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Article

Transcriptome-Wide Identification of Neuropeptides and Neuropeptide Receptors in the Twenty-Eight-Spotted Ladybird Henosepilachna vigintioctopunctata

by
Quanxing Lei
1,
Ziming Wang
1,
Shuangyan Yao
1,
Aili Lin
2,
Yunhui Zhang
1,
Chengxian Sun
1,
Xiaoguang Liu
1,
Mengfang Du
1,
Xiaoming Liu
1,* and
Shiheng An
1
1
Henan International Laboratory for Green Pest Control, College of Plant Protection, Henan Agricultural University, Zhengzhou 450046, China
2
Henan International Joint Laboratory of Taxonomy and Systematic Evolution of Insecta, Henan Institute of Science and Technology, Xinxiang 453003, China
*
Author to whom correspondence should be addressed.
Insects 2025, 16(6), 624; https://doi.org/10.3390/insects16060624
Submission received: 21 April 2025 / Revised: 26 May 2025 / Accepted: 9 June 2025 / Published: 13 June 2025
(This article belongs to the Section Insect Molecular Biology and Genomics)
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Simple Summary

The 28-spotted ladybird beetle (Henosepilachna vigintioctopunctata) is a major pest that damages crops in the potato/tomato family. Neuropeptides and their receptors might help control this pest. By analyzing the beetle’s central nervous system, we identified 58 neuropeptide genes and 31 receptor genes linked to biological processes. The neuropeptide genes are active in the insect’s brain, ventral nerve cord, and gut. This research provides a foundation for developing eco-friendly pest control methods targeting these neuropeptide systems, which could reduce crop damage.

Abstract

The ladybird beetle, Henosepilachna vigintioctopunctata, is an oligophagous pest with significant economic impact. This pest causes considerable economic damage on numerous Solanaceae crops. Neuropeptides, along with their designated receptors, play a pivotal role in regulating diverse biological processes in insects, presenting a promising avenue for innovative pest management strategies. Herein, the transcriptome of the central nervous system (CNS) of H. vigintioctopunctata was sequenced. Overall, our analysis identified 58 neuropeptide precursor genes, from which 98 diverse mature peptides were predicted. Furthermore, 31 neuropeptide receptor genes belonging to three distinct classes were discovered, along with predictions for their potential ligands. Moreover, the expression patterns of these 58 neuropeptide genes across larval brain tissue, ventral nerve cord, and gut were evaluated using quantitative real-time PCR. Collectively, these findings will significantly contribute to future research focused on understanding the physiological functions and pharmacological characteristics of neuropeptides and their receptors in H. vigintioctopunctata. Ultimately, these insights may facilitate the development of targeted neuropeptide-based solutions for managing this pest affecting solanaceous plants.

