2891007 (Christie et al., 2015). From Euphausia crystallorophias I analyzed ERR264582 (Toullec et al., 2013) for the presence of a novel putative neuropeptide that was found in the decapod transcriptomes.

The E. sinensis genome was downloaded from http://gigadb.org/dataset/100186, made into a BLAST database and searched for neuropeptide genes as described previously (Veenstra, 2014).

Data analysis

The fasta files were extracted from the SRAs using the fastq command from the SRA Toolkit from NCBI and then made into BLAST databases using BLAST+ (Camacho et al., 2009). Using the P. clarkii predicted neuropeptide precursors, as well as a few other peptides, as queries those databases were then searched using the tblastn command. A few neuropeptide receptors were also analyzed. Identified reads that appeared to belong to the orthologous gene were extracted from the database and then used to identify similar reads using the blastn command. The latter were used as input for the Trinity program (Grabherr et al., 2011) and resulting transcripts were recursively used as input until either the transcript stopped increasing in length or it was judged to be complete based on the location of in-frame stop codons and/or a signal peptide at the N-terminal of the protein predicted from the transcript. Calculations were run on a desktop computer with a AMD FX™-6100 six-core processor and 15.4 Gb of memory under Ubuntu Linux.

This method is very efficient for the extraction of transcripts from single copy genes. However, when there are several paralog genes that have not evolved a lot since their separation, some paralogs may be missed, particularly when their expression levels are low. In those cases, a selection of the particular neuropeptide precursors from which the non-conserved regions (such as the signal peptides) had been removed was used as a query in a tblastn command and all the obtained reads were then fed as input to the Trinity program. It can not be excluded that some less well expressed paralogs of those genes that exist in multiple copies have been missed. This may concern neuroparsin, CHH (crustacean hyperglycemic hormone), PDH (pigment dispersing hormone) and possibly CFSH (crustacean female sex hormone).

Clustal Omega (Sievers et al., 2011) was used for sequence alignments and those were inspected and, when needed, manually corrected using Seaview (Gouy, Guindon & Gascuel, 2010), which was also used to extract the regions for making phylogenetic trees with FastTree (Price, Dehal & Arkin, 2010).

Distribution

Having all the SRAs it seemed interesting to look at where the various genes might be expressed. Although it is possible to do this for all species involved, some are not very interesting as there is a very limited number of tissues sampled, while in other species the different tissues were sampled on different occasions and analyzed in different fashions, making direct comparisons difficult. However, in the case of C. maenas a single publication reports SRAs for a large variety of tissues (Verbruggen et al., 2015). Therefore, I used this species to look at the expression of the various neuropeptide genes in different tissues. Those neuropeptide receptors for which a contig of a significant size could be obtained and for which a likely ligand could be deduced based on homology to a deorphanized protostomian GPCR (see Veenstra, 2016a) were also included. The actual number of individual reads is often small and quantification of RNAseq reads is tricky due to the PCR amplification protocol used to create these libraries. Furthermore, the actual number of samples remains very small, so care should be taken in the interpretation of these data. Nevertheless, some interesting results are apparent (Table S9). Both the neuroparsins and the CHHs are expressed in virtually every tissue. In the case of the neuroparsin it is the neuroparsin 1 gene that is most abundantly expressed in all tissues, with the other two neuroparsin transcripts present at much lower levels. However, the two identified CHH transcripts are differentially expressed, one hormone is most abundant in the central nervous system and the eye (eyestalk), and the other in the intestine. Other neuropeptides found in the intestine are tachykinin, allatostatin C (Veenstra, 2016b), the B transcript of the calcitonin gene, elevenin, orcokinin, the agatoxin-like peptide and hyrg. The expression of CCHamide 2 and trissin in the hemolymph, presumably in hemocytes, is interesting to note as is the relatively large number of neuropeptides found in the SRA derived from the epidermis.

