In silico prediction of neuropeptides in Hymenoptera parasitoid wasps

Parasitoid wasps of the order Hymenoptera, the most diverse groups of animals, are important natural enemies of arthropod hosts in natural ecosystems and can be used in biological control. To date, only one neuropeptidome of a parasitoid wasp, Nasonia vitripennis, has been identified. This study aimed to identify more neuropeptides of parasitoid wasps, by using a well-established workflow that was previously adopted for predicting insect neuropeptide sequences. Based on publicly accessible databases, totally 517 neuropeptide precursors from 24 parasitoid wasp species were identified; these included five neuropeptides (CNMamide, FMRFamide-like, ITG-like, ion transport peptide-like and orcokinin B) that were identified for the first time in parasitoid wasps, to our knowledge. Next, these neuropeptides from parasitoid wasps were compared with those from other insect species. Phylogenetic analysis suggested the divergence of AST-CCC within Hymenoptera. Further, the encoding patterns of CAPA/PK family genes were found to be different between Hymenoptera species and other insect species. Some neuropeptides that were not found in some parasitoid superfamilies (e.g., sulfakinin), or considerably divergent between different parasitoid superfamilies (e.g., sNPF) might be related to distinct physiological processes in the parasitoid life. Information of neuropeptide sequences in parasitoid wasps can be useful for better understanding the phylogenetic relationships of Hymenoptera and further elucidating the physiological functions of neuropeptide signaling systems in parasitoid wasps.


Introduction
Parasitoid wasps (Order: Hymenoptera) are one of the most species-rich groups of animals, potentially accounting for more than 20% of the insects found globally [1]. Studies on insect parasitoids are important to characterize their biodiversity, understand their evolution and, in some cases, apply their parasitic abilities for practical purposes such as biological control of agricultural pests.
In the present study, the publicly accessible sequence data were mined for identifying putative precursor sequences of neuropeptides in parasitoid wasps, and mature bioactive peptide sequences were predicted using a well-established in silico workflow (e.g. Huybrechts et al. [10], Veenstra [12], Christie [14], Xu et al. [15], Christie et al. [16], Christie [24]). Totally more than 500 neuropeptide precursors were found from 24 parasitoid wasp species belonging to six superfamilies: Chalcidoidea, Ichneumonoidea, Cynipoidea, Chrysidoidea, Orussoidea and Platygastroidea. All of these superfamilies except Orussoidea belong to Suborder Apocrita. All parasitoid taxa from Orussoidea and most of parasitoid taxa from Chrysidoidea are ectoparasitoids or cleptoparasitoids; all parasitoid taxa from Cynipoidea and Platygastroidea are endoparasitoids; and both ectoparasitoids and endoparasitoids are included from two of the largest superfamilies (Chalcidoidea and Ichneumonoidea), as reviewed by Whitfield [25]. Mining of neuropeptides of parasitoid wasps might be helpful for further understanding their physiological roles.

Phylogenetic analysis and sequence alignment
ClustalX software [29] was used to perform multiple sequence alignments, by using the slowaccurate mode with a gap-opening penalty of 10 and gap-extension penalty of 0.1, and applying the default Gonnet protein weight matrix. Alignments were visualized using Bioedit v7.0.5.3. Phylogenetic trees of precursor sequences were constructed in MEGA v6.06 [30] by using the neighbor-joining method and bootstrap analysis with 1000 replicates. Sequence logos of manually aligned homologous neuropeptide sequences were generated using the online tool WebLogo (http://weblogo.berkeley.edu/logo.cgi) [31].