Graphical Abstract

1. Introduction

Neuropeptides are classic signaling molecules produced and released by most major types of neurons that are mainly located in the central nervous system, including the brain and ventral nerve cord (VNC) [1]. They are small proteins with generally several to tens of amino acid residues, are one of the structurally most diverse signaling molecules, and are the most diverse group of signaling molecules in multicellular organisms [2,3]. Neuropeptides act via their respective neuropeptide receptors, most of which belong to G protein-coupled receptors [4,5]. Some neuropeptide receptors are not GPCRs, such as the insulin receptor family, which includes three type-II receptor tyrosine kinases (RTKs) that each have one transmembrane domain [6]. It has been widely reported that neuropeptides and their receptors activate the essential signaling pathways that regulate physiological processes such as growth, development, behavior, reproduction, metabolism, and many other physiological processes in insects [1,2].
For example, pheromone biosynthesis-activating neuropeptide (PBAN) activates the synthesis of sex pheromone in Lepidoptera [7,8]. Neuropeptide F (NPF), like neuropeptide Y (NPY) in vertebrates, regulates feeding and metabolism [9]. Insulin-like peptides are known to regulate growth and development, reproduction, stress resistance, and lifespan [10]. The neuropeptide CCHamide (CCH) regulates feeding intake, sensory perception, and olfactory behavior [11,12]. Neuropeptide Bursicon regulates cuticle metabolism of insects and the transition from summer-form to winter-form of Cacopsylla chinensis [13,14]. Thus, neuropeptides and their receptors could be developed as potential insecticides or targets for a novel generation of pesticides. 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, neuropeptides and their receptors have been studied in some species of pests, such as Nilaparvata lugens [15], Aphis craccivora [16], Phauda flammans [17], Grapholita molesta [18], and Eurygaster integriceps [19]. Although a few studies on neuropeptides and their receptors have been reported in Coleoptera, such as Tribolium castaneum [20], Tenebrio molitor Zophobas atratus [21], and Coccinella septempunctata [22], no such information is available for Henosepilachna vigintioctopunctata.
In this study, we conducted high-throughput sequencing of the central nervous system (CNS), identified members of the neuropeptides and neuropeptide receptors of H. vigintioctopunctata, and compared them with those reported neuropeptides and neuropeptide receptors of other species. We also evaluated the expression level of 58 neuropeptides in different larval tissues. Our results could provide useful information on neuropeptides and their receptors and a theoretical basis for their functional analysis.

2. Materials and Methods

2.1. Insect Rearing and Sample Collection

A H. vigintioctopunctata colony was reared in the laboratory (Lab of Insect Physiology and Biochemistry in Henan Agricultural University, Zhengzhou, China) for six generations on the leaves of Solanum nigrum. They were kept at 26 ± 1 °C, 70 ± 10% relative humidity (RH), under a 14:10 h light: dark photoperiodic regime.
For transcriptome analysis, whole CNS (n = 150 pooled specimens) (Figure 1A) were micro-dissected from second- and third-instar larvae in cold 10 mM phosphate-buffered saline (PBS, pH 7.4). For spatial expression analysis, samples of the brain (Br, n = 150), ventral nerve cord (VNC, n = 150), gut (whole gut including foregut, midgut and hindgut, n = 20), and Malpighian tubule (Mt, n = 30) were dissected from 2-day-old second-instar larvae. All the tissues were immediately pooled in RNase-free tubes and used to extract total RNA.

2.2. RNA Extraction and Transcriptome Sequencing

Total RNA was isolated from CNS samples using RNAiso Plus reagent (Takara Bio, Kusatsu, Japan). RNA quality assessment, cDNA library preparation, and transcriptome sequencing were performed by Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) on the Illumina HiSeq platform, generating 150 bp paired-end reads. Raw sequencing data were subjected to quality filtering to obtain clean reads using fastp (default parameters), which were subsequently de novo assembled into unigenes using Trinity v2.8.5 (k-mer = 31, min contig length = 200 bp) [23]. Assembly completeness was evaluated with BUSCO v3.0.2 [24] against the insecta_odb9 dataset [24]. Functional annotation was conducted through six databases: Pfam (HMMER3 v3.1b2, default parameters); KEGG (KOBAS v2.1.1, default parameters); EggNOG (DIAMOND v0.9.24, E-value ≤ 1 × 105); Nr (DIAMOND v0.9.24, E-value ≤ 1 × 105); GO (BLAST2GO v2.5.0, default parameters); Swiss-Prot (DIAMOND v0.9.24, E-value ≤ 1 × 105).

2.3. Exploration of the Neuropeptides and Their Putative G Protein-Coupled Receptors

Based on known sequences from Tribolium castaneum [20,25], Harmonia axyridis [26], Coccinella septempunctata [22,26], Drosophila melanogaster [27], and Bombyx mori [28,29,30], the neuropeptide precursor and GPCRs genes of H. vigintioctopunctata were identified by tBLASTn analysis (E-value cutoff ≤ 1 × 10⁻⁵ and length filtering: precursor sequences ≥ 30 amino acids) [31].
The neuropeptide precursors of H. vigintioctopunctata were classified into distinct families using NeuroPep (version 1.0) [32]. For neuropeptide precursor characterization, N-terminal signal peptides were predicted with the SignalP 5.0 [33], and putative cleavage sites (RR/RK/KK/KR/R/K) were identified based on criteria established in prior research [34] or analyzed through NeuroPred [35]. Multiple sequence alignments were conducted using Clustal Omega (version 1.2.2) [36]. For GPCR analysis, transmembrane domains (TMDs) were predicted via the TMHMM 2.0 [37], and conserved domains were annotated by querying the Conserved Domain Database [38].