Paralogs

There are several neuropeptide genes that have one or more paralog genes. These are allatostatin C, CHH, moult inhibiting hormone (MIH), CCHamide, eclosion hormone, neuroparsin, PDH, insulin and CFSH. In some cases these are sufficiently different within the same species and sufficient similar between different species, that they clearly derive from different genes. This is the case for allatostatin C, CCHamide, insulin, neuropeptide F and eclosion hormone.

In the case of PDH, it is a bit more complicated. Variable numbers of PDH precursors were found in the seven decapod species. One group of precursors encoding PDH-like peptides distinguishes itself by an insertion of two-amino-acid residues in the predicted mature PDH. Such a predicted peptide was first found in P. clarkii (Veenstra, 2015), but since it was based on a single read in one species, it seemed premature to give it a distinct name. Now that complete precursor sequences are available and this peptide appears to be ubiquitously present in decapods, I propose to call it elongated PDH, or ePDH, to distinguish it from the more classical forms of these peptides (Fig. 2). The ePDH gene is one of the few genes that is present on a single contig of the draft genome from E. sinensis. It consists of three exons of which the first one is non-coding (Fig. 3). Partial sequence for one of the classical PDH genes show the intron between the two coding exons to be conserved.

In the case of neuroparsin, PDH, CHHs and its homolog MIH it is not always as clear that they represent different genes with unambiguous orthologs in different species. In some cases the observed differences could reflect allelic variations of a single gene or recent local gene duplications. Although no decapod genomes have been completely sequenced and the E. sinensis CHH genes are mostly very fragmentary, such local gene duplications are well known for CHH in decapods (Gu & Chan, 1998; Gu, Yu & Chan, 2000; Dircksen et al., 2001; Webster, Keller & Dircksen, 2012) as well as Chelicerates (Veenstra, 2016b) and particularly in decapods the number of CHH genes can be quite large (Webster, Keller & Dircksen, 2012).

CHH/MIH

The CHH/MIH neuropeptide family is characterized by CHH, MIH, mandibular organ-inhibiting hormone (MOIH), vitellogenesis-inhibiting hormone (VIH) and gonad-inhibiting hormone (GIH). These hormones have been identifed by different physiological assays, but are in many cases pleiotropic. For example, although identified in different biological assays, VIH and GIH are the same hormone. These peptides can be subdivided in two subfamilies, the CHHs proper and the other peptides. The precursors from the two groups differ in that the CHHs are produced together with a CHH-precursor related peptide, while the prepropeptides from the other homornes consist exclusively of a signal peptide and the sequence of the mature hormone (Webster, Keller & Dircksen, 2012). Four of the CHH/MIH transcripts identified here defy those rules as they do not have the CHH-precursor related peptide, yet on phylogenetic trees they form a separate branch that is closer to the CHH than to the MIH cluster (Fig. 4). Adding more sequences to the tree does not change this (data not shown). In the E. sinensis draft genome many sequences corresponding to these hormones are located on small scaffolds making it impossible to ascertain whether or not these genes are clustered.

CFSH

The CFSH is a recent discovery (Zmora & Chung, 2014) and consequently we still know very little of this very interesting hormone. In P. clarkii three related proteins were previously identified (Veenstra, 2015). In five of the other six decapod species two to four such hormones were found, but not in H. americanus, where there are no ovary transcriptomes. The primary sequence of these different putative hormones is not very well conserved, but the cysteine residues are (Fig. 5). The phylogenetic tree of these hormones suggests an initial gene duplication giving rise to two types of CFSH, that I have arbitrarily called CFSH 1 and 2 (Fig. 6). In most species both CFSH 1 and 2 were found, but in L. vannamei only three CFSH 1 paralogs were found and no CFSH 2. In the draft genome of E. sinensis CFSH 1 and 2b genes contain a single coding exon. The CFSH gene 2a transcript is incomplete and it is not clear from the genomic sequence what it is. This hormone was initially isolated from the eyestalk of the crab Callinectes (Zmora & Chung, 2014), while it in the crayfish P. clarkii it seemed to be strongly expressed in the ovary. It seemed therefore of interest to see whether these hormones might be expressed in the ovary in other decapods also. No significant expression was found in the ovaries of M. rosenbergii and L. vannamei (1 to 2 reads maximum for each hormone in an SRA), but 9 reads corresponding to L. vannamei CFSH 1c (as well as 1 each for 1a and 1b) are present in SRR2060962 from the L. vannamei testis. In E. sinensis expression is similar to that in C. maenas (Table S9), high expression levels in the eyestalk and a few reads only in the ovary. For S. paramamosain and H. americanus there are insufficient data to answer this question.

