Identification of neuropeptides and neuropeptide receptor genes in Phauda flammans (Walker)

Neuropeptides and neuropeptide receptors are crucial regulators to insect physiological processes. The 21.0 Gb bases were obtained from Illumina sequencing of two libraries representing the female and male heads of Phauda flammans (Walker) (Lepidoptera: Phaudidae), which is a diurnal defoliator of ficus plants and usually outbreaks in the south and south-east Asia, to identify differentially expressed genes, neuropeptides and neuropeptide receptor whose tissue expressions were also evaluated. In total, 99,386 unigenes were obtained, in which 156 up-regulated and 61 down-regulated genes were detected. Fifteen neuropeptides (i.e., F1b, Ast, NP1, IMF, Y, BbA1, CAP2b, NPLP1, SIF, CCH2, NP28, NP3, PDP3, ARF2 and SNPF) and 66 neuropeptide receptor genes (e.g., A2-1, FRL2, A32-1, A32-2, FRL3, etc.) were identified and well-clustered with other lepidopteron. This is the first sequencing, identification neuropeptides and neuropeptide receptor genes from P. flammans which provides valuable information regarding the molecular basis of P. flammans.

Insect neuropeptides as a classic signaling molecule are produced by the neurosecretory cells that are mainly located in the brain and the central nervous system, among others, to reach their distant target organs 1 . They are small proteins with generally about 5-80 amino acid residues, which are one of the structurally most diverse signaling molecules and most diverse group of signaling molecules in multicellular organisms 2,3 . Most neuropeptide receptors belong to the family of G protein-coupled receptor (GPCR), and most of the neuropeptides act via G protein coupled receptors 4,5 . It has been widely reported that neuropeptide and their receptors participate in intercellular information transfer from neurotransmission to intrinsic or extrinsic neuromodulation and essential signaling molecules that regulate physiological processes such as growth, development, behavior, reproduction, metabolism and muscle movement in insects [2][3][4]6,7 .
For now, a plethora of neuropeptides and receptors were investigated in insects, such as myoinhibiting peptides (MIPs) [8][9][10][11][12][13][14] , and so forth. Among these, PBAN, galanin and melanocortin are involved in the control of reproduction 10,15 . NPY is regulating feeding and sleep-wake behavior 16 . Thus, neuropeptides and their receptors could be developed as potential insecticides or targets for a novel generation of pesticides 17 , such as the neuropeptide CCH was proved to be regulates feeding motivation and sensory perception and olfactory behavior 18,19 and the enteroendocrine peptides allatotropin (AT) and GSRYamide have feeding acceleratory effects via controlling intestinal contraction 20 . Therefore, identification and functional characterization of neuropeptides and their receptors from insect pests would enhance our basic understanding of neuropeptide-related signal transduction, and provide important molecular insights for pest management. Up to now, neuropeptide and receptors have been the focus of interest in many species of Lepidoptera, such as Manduca sexta [21][22][23][24] , which are mainly nocturnal moths. While, few researches have been reported on diurnal moth of Lepidoptera except for silkworm and butterfly 25,26 .
The diurnal moth Phauda flammans (Walker) (Lepidoptera: Phaudidae) is a serious defoliator which intermittent outbreaks that threaten ficus plant seriously, especially Ficus microcarpa (Miq.) and F. benjamina L. 27 . It not only influences the urban landscapes and ecological effects, but also affects normal growth and development of ficus plant [28][29][30][31] . This defoliator is abundantly distributed in south and south-east Asia and southern China 32 . At present, most of the researches about P. flammans focus on its morphological characteristics [33][34][35][36][37][38][39][40] . However, the research on neuropeptides and their receptors in P. flammans has been limited in comparison to other lepidopteran insects, due to lack of availability of genomic or transcriptomic information.
In this study, we conducted high-throughput sequencing of head, identified members of the neuropeptide and neuropeptide receptor of P. flammans, and compared them with those reported transcriptome of other www.nature.com/scientificreports/ lepidopteran species for the first time. We also evaluated the expression level of 12 neuropeptides in different adult tissues. Our results could provide useful information of neuropeptide and their receptor and theoretical basis for their functional analysis.