Results and discussion
The in silico mining of neuropeptides of parasitoid wasp species Mining from the publicly accessible databases led to the in silico prediction of a total of 517 precursors from 24 parasitoid wasp species belonging to six superfamilies: Chalcidoidea, Ichneumonoidea, Cynipoidea, Chrysidoidea, Orussoidea and Platygastroidea (S1 Table). All the neuropeptide precursors with the predicted putative mature peptide structures are shown in S1  Table 1. Hauser et al. [23] found 30 precursor genes encoding neuropeptides of N. vitripennis. Allatotropin of N. vitripennis was previously identified by Veenstra et al. [32]. Additional neuropeptide genes of N. vitripennis were confirmed in this study, such as CNMamide (CNMa), FMRFamide-like (FMRFa), ITG-like, orcokinin B and an orthologous gene of ion transport peptide-like (ITPL) (S1 Table,  For comparison of the neuropeptides of parasitoid wasps and other insect species (e.g., A. mellifera, L. migratoria, R. prolixus, T. castaneum, B. mori and D. melanogaster; Fig 1), phylogeny analysis of the identified precursor genes and sequence alignment of the predicted mature peptides of insect neuropeptides were conducted (S2 Fig; Figs 2 -5). Although it should be avoided for conducting phylogenetic trees of insects neuropeptide precursors for high diversity of precursor sequences (except mature peptides) in different insects in most cases (e.g., [33][34]), phylogenetic trees with neuropeptide precursors performed in this study showed that most of neuropeptide precursors from different parasitoid wasps were grouped together, probably because higher conservations occur among neuropeptide precursors from the very close taxa. The numbers of predicted neuropeptide genes from parasitoid wasp species were lower than those in other insect species (Fig 1). Some neuropeptides that are conserved in other insects were still highly conserved among parasitoid wasps, such as adipokinetic hormone (AKH), arginine-vasopressin-like peptide (AVLP), crustacean cardioactive peptide (CCAP), CCHamide, myosuppressin, and SIFamide (Panels ii, vi, x, xi, xxii and xxxiii in S2 Fig). All peptide sequences of AVLP and CCAP from different insect species were 100% identical (Panels vi and x in S2 Fig). Moreover, some interesting patterns regarding evolutionary orphysiological perspectives were found in this study.

Fig 2. Phylogenetic tree of AST-CC precursors and alignment analysis of AST-CC sequences in parasitoid wasps and other insect species.
Chrysidoidea sequences in phylogeny trees are indicated with blue circles; Ichneumonoidea sequences are indicated with light green squares; Chalcidoidea sequences are indicated with red triangles; Cynipoidea sequences are indicated with light blue rhombuses; Orussoidea with empty squares. Numbers above branches indicate phylogenies from amino acid sequences and only values above 50% are shown. Identities in alignments are highlighted in dark (100%) and in grey (80%~100%). (PK), are known to encode three kinds of insect PRXamide peptides: periviscerokinins (PVKs), pyrokinins (PKs) and trypto-PKs [12,[36][37][38]. The encoding patterns of CAPA/PK genes were found to differ among Hymenoptera species and other insect species (Fig 4). CAPA precursors from Ichneumonoidea, Chalcidoidea and A. mellifera encode only one or two PVKs, whereas those in Argochrysis armilla and ants (e.g. Camponotus floridanus) encode two PVKs and one PK peptide. CAPA precursors in R. prolixus, B. mori and D. melanogaster encode PVKs and trypto-PKs; in contrast, those in L. migratoria and T. castaneum encode PVKs, trypto-PKs, and one PK peptide. PK precursors from Hymenoptera species, B. mori and T. castaneum encode trypto-PK and PK peptides, whereas L. migratoria, R. prolixus, D. melanogaster encode only PKs (Fig 4).

