Genome-Wide Identification of Neuropeptides and Their Receptors in an Aphid Endoparasitoid Wasp, Aphidius gifuensi

Simple Summary Neuropeptides and their receptors play important roles in insect physiology and behavior. Due to their specificity and importance, they have been considered as the targets for new environmentally friendly insecticides. Hence, more information on genomics and transcriptomics of neuropeptide and their receptors in more insects, especially in beneficial insects, are needed to avoid the side effects of these environmentally friendly insecticides. Aphidius gifuensis is one of the most well-known aphid parasitoids and has been successfully used to control aphids such as Myzus persicae and Sitobion avenae. In this study, we systematically identified neuropeptides and their receptors from the genome and head transcriptome of A. gifuensis. Meanwhile, we also analyzed their expression patterns in response to imidacloprid exposure and in different tissues. Our results not only laid a preliminary foundation for functional studies on neuropeptide precursors and their receptors but also provided useful information for future pesticide development. Abstract In insects, neuropeptides and their receptors not only play a critical role in insect physiology and behavior but also are the potential targets for novel pesticide discoveries. Aphidius gifuensis is one of the most important and widespread aphid parasitoids, and has been successfully used to control aphid. In the present work, we systematically identified neuropeptides and their receptors from the genome and head transcriptome of A. gifuensis. A total of 35 neuropeptide precursors and 49 corresponding receptors were identified. The phylogenetic analyses demonstrated that 35 of these receptors belong to family-A, four belong to family-B, two belong to leucine-rich repeat-containing GPCRs, four belong to receptor guanylyl cyclases, and four belong to receptor tyrosine kinases. Oral ingestion of imidacloprid significantly up-regulated five neuropeptide precursors and four receptors whereas three neuropeptide precursors and eight receptors were significantly down-regulated, which indicated that these neuropeptides and their receptors are potential targets of some commercial insecticides. The RT-qPCR results showed that dopamine receptor 1, dopamine receptor 2, octopamine receptor, allatostatin-A receptor, neuropeptides capa receptor, SIFamide receptor, FMRFamide receptor, tyramine receptor and short neuropeptide F predominantly were expressed in the head whilst the expression of ion transport peptide showed widespread distribution in various tissues. The high expression levels of these genes suggest their important roles in the central nervous system. Taken together, our study provides fundamental information that may further our understanding of neuropeptidergic signaling systems in the regulation of the physiology and behavior of solitary wasps. Furthermore, this information could also aid in the design and discovery of specific and environment-friendly insecticides.