2.4. Phylogenetic Analysis

Phylogenetic trees of H. vigintioctopunctata neuropeptide precursors and neuropeptide GPCRs were reconstructed using sequences from well-characterized species, including B. mori [30], T. castaneum [25], Harmonia axyridis [26] and other species. Amino acid sequences of neuropeptide precursors and GPCRs were retrieved from the NCBI database. All amino acid sequences were aligned using MEGA version 11.0 [39]. The neighbor-joining phylogeny was constructed in MEGA version 11.0 with a p-distance model. Branch robustness was evaluated through 1000 bootstrap replicates, and missing data/gaps were treated via pairwise deletion. The metabotropic glutamate receptor of Neocloeon triangulifer (NtrGluR, XP_059491291) was designated as the outgroup for GPCR phylogeny. Final dendrograms were annotated and color-coded using FigTree1.4.4 (http://tree.bio.ed.ac.uk) (accessed on 6 March 2025).

2.5. Spatial Expression

Total RNA was extracted from pre-processed tissues using the method described in the preceding section. First-strand cDNA synthesis was carried out with the PrimeScript RT Reagent Kit with gDNA Eraser (Takara Bio, Japan). Quantitative Real-Time PCR (qRT-PCR) reactions were conducted in 20 μL volumes using TB Green™ Premix Ex Taq™ (Takara Bio, Japan) on a CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), with the following thermal cycling parameters: initial denaturation at 95 °C for 30 s; 40 cycles of 95 °C for 5 s, 60 °C for 30 s; and a melt curve analysis from 65 °C to 95 °C with 0.5 °C increments per 5 s. Two ribosomal protein genes (HvpRPS18 and HvpRPL13) were employed as endogenous controls [40]. Gene-specific primers for 58 neuropeptide precursor genes (Table S3) were designed using the Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA, USA), with primer efficiencies (80–120%), standard curves, amplification plots, and melting curves validated prior to expression analysis (Table S6; Figure S3). Experimental design included triplicate biological replicates. The relative quantification was calculated using the method with two reference genes [41,42,43]. The expression level in the brain sample was used as the calibrator.

2.6. Statistical Analysis

Statistical analysis was performed using IBM SPSS21 (IBM Corp., Armonk, NY, USA). Tissue-specific expression data (mean ± SEM) were analyzed by one-way ANOVA with post hoc Tukey’s HSD test. Statistical significance was defined as p < 0.05.