Materials and methods
Insect rearing and tissue collection for RNA-seq. The  Transcriptome data analysis. The unigene expression was calculated and normalized to RPKM (Reads Per kb per Million reads) 41 and the relative expression of differential expressed genes were viewed by volcano plot. Unigene sequences were aligned by BLASTx and TBLASTx searches against the protein database (http:// blast. ncbi. nlm. nih. gov/) such as NCBI non-redundant protein (Nr) database, SwissProt database, KEGG Ontholog database (KO) and Gene Ontology (GO) for annotation information. The transcriptomic (RNA-seq) data derived from P. flammans were used for identification of the neuropeptides and receptors.
Sequence analysis and phylogenetic tree analysis. Transmembrane domains (TMDs) were calculated using the TMHMM 2.0 prediction software (http:// www. cbs. dtu. dk/ servi ces/ TMHMM/). The presence of signal peptide was predicted using SignalP software version 4.1 (http:// www. cbs. dtu. dk/ servi ces/ Signa lP/). The splice sites were predicted using the Known Motif and Insect Models methods of NeuroPred (http:// stagb eetle. animal. uiuc. edu/ cgi-bin/ neuro pred. py) and were corrected based on the processing procedures of known insect neuropeptide precursors. Thesequence alignments were done using CLUSTALW, the result were implemented in MEGAv7.034 and GeneDoc software. With tBLASTn, the available sequences proteins from lepidoptera species were used as queries to identify candidate unigene involved in neuropeptides and neuropeptide receptor genes in P. flammans. To construct an evolutionary tree of neuropeptides and receptors, the amino acid sequences of the Atrijuglans hetaohei, Bombyx mori, Chilo suppressalis, H. armigera, Grapholita molesta, Ostrinia furnacalis, Papilio machaon and Pl. xylostella were downloaded from the NCBI database and performed in MEGA7 and the tree was constructed using the Neighbor-Joining method with 1000 bootstraps.
Tissue expression profile via quantitative PCR. The head (without antennae), thoraxes (without legs), abdomens were dissected from 15 virgin 1-day-old of females or males, respectively. These tissues were immediately transferred into 1.5 mL RNA-free tube, super-cooled via liquid nitrogen, and then stored at − 80 °C freezer. These tissues were used for RNA extraction with RNAiso Plus (TAKARA, 9109, Dalian, China) and then cDNA synthesis with A Prime Script RT reagent Kit with gDNA Eraser (TAKARA, RR047, Dalian, China). The quantitative PCR reactions were conducted on an ABI QuantStudioTM 6 Flex system (Thermo Fisher Scientific, Massachusetts, USA). The PCR reaction was performed with each reaction was performed with Green Premix Ex Taq II Kit (TAKARA, RR820A, Dalian, China) and prepared as introduced 42 . The expression level of target gene was normalized with reference gene TUB1 (α-tubulin) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) via 2 -∆∆CT method according to our previous works 39,42 . The primers used in this research were listed in the Table S1.

Differentially expressed genes (DEGs) between female and male heads.
The results of differential expression analysis of genes in the heads of male and female adult P. flammans showed that a total of 217 differentially expressed genes were screened, with 156 genes up-regulated and 61 genes down-regulated, using FDR < 0.05 and |log2FC|> 1 as screening criteria (Fig. 2). The detailed information about these DGEs were listed in the supporting information 1.   The PfSNPF precursor had an N-terminal signal peptide of 19 amino acids and 3 mature SNPF were generated by sulfidation modifications. The PfSNPF precursor contained the -RLRF sequence, which belongs to the C-terminal motif unique to the SNPF family. Thereafter followed an amidation site (G) and a dibasic cleavage site (RR). The multiple alignments also showed that the SNPF of P. flammans had a higher similarity with other lepidopteron (Fig. 3).
The 152 reported neuropeptide receptor sequences of B. mori, C. suppressalis, H. armigera, G. molesta and Pl. xylostella from lepidopteran and the identified neuropeptide receptors of P. flammans were used to construct an interspecies phylogenetic tree (Fig. 5). The results showed that A26 of P. flammans was clustered together with the A26 of B. mori, C. suppressalis and Pl. xylostella in 100; B3-1 of P. flammans was clustered together with the B3 of B. mori and C. suppressalis; A6-b of P. flammans was clustered together with the A6 of H. armigera. A19-2-, A10-1, A32-2, CCH1R2, B3-2 and FRL2 were individually clustered together. A21-1, A21-2, A21-3 and A21-4 were individually clustered together, and it's the same with CCHIR-1, CC1R1, CPR2 and CPR3. It showed that neuropeptide receptor emerged highly differentiation in P. flammans. The remaining receptors were clustered together with the orthologs from other lepidopteran insects in the same clade.