Introduction
Neuropeptides are 3 to 100 amino acid polypeptides and have been identified as involved in various physiological processes in insects, such as behavior regulation, reproduction, and circadian rhythm [1][2][3][4][5]. For example, in Drosophila melanogaster, neuropeptide F (NPF) regulates their feeding behaviors thereby influences their reproductive capacity [2]. In Schistocerca gregaria, NPF plays a critical role in reproduction and ecdysteroidogenesis [6]. Silencing insulin-like peptide 2 leads to smaller male mandible size in Gnatocerus cornutus [7]. A neuropeptide is encoded by a larger precursor gene (called pre-propeptide) that produces one or more mature peptides by a series of cleavages and modifications [1,8]. Mature peptides exert their biological functions by binding to their corresponding receptors in the extracellular environment [1,9].
Neuropeptide corresponding receptors belong to G protein-coupled receptors (GPCRs), receptor guanylyl cyclases (RGCs), and receptor tyrosine kinases (RTKs) [9][10][11]. Neuropeptide corresponding GPCRs belong to the A-family (or rhodopsin-like), the B-family (or secretin-like), or leucine-rich repeat-containing GPCRs (LGRs) [11]. Insect family A GPCRs include opsins, biogenic amine receptors and neuropeptide and protein hormone receptors [12]. Generally, the B family GPCRs have a long N-terminal domain and contain four types of receptor: corticotrophin-releasing-factor-related diuretic hormone receptor, calcitonin-like diuretic hormone receptor, pigment-dispersing factor receptor and parathyroid hormone receptor-like [13]. According to the numbers of leucine-rich repeat motifs, receptors in LGRs have three types: type A, B and C [14]. Receptors of prothoracicotropic hormone (PTTH), insulin-like peptides and neuroparsin are not GPCRs, but belong to RTKs [15,16]. In Tribolium castaneum, knockdown of dopamine-2 receptor results in over 90% mortality during larval-pupal metamorphosis [17]. Interestingly, injected dopamine and its agonists in Locusta migratoria cause gregarious behavior in the migratory locust [18]. Injection of dsDop1 (dsRNA of dopamine-1 receptor) and Dop1 antagonist impair the olfactory preference of gregarious locusts to their odors [19]. Notably, the injection of Dop1 agonist in solitarious locusts initiates preference to gregarious odors [19]. In Helicoverpa armigera, dopamine receptor is also serviced as a membrane receptor for 20-hydroxyecdysone [20]. Knockdown of CCHamide2 receptors in A. pisum influenced its feeding behaviors and thus reproductions [21]. Silencing PTTH receptor torso in the prothoracic gland prolongs the third-instar larval stage of D. melanogaster [16]. Topical application of 1895 and CAPA-PVK analogue 2315 (ASG-[β 3 L]-VAFPRVamide) cause 72% mortality in Myzus persicae, while having no influence on beneficial insects including Bombus terrestris, Chrysoperla carnea, Nasonia vitripennis and Adalia bipunctata [22]. Due to the critical role of neuropeptides and their receptors in insect survival, they have been considered as attractive targets for developing new environmentally friendly pesticides to control the economically important pests [5,[23][24][25]. Meanwhile, the conservation of these pesticides target genes between target pests and beneficial insects should also be considered to avoid adverse side effects on these beneficial insects [1,24]. Thus, genetic information about neuropeptides and their corresponding receptors in beneficial insects are needed [26][27][28].
Aphidius gifuensis has been selected as a potential biological agent of the green peach aphid Myzus persicae Sulzer, one of the main pests of various crops in China and Japan, and it has been successfully used to control M. persicae on tobacco in Yunnan and many other regions of China [29][30][31][32][33]. A. gifuensis is non-host feeding, and a female parasitizes about 60 aphids per day [34]. A. gifuensis female is able to discriminate the plant volatiles that released from healthy, mechanically damaged or aphid infested plant [35]. Furthermore, when provided with various aphid species, the A. gifuensis female can adjust its parasitic strategy by laying fewer eggs in maternal aphid species and more eggs in other aphid species [30,36]. Interestingly, both virgin and mated A. gifuensis females show high prefer-ence of aphid alarm pheromone, E-(β)-farnesene [37]. All of these results suggested that A. gifuensis has evolved a comprehensive chemosensory and nervous system to help it find a suitable host and conduct the right parasitic strategy. The identification and expression of chemosensory genes have been well documented at genomic and transcriptomic levels, whereas neuropeptides and their corresponding receptors were ignored [37][38][39]. In our recent study, we found that exposure of imidacloprid (IMD) significantly disrupted the olfactory and parasitism behavior by influencing the expression of related genes such as detoxification genes, chemosensory genes, and acetylcholine receptors [40]. As for neuropeptide and their receptors, we only analyzed the expression of DopR1, OAR, TAR and NFR [40]. In D. melanogaster, neonicotinoids disturbed learning memory, circadian behavior and sleep by preventing the accumulation of pigment dispersing factor neuropeptides [41]. Thus, a comprehensive analysis of the influence of IMD on the expression of neuropeptides and their receptors in A. gifuensis is needed. In this study, we predicted the neuropeptide precursors and their receptors using the publicly accessible genome data and our newly sequenced head transcriptome data [38]. Furthermore, we also analyzed the expression of these genes in different tissues and their responses after the exposure of IMD based on our previous study [40]. We thought that the identification and expression of neuropeptides and their receptors provides fundamental information that may further our understanding of neuropeptidergic signaling systems in the regulation of physiology and behavior of solitary wasps. Furthermore, this information could also aid in design and discovery of specific and environment friendly insecticides.