3. Results

3.1. Summary of the Transcriptome Analyses

The RNA-seq dataset yielded 11.78 Gb of raw reads, with 11.55 Gb retained as high-quality clean reads after filtering (Table S1). The clean reads exhibited Q30 and Q20 scores of 96.62% and 98.95%, respectively (Table S1). De novo assembly generated 23,040 unigenes with an average length of 1285 bp and a N50 value of 2476 bp (Table S2). The assembly completeness, assessed via BUSCO analysis, reached 96.1%, comprising 94.7% single-copy orthologs and 1.4% duplicated BUSCOs (Table S2). Comparing unigenes to six major functional databases for functional annotation, 10,253 (44.50%), 8224 (35.69%), 11,625 (50.46%), 13,599 (59.02%), 8981 (38.98%), and 9534 (41.38%) unigenes had homologous sequences in the GO, KEGG, eggNOG, NR, Swiss-Prot and Pfam, respectively (Figure S1). A Venn diagram revealed 3909 unigenes shared across NR, Swiss-Prot, Pfam, and KEGG databases (Figure 1B). Comparative analysis against the NR database revealed taxonomic distribution patterns of H. vigintioctopunctata transcripts, with the highest annotation proportions observed in Coccinella septempunctata (43.76%), followed by Harmonia axyridis (21.73%), Tenebrio molitor (1.46%), and Tribolium castaneum (1.18%) (Figure 1C).
Functional annotation classified 10,253 unigenes (44.50%) into 55 Gene Ontology (GO) terms spanning three primary categories: biological processes, cellular components, and molecular functions. Within these categories, the majority of annotated unigenes were associated with the biological processes “metabolic process” and “cellular process”, the cellular components “cell part” and “membrane part”, and the molecular functions “binding” and “catalytic activity” (Figure S2). KEGG pathway analysis assigned 8224 unigenes (35.69%) to six functional groups: Environmental Information Processing, Human Diseases, Organismal Systems, Cellular Processes, Genetic Information Processing, and Metabolism. ‘Signal transduction’ (1083), ‘Cancers: Overview’ (824), ‘Endocrine system’ (565), ‘Transport and catabolism’ (562), ‘Translation’ (456), and ‘Carbohydrate metabolism’ (349) were the dominant pathways in each group, respectively (Figure 2A). In the pathway ‘Nervous system’, 325 unigenes were mapped to 10 KEGG pathways, with “Retrograde endocannabinoid signaling” (112), “Dopaminergic synapse” (88), and “Glutamatergic synapse” (74) representing the most enriched pathways (Figure 2B). In the pathway ‘Endocrine system’, 565 unigenes were annotated to 23 KEGG pathways, dominated by “Thyroid hormone signaling pathway” (98), “Aldosterone synthesis and secretion” (97), and “Oxytocin signaling pathway” (97) (Figure 2C).