Neuroparsins and receptors

Three to four neuroparsin transcripts were identified in each of the seven decapod species. Three of the E. sinensis genes were found in the draft genome, two of these (neuroparsins 3 and 4) are on the same scaffold in a tail to tail configuration, where the start and stop codons of the two genes are separated by 11,960 and 9,045 nucleotides respectively (Fig. 7). These are the two E. sinensis genes that are most similar to one another, suggesting that they may reflect the most recent neuroparsin gene duplication in this species. As both these genes seem to have direct orthologs in S. paramamosain and C. maenas, that particular gene duplication possibly occurred in a common ancestor of the three crab species (Fig. 8). The neuroparsin receptor was recently identified as a venus kinase receptor (Vogel, Brown & Strand, 2015); two such receptors are found in all seven decapod species (Table S8). The phylogenetic tree made of the various venus kinase receptors suggests that the other arthropods venus kinase receptors are equally similar to both decapod receptors (Fig. 9).

Insulin-like peptides and receptors

Three different insulin-related peptides were identified. These are the well known androgenic insulin-related peptide (Fig. 10), an insulin-like peptide (Fig. 11) that seems most similar to the Drosophila insulin-like peptides 1–6 (Nässel & Vanden Broeck, 2016), and a peptide that is orthologous to Drosophila insulin-like peptide 7 and that has been called relaxin (Fig. 12). The latter was previously identified in Sagmariasius and P. clarkii (Chandler et al., 2015; Veenstra, 2015). As can be seen from the figures, the androgenic insulin-like peptide is the least conserved of those three (Figs. 10–12). Insulin-related peptides use two different types of receptors, the typical tyrosine kinase receptor and GPCRs. Insects typical have one gene coding an insulin tyrosine kinase receptor and have one or two GPCRs that are related to the vertebrate relaxin receptors RXFP1 and RXFP2. Given the interest in the androgenic insulin-like peptide both for its intriguing physiology as a peptidergic sex hormone and for its commercial potential (Ventura and Sagi, 2012), I also analyzed the likely insulin receptors.

The typical insulin tyrosine kinase receptor, similar to the one recently described from M. rosenbergii (Sharabi et al., 2016), was also found in the other six decapods (Table S8). Two receptors similar to the vertebrate relaxin receptors RXFP1 and RXPF2 were identified. Those two GPCRs are most similar to the Drosophila receptors CG31096 and CG34411, also known as leucine-rich repeat containing GPCR- 3 and 4 (LGR3 and LGR4) respectively. However, the much weaker expression of those receptors made it impossible to deduce their complete cDNA sequences and in some cases no contigs could be obtained. Interestingly the SRA from the E. sinensis accessory gland (SRR2170964) not only shows very large number of reads for the androgenic Insulin-like peptide, but also very significant expression of the insulin tyrosine kinase receptor and a somewhat lower expression of the ortholog of Drosophila LGR3.