RNA Sequencing and Gene Identification
The heads of A. gifuensis were cut from newly emerged adult male and female wasps (1-2 days old), and then frozen in liquid nitrogen. Total RNA of the heads was extracted from 40 heads using RNAiso reagent (Takara Bio, Dalian, China) following manufacturer's instructions. The RNA integrity and quality were verified by 1% agarose gel electrophoresis and Nanodrop ND-2000 spectrophotometer. A high-quality RNA sample was sent to Novogene Bioinformatics Technology Co., Ltd. for cDNA and Illumina library generation. Transcriptome de novo assembly was carried out as our previously published paper [39,40].
The identification of neuropeptide precursors and their receptors in A. gifuensis were predicted by local BLAST searching in the genome and transcriptome of A. gifuensis with the amino acid sequences of neuropeptide precursors and their receptors from Pteromalus puparum, Habrobracon hebetor, D. melanogaster and Nasonia vitripennis [26][27][28]38,42].

Expression Analysis
Heat maps of neuropeptide precursors and their receptors were performed by pheatmap in R 4.0.4 (https://www.r-project.org/, accessed date (15 February 2021)). Tissue-specific expressions of several target genes were determined by RT-qPCR. Total RNA of heads and bodies (without heads) were extracted using RNAiso reagent (Takara Bio, Dalian) following manufacturer's protocol. To remove the genomic DNA, the total RNA was treated by Dnase I (42 • C 2 min). These total RNAs were subsequently used to produce cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time, Takara Bio, Tokyo, Japan). Specific primers were designed by Primer Premier 5 (PREMIER Biosoft International, Palo Alto, CA, USA), and are provided in Table S2. qPCR was performed in iQ5 (Bio-rad, Hercules, CA, USA) in 20 µL reactions. The qPCR reaction condition and normalization of these genes were conducted as in our previously published paper [39,46]. Relative expressions of these genes were normalized by 18S RNA, and the normalization of each gene was compared with the expression level in female heads. Each sample had three technical replicates and three biological replicates (50 heads or 20 bodies per replicate). Differences among these samples were subjected to one-way analysis of variance (ANOVA) with Bonferroni's test at p < 0.05.

Transcriptome Sequencing, Assembly, and Annotation
We sequenced the head of A. gifuensis and obtained 48,430,060 clean reads (Table S1). GC content was 28.32% (Table S1). N50 of the head transcriptome was from 546 (transcripts) and 370 (unigenes) ( Table S1). Mean length was from 351 (transcripts) and 293 (unigenes) (Table S1). Gene ontology (GO) analysis showed that genes involved in cellular process, metabolic process, single-organism process, binding and catalytic activity were predominantly expressed in head ( Figure S1). Notably, signal transduction, translation and endocrine system were the major enrichment pathways ( Figure S2).

Identification of Neuropeptide Precursors
Based on the A. gifuensis genome and transcriptome data, we predicted and annotated 35 neuropeptide precursors from A. gifuensis genome and 27 neuropeptide precursors from A. gifuensis transcriptome ( Figure 1; Table 1 and Table S2). Adipokinetic hormone, allatostatin B, allatostatin C, RYamide, sulfakinin and tachykinin were missing in A. gifuensis (Table 1). Allatostatin A, ecdysis triggering hormone, insulin-like peptide (ILP), leucokinin, natalisin, proctolin, SIFamide and trissin were only observed in the genome of A. gifuensis, while they were not found in the head transcriptome ( Figure 1a). For the allatostatin neuropeptide family, allatostatin CCC was most conserved whereas allatostatin A was the most variable of the three allatostatin neuropeptides ( Figure 2).