Tissue expression profile in female and male adults. The expression profiles of 12 neuropeptides of P. flammans in heads, thoraxes, and abdomens of male and female adults were showed in Fig. 6. The expression of CA, LM, Ast, F1b, and NPLP1 were significantly higher in heads than other two body parts in both female and male. While the expression level of AR, DP3, and NP28 showed no significant difference in these three body parts in both sexes. All these neuropeptides showed no difference in female and male heads except for CCH2.

Discussion
Neuropeptides and receptors regulate a wide range of physiological processes in insects. Transcriptome sequencing is fundamental to dentification of genes, and identification of neuropeptides and their receptors is the first and foremost step of deep function depth studies in physiological processes. However, the types and expression of neuropeptides and their receptors in P. flammans are unavailable. Therefore, a sequencing analysis was performed of head in P. flammans. After high-throughput sequencing, among the 99,386 unigene acquired by the assembly program Trinity, 40.38% could be annotated through NR, KEGG, Swiss-Prot, KOG and GO databases, implies that not all unigene contain annotated genes. Some unigene may be non-coding which do not BLAST with the non-redundant protein/nucleotide database. Compared with the transcriptome data of head from Mythimna separata 43 and H. armigera 44 , the P. flammans had a similar result. Q20 and Q30 values were all > 93%, and GC content was similar, indicating that the data was accurate and reliable. In Nr databases, the number of homologous sequences most with P. flammans include E. japonica, B. mori, G. mellonella, which all  www.nature.com/scientificreports/ order of Lepidoptera, suggesting that the transcriptome was commendably sequenced and annotated. Overall, the assembly quality of transcriptome was adequate. Basically, the number of achieved target gene should be closely related to the sample resource and expression abundance in addition to sequencing depth with species specificity. The same was true for neuropeptides and neuropeptide receptors in P. flammans. Totally, 15 neuropeptides and 66 neuropeptide receptors were identified from head of adult P. flammans, which was different with other lepidopteran species [44][45][46][47] and should partly be relevant with their differences in sample physiological status. For example, in B. mori, 32 neuropeptide genes and 6 neuropeptide-like precursor genes were identified from larval and pupal brain 45 . In C. suppressalis, 43 neuropeptide precursors and 51 putative neuropeptide G protein-coupled receptors were identified the fifth instar larval central nervous system including brain, suboeophageal ganglion, thoracic ganglion, and the abdominal ganglion 46 . In H. armigera, 34 neuropeptides and peptide hormones, 17 neurotransmitter precursor processing enzymes, and 58 neurotransmitter receptors were identified from mixed pupa and adult head 44 . It seems that more sophisticated sampling would yield a larger number of neuropeptides and receptor genes. In addition, the number of identified genes might also have species specificity. The number of identified neuropeptides of P. flammans was less than the number of some other lepidopteran species such as from the transcriptome data of head, such as A. hetaohei 47 .
There are several factors that may account for the difference in the number of identified genes of specific functions which has been discussed 48,49 . Firstly, the head used as the sequenced samples did not cover complete the individual and all stages of life cycle. Secondly, some genes with small expression levels made it impossible to quantitatively measure the gene expressions in samples presented a not expression state, or them may not have been expressed at all. And then, due to does not involve the modification of corresponding protein-coding regions, many genes lack strong sequence conservation, their clear orthologs could not be found in P. flammans based on homology searches. The neuropeptides may truly present with small quantity in P. flammans because of highly species specificity which needs further investigation.