Distinct patterns of neuropeptides between different groups of parasitoid wasps
Interestingly, sulfakinin (SK) was only found in cleptoparasitic wasps (Chrysidoidea: Argochrysis armilla and Chrysis viridula ; Fig 1; Panel xxxiv in S2 Fig), and was not found in any other wasp groups based on BLAST results in NCBI. After the receptor genes for SK were checked based on BLAST analysis in NCBI, no gene encoding SK receptor was found from any parasitoid wasp species except from Chrysidoidea wasps. SK was first isolated from Leucophaea madera and was shown to stimulate hind gut contractions [39][40]. SKs are multifunctional neuropeptides found in many insects (e.g., A. mellifera, Camponotus floridanus, L. migratoria, R. prolixus, T. castaneum, B. mori and D. melanogaster; Fig 1) and are involved in food uptake [41]. It seems that the obvious lack of SK in endoparasitoid taxa could be related to the distinct food patterns in the parasitoid life. However, further studies are warranted to determine whether the absent of SK in endoparasitoid taxa is related to the distinct parasitoid life form or limited transcriptome data.
In particular, a few neuropeptides (e.g., elevenin, kinin, natalisin, neuropeptide-like precursor 1 (NPLP1), and trissin) were not found in any species of Chalcidoidea, but were present in other parasitoid wasp species and other insect species (Fig 1; Panels xvi, xxi, xxiii and xxvii S2  Fig). Among them, three neuropeptides, kinin, NPLP1, and trissin, were reported to be involved in insect feeding progress [3,18,42]. Insect kinins are small neuropeptides that function as myotropic, neuromodulatory, and diuretic hormones in the Malphigian tubules of insects [3]. NPLP1 was identified in the salivary glands of R. prolixus [18], suggesting that it plays a role in the hormonal control of salivary secretion. Trissin is dominantly expressed in the frontal ganglion of B. mori [42], indicating its possible role in the regulation of foregutmidgut contractions and food intake. Whether the lack of these neuropeptides in parasitoid wasps is related to their distinct feeding patterns or limited transcriptome data needs to be investigated in the future.
Elevenin was first identified as a neuropeptide from the abdominal ganglion of the gastropod mollusk Aplysia californica [43]. Similar neuropeptide precursors have been identified from many insect species [12,13]. At present, only one report is available regarding the physiological role of elevenin [44]. In the planthopper Nilaparvata lugens, elevenin is known to regulate body color via a G protein-coupled receptor NlA42, which is expressed in the abdominal integument; this might indicate the direct action of elevenin on the melanization of the cuticles of N. lugens [44]. In the present study, elevenin was not found in any Chalcidoidea species, as well as in D. melanogaster and B. mori (Fig 1; Panel xvi in S2 Fig). Phylogenetic analysis of insect elevenin precursor genes showed a significant divergence between Hymenoptera and other insects (Panel xvi in S2 Fig). Sequence alignment of insect elevenin peptides showed high variations among different insect species, which only share a C-terminus motif CRGXXX and two cysteine residues (Panel xvi in S2 Fig). However, the elevenin gene sequences were highly conserved within the same subfamily of parasitoid wasps, e.g., Microgastrinae, Opiinae and Chrysidini, that they can be used a molecular marker for species identification between different subfamilies of small parasitoid wasps (e.g., of Braconidae family; Panel xvi in S2 Fig). Like mentioned before, information reagrding insect elevenin is limited, and hence the determination of whether elevenin was not found in Chalcidoidea wasps because of their distinct evolutionary/physiological progress, or the remarkable diversity in these wasps, or limited transcriptome data is not possible. Natalisin was first identified as a functional neuropeptide associated with sexual activity and fecundity in insects [45]. In the present study, natalisin was found in five Ichneumonoidea species and other Hymenoptera species (e.g., Athalia rosae and Camponotus floridanus), but not in any other parasitoid species except Ichneumonoidea (Panel xxi in S2 Fig). High variants in copy numbers and peptide sequences of natalisin occur between Hymenoptera species and other insects, as well as among different species from Ichneumonoidea, suggesting that natalisin was not found in some parasitoid wasps because of the remarkable diversity of natalisin in parasitoid wasps and limited transcriptome data.
Several neuropeptides showed vast sequence differences between between Ichneumonoidea and Chalcidoidea, the two major superfamilies of parasitoid wasps. Short neuropeptide F (sNPF) was first identified in Aedes aegypti [46]. The main functions of sNPF is likely the regulation of feeding behavior [47]. sNPFs are widespread among parasitoid wasps. sNPF precursors were found in 17 species of six superfamilies (Figs 1 and 5). This neuropeptide is conserved in parasitoid wasps and possesses a C-terminal motif-RSPSL/YRLRFamide (Fig 5). Two distinct peptides were predicted from sNPF precursors in all six Chalcidoidea species, whereas only one sNPF peptide was found from each of the other 11 parasitoid species. All the predicted precursors of parasitoid wasp species except for the six Chalcidoidea species possess the same or similar C-terminal motifs as A. mellifera_sNPF (-SQRSPSLRLRFamide; Fig 5). High variations in the N-terminal sequence of sNPF peptides were found between Chalcidoidea species and other Hymenoptera insects.
Eclosion hormone (EH) and ecdysis triggering hormone (ETH) are two of the major components of the peptidergic circuit controlling ecdysis in insects [48]. ETH peptide is highly conserved among Hymenoptera species (Panel ix in S2 Fig), whereas EH peptides remarkably differed among Hymenoptera species, especially between Ichneumonoidea and Chalcidoidea (Panel xv in S2 Fig). EH is a long peptide hormone with 6 cysteine residues forming three disulphide bridges in most insects. However, only 4 cysteine residues were found in the EHs of Chalcidoidea species. A low level of identity was found for putative EH sequences between Nasonia vitripennis and Fopius arisanus (Panel xv in S2 Fig), with an identity score of 45%, which was calculated using GeneDoc.
The phylogenetic and alignment analyses of the above neuropeptides (elevenin, kinin, natalisin, NPLP1, trissin, sNPF, and EH), suggested that some of these neuropeptides not found in Chalcidoidea or having considerably diverged between those in Chalcidoidea and other Hymenoptera species, might be related to different evolutionary or physiological patterns in Chalcidoidea species. However, further studies are warranted to explore the relationships of sequence patterns and functional roles of these neuropeptides in parasitoid wasps.