Neuropeptide Receptors in A. gifuensis
Based on the genomics and transcriptomics analyses, we also predicted 49 neuropeptide receptor genes (Table S1), including 35 A-families GPCRs, four B-families GPCRs, two LGRs, four RGCs, and four RTKs. Receptors for PDF, sNPF, CAPA and CCAP were only

Neuropeptide Receptors in A. gifuensis
Based on the genomics and transcriptomics analyses, we also predicted 49 neuropeptide receptor genes (Table S1), including 35 A-families GPCRs, four B-families GPCRs, two LGRs, four RGCs, and four RTKs. Receptors for PDF, sNPF, CAPA and CCAP were only found in the genome of A. gifuensis (Figure 1b).

Expression Profiles of Neuropeptides and Their Receptors
Based on previously published and our newly sequenced transcriptome data, we analyzed the expression profiles of neuropeptides and their receptors in response to IMD exposure (Figure 8

Expression Profiles of Neuropeptides and Their Receptors
Based on previously published and our newly sequenced transcriptome data, we analyzed the expression profiles of neuropeptides and their receptors in response to IMD exposure ( Figure 8 Furthermore, we also analyzed their expressions in different tissues by RT-qPCR. We found that sNPF was highly expressed in the head, while the expression of sNPF in the female head was significantly higher than that in the male head ( Figure 9). The expression trends of DopR1, DopR2, AstAR, FMRFR (FMRFamid receptor), TAR, CAPAR and OAR were similar with sNPF ( Figure 9). SIFaR (SIFamide receptor) was predominantly expressed in the head, and there was no significant difference between female and male ( Figure 9). There was no tissue-specific expression of ITP (ion transport peptide; Figure  9). Furthermore, we also analyzed their expressions in different tissues by RT-qPCR. We found that sNPF was highly expressed in the head, while the expression of sNPF in the female head was significantly higher than that in the male head ( Figure 9). The expression trends of DopR1, DopR2, AstAR, FMRFR (FMRFamid receptor), TAR, CAPAR and OAR were similar with sNPF ( Figure 9). SIFaR (SIFamide receptor) was predominantly expressed in the head, and there was no significant difference between female and male ( Figure 9). There was no tissue-specific expression of ITP (ion transport peptide; Figure 9).

Discussion
In the present study, we systematically identified neuropeptide precursors and their receptors of A. gifuensis from the previously released genome and our sequenced head transcriptome databases. The numbers of our identified neuropeptide precursors and their receptors in A. gifuensis genome are similar to those found in other parasitic wasps such as P. puparum, H. hebetor and N. vitripennis [26][27][28]. However, the numbers are less than those in D. melanogaster, Bombyx mori, N. lugens, A. aegypti, L. migratoria, Rhynchophorus ferrugineus, Aphis mellifera and T. castaneum, which might be due to the parasitic life of parasitic wasps [8,42,[47][48][49][50][51]. Notably, there were three very conservative neuropeptide precursors (adipokinetic hormone, RYamide and tachykinin) not found in the A. gifuensis genome and transcriptome. RYamide was firstly found in N. vitripennis with the C-terminal consensus sequence FFXGSRY-amide [28]. In D. melanogaster and B. mori, RYamide or its receptor was found to be expressed in the brain, terminal abdominal ganglion and gut (enteroendocrine cells: EEs) [52,53]. Injection of RYamide significantly depressed the proboscis extension reflex of Phormia regina to sucrose [54]. Tachykinin is a multifunctional neuropeptide with a characteristic conserved C-terminal sequence FXGXR-amide [55,56]. In Bactrocera dorsalis, tachykinin modulates the olfactory sensitivity to ethyl acetate (avoid aversive odor) [57]. Knockdown of tachykinin significantly decreased the electrophysiological responses to ethyl acetate and suppressed the avoid behavior for ethyl acetate [57]. Furthermore, tachykinin is one of the most abundant secreted peptides expressed in midgut EEs [55]. In D. melanogaster and Plutella xylostella, tachykinin has been found to play a critical role in lipid metabolism [55,58]. Therefore, we speculate that the absence of some neuropeptide precursors might be due to a method as some specific neuropeptide genes that might not be identified by homolog searching. In the future, peptidomics is an alternative method to be used for identifying the neuropeptide genes and their products. Furthermore, it also can be used for confirming the predicted neuropeptide genes from genome and transcriptome.
Generally, expressions of genes in different tissues reflect their functions. In this study, sNPF, DopR1, DopR2, AstAR, FMRFR, TAR, CAPAR and OAR predominately expressed the head, while ITP was widely expressed among these samples. Consistent with