Splice variants

There were a number of neuropeptide encoding cDNAs that revealed splice variants. Those that concerned the untranslated regions were ignored, but there are five neuropeptide genes that have alternative transcripts producing different precursors: the CHHs, CNMamide, neuropeptide F 1, calcitonin and the agatoxin-like peptide. In the case of neuropeptide F 1, there is an extra exon sliced into the sequence of the peptide, as described previously from insects and D. pulex (Roller et al., 2008; Nuss et al., 2010; Dircksen et al., 2011). The CNMamide gene in the termite Zootermopsis contains five coding exons, the last two of which are alternatively added to the first three and then produces a different CNMamide-like peptide. In four of the seven decapods, similar alternative splice products were found for the CNMamide precursor. However, while the mature peptide derived from the major splice form is well conserved, the second is much less so (Fig. 13). Two to four splice variants (Fig. S2) were found for the recently discovered μ-agatoxin-like peptide (Sturm et al., 2016). As in some insects (Veenstra, 2014), the calcitonin gene produces two different transcripts, encoding different types of calcitonin, that are similar to the insect calcitonins (Fig. S3). In L. vannamei, M. rosenbergii, H. americanus and P. clarkii the second transcripts are predicted to produce a calcitonin-like peptide that does not have one but two cysteine bridges at is N-terminus (Fig. 14). The calcitonin gene is absent from the E. sinensis draft genome, and hence it is impossible to compare the insect and decapod calcitonin gene structures.

Other peptides

In several cases novel neuropeptides have been detected by mass spectrometry. These are often structural variants of well known neuropeptides such as the RFamides, tachykinins and allatostatins A or B (e.g., Ma et al., 2008; Ma et al., 2009; Ma et al., 2010). However, not all peptide sequences identified this way belong to known neuropeptides. From H. americanus, C. maenas and L. vannamei other peptide sequences have been reported. The ones from C. maenas have previously been suggested to represent fragments of cryptocyanin (Ma et al., 2009), and this was confirmed (Fig. S4). Several of the peptides from H. americanus are shown here to represent fragments of thymosin, actin or histone 2A, however the origins of others remain unclear (Fig. S4). The one peptide reported from L. vannamei, L/IPEPEDPMAEAGHEL/I (Ma et al., 2010), is more interesting, as it could potentially be part of a novel neuropeptide (precursor). This sequence is part of a small protein that has a signal peptide followed by a peptide containing a small piece that is very well conserved (Fig. 15). However, it lacks the classical convertase cleavage sites that one usually finds in neuropeptide precursors and hence its status as a neuropeptide is unclear. Such proteins are also found in the other decapods. Although it was not possible for Trinity to produce a complete contig for S. paramamosain, a similar sequence is present in the databanks for Scylla olivacea. I was unable to find similar proteins in insects, but an orthologous protein was detected in the SRA from Euphausia crystallorophias. The latter sequence shows that the only conserved part is the same as in the decapods (Fig. 15). This peptide was called hyrg (pronounced hirg), for four of the conserved amino acids. Interestingly, the eyestalk seems to be the tissue where expression of hyrg is the highest (Table S9), thus suggesting that it is likely a neurohormone.

Neuropeptide evolution

It thus appears that the neuropeptidome of decapods has been remarkably well conserved during evolution. Differences that are found between the insect and decapod neuropeptidomes are the loss or the gain of a neuropeptide. Although there possibly still remain arthropod neuropeptides to be discovered, it appears that the loss of neuropeptides in decapods is limited to a single neuropeptide, i.e., allatotropin. Allatotropin is present in mollusks, annelids as well as chelicerates (Veenstra, 2010a; Veenstra, 2011; Veenstra, 2016a) and hence, it must have been present in the arthropod ancestor. Small peptides are sometimes hard to find using the BLAST program and allatotropin is no exception to this rule (Veenstra, Rodriguez & Weaver, 2012). Nevertheless, as I was neither able to find even a single read correponding to its receptor, including in the very abundant number of transcriptome reads from H. americanus, I conclude that this peptide was most likely lost. In the termite and the fruit fly on the other hand, more neuropeptides are missing, particularly in Drosophila. At first sight insects, as a group, lack EFLamide, the androgenic insulin-like hormone, CFSH and ePDH. However, the recent identification of an EFLamide receptor in Platynereis dumerlii as a TRH GPCR ortholog (Bauknecht & Jékely, 2015) and the presence of such a GPCR in Nilaparvata lugens (Tanaka et al., 2014) suggests that some insects may have such a gene. As described below, it is plausible that the androgenic peptide has an insect ortholog. What seems really different is that many insect species, in particular holometabolous species, have lost several neuropeptides (Derst et al., 2016). Thus Drosophila no longer has genes for elevenin, vasopressin, allatotropin, allatostatin CCC, EFLamide, neuroparsin, calcitonin, ACP, eclosion hormone 2, neuropeptide F 2 and it also lost the possibility to produce alternative transcripts from the CNMamide and neuropeptide F1 genes. The beetle Tribolium castaneum on the other hand still has most of those neuropeptides, but lost allatostatin A, corazonin and leucokinin.