In this analysis, female and male head transcriptome in P. flammans was performed with focus on the feeding behavior regulation and sexual difference. Only a total of 217 differentially expressed genes were screened, with 156 genes up-regulated and 61 genes down-regulated. Approximately 12% of these DGEs were olfactory association related genes (Supporting information 1), while no neuropeptide or neuropeptide receptor were found. Moreover, some neuropeptide and neuropeptide receptors have reported to induce sex pheromone biosynthesis and feeding behaviors 50,51 . Therefore, the small number of neuropeptides and neuropeptide receptors from head in P. flammans might lead to those gene tightly to olfactory regulation and reduce workload in targeting behavior regulation gene. For instance, there were 19 unigenes which located in the Ko00981, the insect hormone biosynthesis pathway, where only unigene0063695 and unigene0024395 were significantly differential expressed and annotated as gene cytochrome P450 18a1 (CYP18A1) and farnesol dehydrogenase-like (FoLDH), respectively (Fig. S1). CYP18A1 played a controlling role in 20-hydroxyecdysone inactivation in B. mori 52 , and were reported to function in development, especially to regulate dimorphic metamorphosis via by insect hormones 53,54 . FoLDH could induce oxidation of farnesol to farnesal and produce the second branch of JH III in Pl. xylostella 55 . In www.nature.com/scientificreports/ addition, DGE Unigene0010507 was annotated as juvenile hormone binding-like protein (Supporting information 1) and how the relationship between it and insect hormone biosynthesis pathway attracted our attention. Therefore, the functions of these DGEs require further analysis and validation in P. flammans. Neuropeptides and neuropeptide receptors identified from the head of P. flammans showed no significant difference between male and female adults, however, they are crucial in regulating a range of physiological functions, including development, reproduction and feeding 56 . Therefore, identification and analysis neuropeptides and their receptors are still necessary and meaningful. In the aspects of feeding behavior, for example, short neuropeptide F peptide is expressed in the nervous system and it regulates food intake and body size by overexpression of SNPF with regulate expression of insulin-like peptides in Drosophila 57 . Another example, NPF as a pleiotropic factor, is well known for its role in the regulation of feeding 58 , through activating neuropeptide G protein-coupled receptor to regulate feeding and growth in B. mori 59,60 , which is also a daily oligophagous species www.nature.com/scientificreports/ that might provide some references for P. flammans. In the aspects of sexual difference, the release of SIFamide in the brain could inhibit sexual behavior until the flies encounter the right physiological conditions 61 , which might also function in sexual differences. All these deductions need further confirmation far and away via quantitative PCR, tissue localization, function inhibition and so on. Neuropeptides were less abundant in this study and easier to target their expression in different tissues. From a general view, all the measured neuropeptides were expressed highly or moderately in heads where they were identified from transcriptome (Fig. 6). As mentioned above, the neuropeptide CCH2 and the neuropeptide receptor CCH1R-1 could be identified, but them were no significantly different expressed in the head of females and males (Supporting information 1), while quantitative PCR results showed a slightly significant difference in CHH2 (Fig. 6C). Similar results were also found in CCHamide 1 and CCHamide 2 which were significantly different expressed in the head of females and males of A. hetaohei 47 . Fold changes in CHH2 expression in female and male heads by QPCR was minor, and therefore the conflicting point shall result from the sensitiveness of QPCR and RNA-Seq methods. In addition, SIFamide a highly conserved neuropeptide and has been reported to The drawbacks of the adopted second generation sequencing were undoubted. However, we did obtain a mass of valuable genetic data for P. flammans with a tight fund, especially in neuropeptides and their receptors. Novel neuropeptides could be supplemented via Genomics-and peptidomics -based discovery in the future 63 . Moreover, association of multiple omics, such as full-length transcriptome, proteome and metabolome might be needed 64 , which would contribute to the feeding and sexual behavior regulation researches in this diurnal moth P. flammans by outlining a chain with cascaded neuropeptide, neuropeptide receptor, pheromone metabolism and behavior.

Conclusion
In this study, 15 neuropeptides and 66 neuropeptide receptors were identified from P. flammans, and the genes exhibited no significantly different expression in head between female and male. Phylogenetic analyses tree with neuropeptides and receptors of other lepidopteran species illustrated clear interspecies relationships and contributed to further function understanding. Our findings enriched neuropeptides and neuropeptide receptor gene database, which provide a theoretical support for pest management strategies and physiological and biochemical researches in P. flammans. Figure 6. Tissue expression of neuropeptides in both sexes of P. flammans. Data are expressed as mean ± standard error (SE). Values followed by different letters are significant (P < 0.05) analyzed by Tukey's honestly significant difference (HSD) multiple test.