Discussion
In the present study, we systematically identified neuropeptide precursors and their receptors of A. gifuensis from the previously released genome and our sequenced head transcriptome databases. The numbers of our identified neuropeptide precursors and their receptors in A. gifuensis genome are similar to those found in other parasitic wasps such as P. puparum, H. hebetor and N. vitripennis [26][27][28]. However, the numbers are less than those in D. melanogaster, Bombyx mori, N. lugens, A. aegypti, L. migratoria, Rhynchophorus ferrugineus, Aphis mellifera and T. castaneum, which might be due to the parasitic life of parasitic wasps [8,42,[47][48][49][50][51]. Notably, there were three very conservative neuropeptide precursors (adipokinetic hormone, RYamide and tachykinin) not found in the A. gifuensis genome and transcriptome. RYamide was firstly found in N. vitripennis with the C-terminal consensus sequence FFXGSRY-amide [28]. In D. melanogaster and B. mori, RYamide or its receptor was found to be expressed in the brain, terminal abdominal ganglion and gut (enteroendocrine cells: EEs) [52,53]. Injection of RYamide significantly depressed the proboscis extension reflex of Phormia regina to sucrose [54]. Tachykinin is a multifunctional neuropeptide with a characteristic conserved C-terminal sequence FXGXR-amide [55,56]. In Bactrocera dorsalis, tachykinin modulates the olfactory sensitivity to ethyl acetate (avoid aversive odor) [57]. Knockdown of tachykinin significantly decreased the electrophysiological responses to ethyl acetate and suppressed the avoid behavior for ethyl acetate [57]. Furthermore, tachykinin is one of the most abundant secreted peptides expressed in midgut EEs [55]. In D. melanogaster and Plutella xylostella, tachykinin has been found to play a critical role in lipid metabolism [55,58]. Therefore, we speculate that the absence of some neuropeptide precursors might be due to a method as some specific neuropeptide genes that might not be identified by homolog searching. In the future, peptidomics is an alternative method to be used for identifying the neuropeptide genes and their products. Furthermore, it also can be used for confirming the predicted neuropeptide genes from genome and transcriptome.
Generally, expressions of genes in different tissues reflect their functions. In this study, sNPF, DopR1, DopR2, AstAR, FMRFR, TAR, CAPAR and OAR predominately expressed the head, while ITP was widely expressed among these samples. Consistent with our results, three dopamine receptors in Chilo suppressalis were also found to be significantly highly expressed in the central nervous tissues [59]. In Agrotis ipsilon, brains and antennal lobes predominately expressed DopR1 that is involved in sex pheromone responsiveness [60]. Similarly, TAR1 is highly expressed in the antennae, and silencing TAR1 reduced the sensitivity of Halyomorpha halys to its alarm pheromone (E)-2-decenal [61]. In L. migratoria, dopamine and octopamine signaling mediated the olfactory behavior and ultimately influenced the phase change [18,43,62]. Silencing DopR1/2 and OARα1 disrupted the attractive and repulsive behavior of L. migratoria to their odors [18,19,62,63]. For FMRFR, RNAi silencing of FMRFR decreased the locomotor and flight activities in D. melanogaster [64,65]. In previous studies, A. gifuensis showed higher parasitoid preference and suitability on its maternal host aphids than other aphid species [30]. Natal rearing experience affects the host discrimination and oviposition decision of A. gifuensis [36]. Due to the importance of neuropeptides and their receptors in the modulation of insect behavior, all of these results indicated that neuropeptides and their receptors in A. gifuensis may play a critical role in the odor perception and successful parasitism.