New neuropeptides

Since the last common ancestor of decapods and insects—estimated to have lived 596 Mya (Hedges et al., 2015)—very few neuropeptides seem to have been added to either of the two lineages. Novel neuropeptide genes that have appeared seem all to have originated by duplication from existing ones and are easily recognized as the paralogs of the parent genes. Examples of such genes are the various paralogs of CHH and MIH, PDH and neuroparsin in crustaceans and in insects the typtopyrokinin and SIFamide paralogs as well as the great variety of adipokinetic hormones (all orthologs of crustacean RPCH). The only exception may be hyrg, the precursor for the peptide initially identified from L. vannamei (Ma et al., 2010). This peptide, that is well expressed in the eyestalk and the midgut, has a distribution typical of a neuroendocrine peptide. As I was unable to find it outside of crustaceans, it could be a novel invention of this group. The structure of this putative neuropeptide precursor is somewhat reminiscent of limostatin, a small neuroendocrine protein discovered in Drosophila that intereacts with a GPCR (Alfa et al., 2015) previously identified as the receptor for neuropeptide pyrokinin 1 (Cazzamali et al., 2005). The similarity between limostatin and hyrg resides in the apparent absence of conventional convertase sites in these putative neuropeptide precursors (those postulated to function in the limostatin precursor (Alfa et al., 2015) seem highly unusual (Veenstra, 2000)). In the same context the Drosophila sex peptide comes to mind, as it also acts on a neuropeptide receptor without having neither a well conserved structure nor the typical neuropeptide convertase cleavage sites (Kim et al., 2010). Perhaps one or more of these proteins represent newly evolved ligands for existing neuropeptide receptors that could potentially become novel neuropeptides.

Missing neuropeptides

Many decapod neuropeptides have been identified by mass spectrometry over the years (e.g., Stemmler et al., 2007a; Stemmler et al., 2007b; Stemmler et al., 2010; Christie et al., 2008; Ma et al., 2008; Ma et al., 2009; Ma et al., 2010; Dickinson et al., 2008; Dickinson et al., 2009a; Dickinson et al., 2009b). Most of those were identified in the various SRAs, although not always in exactly the same molecular form. In particular, I was unable to find some of the analogs of SIFamide that have been reported (e.g., Hui et al., 2012). I could neither find [Val¹]-SIFamide in any species, however this peptide seems to be present in the stomatogastric nervous system (Christie et al., 2006) and this might explain its absence from the various SRAs. Several of the peptides previously described from these data that did not appear to be neuropeptides could be identified as being part of well known proteins and it also allowed me to identify the hyrg trancript. However, there are three neuropeptides that either have been reported or suggested to be present in decapods that were not found in any of the SRAs from the seven decapod species studied here. These are a pituitary adenylate cyclase activating polypeptide (PACAP) from L. vannamei (Lugo et al., 2013), a GnRH-like peptide from P. clarkii (Guan et al., 2014) and two kisspeptins from M. rosenbergii (Thongbuakaew et al., 2016). None of these peptides could be found in any of the SRAs, neither those from the species from which they were reported, nor from any of the other species. In two cases (PACAP and GnRH), the amino acid sequences of the peptides have been published from the same species used here, so my inability to find these peptides is not due to significant sequence differences between the species used for bioinformatic analysis and those from which the peptides were identified. I was neither able to find evidence for the receptors for such peptides in any of decapods. The GnRH receptor identified from the ovary of the oriental river prawn Macrobrachium nipponense is the corazonin receptor ortholog (Du, Ma & Qiu, 2015), implying that corazonin is its ligand. Given the strong conservation of the decapod neuropeptidome described here, I conclude that is highly unlikely that any of those three peptides is present in decapods.