Conclusions
In the present study, publicly accessible databases and a well-established workflow were used for the prediction of neuropeptide sequences. In all, 517 precursors from 24 parasitoid wasp species were identified. Among them, five neuropeptides, i.e., CNMa, FMRFa, ITGlike, ITPL and orcokinin B, were identified for the first time in parasitoid wasps, to our knowledge.
Comparisons of neuropeptides among parasitoid wasps and other insect species revealed some interesting patterns regarding the evolutionary or physiological perspectives and might be useful for investigating the phylogenetic and divergence relationships among the Hymenoptera and other insect groups. Phylogenetic analysis of C-type ASTs suggested the divergence of AST-CCC within Hymenoptera. Further, the encoding patterns of CAPA/PK family genes were different between Hymenoptera species and other insect species. Some neuropeptides that were not found or were considerably divergent in some superfamilies of parasitoid wasps might be related to distinct feeding habits or other physiological processes in some parasitoid groups. Sulfakinin was not found in any parasitoid wasp species except cleptoparasitic wasps. A few neuropeptides (e.g., Elevenin, kinin, NTL, NPLP1, and Trissin) were not found in any species of Chalcidoidea but were present in other parasitoid wasp species and other insect species. Several neuropeptides (e.g., sNPF and EH) sequences show considerable difference between Chalcidoidea and other Hymenoptera insects. However, further studies are warranted for determining whether these patterns are due to the distinct parasitoid life or limited transcriptome data.
Analysis of neuropeptidomes in parasitoid wasps can be useful for better understanding the phylogenetic evolution of Hymenoptera and for conducting in-depth analysis of the physiological roles of neuropeptide signaling systems in parasitoid wasps. Cynipoidea sequences are indicated with light blue rhombuses; Orussoidea with empty squares; Platygastroidea with empty rhombuses. Numbers above branches indicate phylogenies from amino acid sequences and only values above 50% are shown. The numbers of the paracopies carrying the motif are shown by the repeat numbers, and the numbers in parentheses means the numbers of the paracopies predicted from a partial precursor. Identities in alignments are highlighted in dark (100%) and in grey (80%~100%). (PPTX)