Based on previously published work, we analyzed the expression of neuropeptide precursors and their receptors in response to IMD [40]. The expressions of AstCC, DH44, Ele, NVP and SIFamide were up-regulated by IMD whereas FMRFamide, Natalisin and PDF were down-regulated. In D. melanogaster, PDF mutants showed a decreased male sex pheromones and increased frequency of remating behaviors [66]. Exposure of IMD disrupted the learning, behavioral rhythmicity and sleep of D. melanogaster [41]. Further investigation revealed that IMD prevented the accumulation of PDF in the dorsal terminals of clock neurons [41]. SIFamides are highly conserved neuropeptide precursors and have been identified to be involved in the regulation of sexual behavior, feeding and sleep [1,67,68]. Knockdown SIFamide in Rhodnius prolixus caused smaller blood meal consumption, while the injection of Rhopr-SIFa resulted in a larger blood meal [67]. Surprisingly, mutations and silencing (RNAi) of SIFamide in D. melanogaster led to male-male courtship behavior whilst the activation of SIFamidergic neuron enhances appetitive behavior [68]. Our previous work reported that some of the IMD-exposed females A. gifuensis bent their abdomens forward as if they were mating or attacking aphids, while no male A. gifuensis and aphids were present [40]. All of these results indicated that this abnormal behavior might be caused by the impact of IMD on these neuropeptide precursors that eventually leads to nervous disorder.
In the pharmaceutical industry, GPCRs are one of the therapeutic targets for the development of new drugs [23,24]. About 40-50% of all medications exert their effects via this family of proteins such as losartan, olanzapine and cinacalcet [9,23]. Due to the important roles of GPCRs in modulating biology, physiology and behavior in insects, GPCRs are also considered as targets for next generation insecticides. In contrast to the pharmaceutical industry (knowledge-driven drug design), research on insect GPCRs especially for new pesticide discovery is lacking [18,24]. Amitraz is the only pesticide that targets the octopamine receptor and tyramine receptor [69,70]. Recently, a research group provided a novel approach "proof-of-concept" for insecticide discovery using genome sequence data for functional characterization and chemical molecules screening of GPCRs [71]. They successfully found two lethal molecules (amitriptyline: 93% mortality and doxepin: 72% mortality) for A. aegypti [71]. In this work, we found that exposure of IMD significantly influenced the expression of GPCRs in A. gifuensis including dopamine receptor and FMR-Famide receptor, which means random ligand screening for novel insecticide discovery might impair beneficial insects including natural enemies of insect pests and pollinators. Thus, genetic information of these beneficial insects will contribute to our current and future efforts in rational insecticide discovery and design.
Taken together, in this study, we systematically identified the neuropeptide precursors and their receptors in A. gifuensis. Meanwhile, we also analyzed their expression patterns in response to IMD exposure and different tissues. Our results not only laid a preliminary foundation for the functional studies on neuropeptide precursors and their receptors but also provided useful information for future pesticide development.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/insects12080745/s1, Figure S1: Functional annotation of A. gifuensis transcripts based on gene ontology (GO) categorization, Figure S2: Top 20 enriched Kyoto Encyclopedia of Genes and Genomics (KEGG) pathways of A. gifuensis, Table S1: The amino acid sequences of neuropeptide receptors, Table S2: Primers used in this study, Table S3: Summary of head transcriptome, Table S4: The amino acid sequences of neuropeptide precursors. Predicted signal peptides (highlighted in yellow), cleavage signals (red), putative bioactive mature peptides (light blue), amidation signals (pink), and cysteine residues (deep yellow) are indicated.