Functional aspects

Conservation of structure does not necessarily imply conservation of function. The function of crustacean RPCH and its insect ortholog AKH are distinctly different. A neuropeptide sequence does not reveal its function, but the distribution of its receptor may give some clues. FMRFamide is known to effect muscle contraction in decapods (Worden, Kravitz & Goy, 1995), the expression of its putative receptor in muscle, heart and the epidermis (that contains muscle as well) suggests that it has similar effects. The simultaneous expression of elevenin and a putative elevenin receptor in the midgut suggests that is has a digestive function. The hormone GPA2/GPB5 was suggested to be an antidiuretic hormone in Drosophila (Sellami, Agricola & Veenstra, 2011) and was subsequently shown to stimulate sodium reabsorption in the mosquito hindgut (Paluzzi, Vanderveken & O’Donnell, 2014). The very abundant expression of its receptor in the gill suggests that its function C. maenas may well be similar. An interesting difference between insects and decapods is the presence of ecydysis triggering hormone in the decapod nervous system and eye(stalk); in insects this peptide seems to be exclusively present in cells associated with the tracheal system and absent from the central nervous system (Roller et al., 2010). It will be interesting to know whether the function of ecdysis triggering hormone within the decapod nervous system is related to ecdysis behavior.

Intestine

Neuropeptides in the intestine are typically produced by enteroendocrine cells. CHH (Chung, Dircksen & Webster, 1999), SIFamide and tachykinin immunoreactive enteroendocrine cells (Christie et al., 2007) have been previously described from decapods. No SIFamide reads were found in the C. maenas intestine SRA, but allatostatin C, calcitonin-B, elevenin, orcokinin and hyrg were all present in seemingly significant numbers of reads (Table S9). This ensemble of gut neuropeptides differs significantly from what is known from the Drosophila midgut (Veenstra & Ida, 2014), even though tachykinin, allatostatin C and orcokinins are present in both, while the calcitonin B transcript is abundant in phasmid midgut SRAs (Veenstra, 2014).

CHH and MIH

The neuropeptides related to CHH are amongst the best known crustacean hormones (excellent review by Webster, Keller & Dircksen, 2012). As was expected based on the literature, several molecular forms were found. There are reasons to think there may be more of these hormones than reported here. First of all, the few decapod CHH genes that have been identified are typically present in clusters and in Metapaeneus ensis 16 such genes have been found (Gu & Chan, 1998). Secondly, as shown here and elsewhere (e.g., Hsu et al., 2006; Li et al., 2010; Ventura-López et al., 2016) some of these genes are differentially expressed. Thus, if a gene is predominantly expressed in a tissue not included in the analysis, it may not be found. Finally, since these hormones are similar in structure, it is possible that Trinity would have problems producing all contigs.

The biological activities of these hormones vary widely and the hormones with very similar sequences may have quite different physiological effects (e.g., Webster, Keller & Dircksen, 2012; Luo et al., 2015). It is for this reason impossible to interpret the meaning of the four predicted hormones that defy classification as either a CHH-like or MIH-like hormone (Fig. 5).

PDH

There are generally within the same species several precursors coding the shorter, more classical, PDHs, those different precursors code sometimes for the same mature peptide. It seems plausible that some of these differences reflect either allelic variations of a single gene or recent local gene duplications. Most of the species have two or more different predicted mature PDH peptides. It has previously been shown that the two PDHs from the crab Cancer productus have different functions, one is released as a hormone into the hemolymph, while the other is used within the central nervous system (Hsu et al., 2010). As the tissue used for the S. paramamosain transcriptome did not include the eyestalk it is thus not surprising that the hormonal PDH is lacking from the deduced transcriptome in this species. ePDH is not expressed in the eyestalk and one might therefore be tempted to think it is not released into the hemolymph. However, it is present in the L. vannamei transcriptome that was produced from abdominal muscle, hepatopancreas, gills and pleopods (Ghaffari et al., 2014) and thus is likely produced somewhere in the periphery (this transcriptome contains relatively few neuropeptides as it includes neither the central nervous system nor the intestine).

CFSH

CFSH was discovered very recently in the crab Callinectes sapidus (Zmora & Chung, 2014) and consequently we know still very little of this extraordinarily interesting hormone. I previously reported the presence of both CFSH and two homologous proteins in P. clarkii (Veenstra, 2015). Now that there are a few more sequences available, it appears that this gene commonly has several paralogs. Some of these seem to have a relatively recent origin, as the most closely related sequence comes from the same species (Fig. 6). The independent gene duplications of these proteins as well the great sequence variability between and within species may indicate that all these hormones act on the same receptor. Given the relatively large size of these hormones one might expect a leucine rich repeat G-protein coupled receptor or a dimeric protein kinase, perhaps one of the two venus kinase receptors, but this remains speculation. The primary structure of CFSH is not very well conserved and its receptor is unknown. Hence, we don’t know whether an orthologous hormonal regulatory system might be present in other arthropods, like e.g., insects (given the great similarity in their neuropeptidomes this seems a distinct possibility, at least in the more primitive insects). It seems that the expression of this hormone in the ovary of P. clarkii (Veenstra, 2015) is unusual, as it was not found in any of the other decapods for which an ovary SRA is available.

Insulin and neuroparsin

Other intriguing neuropeptides are the neuroparsins and the insulin-related hormones. There are three different insulin-like hormones. There are also three different insulin receptors, the classical tyrosine kinase and two G-protein coupled receptors. What I have called insulin is the hormone most similar to the Drosophila insulin-like peptides 1–6, which function as growth hormones and are also important for reproduction and that signal through the classical tyrosine kinase receptor (Nässel & Vanden Broeck, 2016). The same receptor is also present in decapods as shown here and elsewhere (Veenstra, 2015); it has recently been characterized in two decapods (Aizen et al., 2016; Sharabi et al., 2016). Both insulin and neuroparsins activate tyrosine kinase receptors. However, whereas the actions of insulin in insects are relatively well understood due to very extensive research on these peptides in Drosophila (Nässel & Vanden Broeck, 2016), the function of neuroparsin is less clear, as it is absent from Drosophila melanogaster (Veenstra, 2010b). It is interesting to note that some species have several insulin genes and few if any neuroparsin genes (Drosophila, Acyrthosiphon, Zootermopsis), while decapods and Locusta have several neuroparsin transcripts and only a single insulin gene, suggesting some complementation between these two hormones. Indeed, in some cases, such as vitellogenesis in the mosquito both hormones have synergistic effects (Brown et al., 1998; Dhara et al., 2013), however in the migratory locust they act antagonistically (Badisco et al., 2011). Initially isolated from the migratory locust L. migratoria (Girardie et al., 1989) neuroparsin was shown to have strong anti-juvenile hormone effects, effecting both reproduction and metamorphosis (Girardie et al., 1987). It has been shown that neuroparsin RNAi also inhibits vitellogenesis, and hence reproduction, in the decapod Metapenaeus ensis (Yang et al., 2014). The receptor for this hormone was recently identified in mosquitoes as a venus kinase receptor (Vogel, Brown & Strand, 2015), a type of receptor that was lost in chordates during evolution (Dissous, 2015). Although orthologous venus kinase receptors are present in other arthropod genomes (notably Limulus, Strigamia and Stegodyphus, Table S8) as well as mollusks (Vanderstraete et al., 2013), no neuroparsin orthologs could be found in those species. The evolutionary origin of neuroparsin is therefore unclear and it is not known whether species that seem to lack neuroparsin need a hormone ligand to activate the venus kinase receptor (Dissous, 2015). The presence of two such receptors in decapods is intriguing, but has also been found in Lepidoptera and trematodes (Dissous, 2015).

The other insulin-like peptides

Insects and decapods share many neuropeptides and it is not surprising that the various decapod insulin-related hormones also have insect orthologs. The insulin-like hormone I have called relaxin is an ortholog of Drosophila insulin-like peptide 7. This hormone is not only present in insects, but also in ticks, spider mites, mollusks and even acorn worms (Veenstra, 2010a; Veenstra, Rombauts & Grbić, 2012). As previously pointed out, this hormone is only present in the genomes of those species that also have an ortholog of Drosophila gene CG34411, encoding LGR4 that is homologous to vertebrate relaxin GPCRs (Veenstra, Rombauts & Grbić, 2012; Veenstra, 2014). This suggests that this GPCR functions as a receptor for the arthropod relaxins. It must be noted that this does not exclude the possibility that arthropod relaxins may also signal through the classical insulin tyrosine kinase receptor. In fact, there is evidence from Drosophila that this is so (Linneweber et al., 2014).

Drosophila has an eighth insulin-like hormone that was initially discovered because it is secreted by the imaginal discs (Colombani, Andersen & Léopold, 2012; Garelli et al., 2012). However, data from fly atlas (Chintapalli, Wang & Dow, 2007) show that it is also expressed by the ovary. This hormone was suggested to be acting through the GPCR encoded by Drosophila gene CG31096 encoding LGR3 (Veenstra, 2014) and this has now been confirmed (Vallejo et al., 2015; Garelli et al., 2015). LGR3 is also related to vertebrate GPCRs binding relaxin. As reported previously it has a P. clarkii ortholog (Veenstra, 2016a), and as shown here is generally present in decapods. Combined these data suggest that LGR3 is the receptor for the androgenic insulin-like peptide from the accessory gland. The absence of clear sequence homology between the Drosophila and decapod peptides is not surprising, as the primary sequence of this hormone is poorly conserved in both decapods (Fig. 10) and insects (other insects almost certainly have such a peptide, since they have the receptor, but only within flies is it possible to find orthologs using the BLAST program). Interestingly, both these hormones are produced by gonads or associated accessory glands. At first sight it seems that in crustaceans it is predominantly the male that produces it, while in adult flies it is the female. However, work on the expression of LGR3 in Drosophila shows it to be important for the development of both male and female specific sexual characters (Meissner et al., 2016) and it is perhaps better considered a (sexual ?) maturation hormone for both sexes. This would also make it easier to understand how during evolution it was coopted by the imaginal discs. In decapods the male has two Z chromosomes and is the default sex (Parnes et al., 2003; Staelens et al., 2008; Ventura et al., 2011; Cui et al., 2015). Therefore, one would expect females to have a mechanism (not necessarily hormonal) to escape becoming a male and might thus expect a gynogenic rather than an androgenic hormone (this is one of the reasons why CFSH is so interesting). Even in decapods there is now evidence that the androgenic insulin-like peptide is not specific for males. Thus, in S. paramamosain it is also expressed by the ovary and at higher levels at the end of vitellogenesis (Huang et al., 2014). While the relative levels of expression may seem low as compared to those of actin (Huang et al., 2014), the actual quantities of peptide produced may well rival those made by the accessory gland, considering that the ovary is so much larger (could the accessory gland be the remnants of an embryonic ovary anlage ?). Given the effectiveness of RNAi in crustaceans and the strong phenotypes obtained in the absence of androgenic peptide (Ventura et al., 2009), the hypothesis that LGR3 is important in the transduction of the androgenic peptide signal can be tested. As with relaxin, a GPCR specifically activated by the androgenic insulin-like peptide does not exclude the possibility that it may also act on the classical insulin tyrosine kinase receptor, as shown by recent experiments in the decapod Sagmariasus (Aizen et al., 2016). Possible relations between the decapod insulin-related peptides and their receptors are illustrated in Fig. 16.

It is of interest to note that the mammalian GPCR most similar to LGR3 is RXFP2, the receptor for insulin-related peptide 3. The latter hormone was initially discovered from the testis and is important not only to insure testicular descent (Adhama, Emmen & Engel, 2000) but also in the female reproductive system (Satchell et al., 2013). Thus the data suggest that not only the structures of the receptor and its ligand are recognizably similar, but so might be their function. This is rather interesting, as most neuropeptides with orthologs in both proto- and deuterostomia have quite different functions in these two groups.