3.2. Analysis of Neuropeptides

A total of 58 neuropeptide precursor genes were identified in H. vigintioctopunctata, classified into 37 evolutionarily conserved families (Table 1). All genes contained complete open reading frames (ORFs), encoding predicted proteins ranging from 69 amino acids (aa) for calcitonin-like diuretic hormone 31 (CL-DH31) gene to 371 aa for neuropeptide-like precursor 1 (NPLP1) gene, with signal peptide cleavage sites predicted between residues 16 and 34 (Table 1). In silico analysis of post-translational processing predicted 98 unique mature peptides derived from these precursors (Table S3).
Comparative analysis revealed broad conservation of H. vigintioctopunctata neuropeptide precursors across multiple insect species, including Tribolium castaneum, Coccinella septempunctata, Tenebrio molitor, Grapholitha molesta, Harmonia axyridis, Carabus violaceus, and Zophobas atratus (Table 1). Most neuropeptide families were successfully identified in H. vigintioctopunctata (Table S4). Among these precursors, part of them were prevalent over species of five different orders (Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera), such as adipokinetic hormone 2 (AKH 2), allatostatin CC (Ast CC), allatotropin (AT), bursicon, capability (CAPA)/periviscerokinin (PVK)/cardioacceleratory peptide 2b (CAP2b), crustacean cardioactive peptide (CCAP), CCHamide (CCH), Calcitonin-like diuretic hormone 31 (CL-DH31), eclosion hormone (EH), ecdysis triggering hormone (ETH), FMRFamide (FMRF), IDLSRF-like peptide (IDLSRF), insulin-like peptide (ILP), ion transport peptide (ITP), ITG-like (ITG), myosuppressin (MS), Natalisin (NTL), Neuropeptide-like precursor 1 (NPLP1), orcokinin (OK), pigment-dispersing factor (PDF), Pyrokinin (PK)/Pheromone biosynthesis activating neuropeptide (PBAN), ryamide (RY), short neuropeptide F (sNPF), SIFamide (SIF), sulfakinin (SK), and tachykinin (TK) (Table S4). Notably, AKH/corazonin-related peptide (ACP), allatostatin-C (AstC), elevenin, insulin-like growth factor-like peptide (IGFLP), and IMFamide (IMF) were absent in Coccinellidae species, while corazonin (Crz), allatostatin A (AstA), leucokinin (LK), and LQDVamide (LQDV) remain unreported in Coleoptera genomes (Table S4). Additionally, calcitonin and corticotropin-releasing factor-like diuretic hormone 47 (CRF-DH47) were not detected in H. vigintioctopunctata (Table S4). We identified six Insulin-like peptides (Table 1). Just one adipokinetic hormone precursor was identified in this species (Table 1). Three groups of osmoregulatory neuropeptide genes were identified in H. vigintioctopunctata: One gene encoding an arginine vasopressin-like peptide (AVPL), three genes encoding antidiuretic factors (ADFs), and two genes encoding CRF-like diuretic hormones DH37 and DH44, respectively (Table 1).
Prepropeptide architectures displayed a conserved organization of structural motifs across diverse precursors, such as signal peptides, cleavage sites, and mature peptides. For instance, in AKH, proctolin, SIFamide precursors, and short mature peptides (5–12 aa) were positioned immediately downstream of the signal peptide, followed by longer associated peptides (37–51 aa) (Table S3). The ecdysis-triggering hormone (ETH) precursor contained two consecutive mature peptides following the signal peptide sequence (Table S3). Repetitive motifs separated by proteolytic cleavage sites characterized precursors such as FMRFamide (six repeats), allatostatin-B (six), CAPA (two), PBAN (two), and tachykinin (TK) (eight) (Table S3). Transcript variants were observed in AstCC, CRF-DH37, orcokinin (OK), NPLP1, and neuropeptide F1 (NPF1). Specifically, NPF1 generated two isoforms: a truncated NPF1a lacking a 39-aa fragment present in the full-length NPF1b (Table S3).
Multi-sequence alignments demonstrated varying identity levels among precursors (Figure S4–S53). Highly-identity sequences included agatoxin (Figure S5), bursicon (Figures S15 and S16), ILDSRF (Figure S31), PDF (Figure S43), and Pro (Figure S45), whereas PTTH (Figure S44), PY/PBAN (Figure S46), and tachykinin (TK) (Figure S52) displayed lower sequence identity. Phylogenetic reconstruction confirmed orthology, with all H. vigintioctopunctata neuropeptides clustering into clades shared with other insect homologs (Figure 3).

3.3. Spatial Expression Patterns of Neuropeptide Precursors

To explore the potential characteristics and roles of these neuropeptides in H. vigintioctopunctata, the spatial expression profiles of the 58 neuropeptide precursor genes were examined across larval tissues of the brain, ventral nerve cord (VNC), gut, and Malpighian tubules (MT). Among these, most neuropeptide precursor genes were expressed at higher levels in the brain and VNC tissues. For brain-enriched precursor genes: fourteen genes exhibited predominant expression in the brain, including AKH, EH, ILP2, ILP3, ILP4, ILP5, MS, NTL, NPA, PDF, PTTH, SIF, SK, and TK (Figure 4A). For VNC-enriched precursor genes: fifteen genes showed elevated expression in the VNC, such as AstB, AstCC-X1, AstCC-X2, AVPL, Burβ, CAPA, CCAP, CCH1, CCH2, ETH, FMRF, ILP7, NPLP1-X2, Pro, and PK (Figure 4B). For brain-VNC co-expressed precursor genes: twenty-two genes were highly expressed in both tissues, including ALP, AstCCC, AT, Baratin, Burα, CL-DH31, CRF-DH37-X1, CRF-DH37-X2, GPA2, GPB5, Hansolin, IDLSRF, ILP1, ITP, ITG, NPF1b, NPLP1-X1, OK-A, RF, RY, and sNPF (Figure 4C). For non-neural tissue expression: ADF-b1, ADF-b4, and ADF-b5 were enriched in MT, with additional expression in the brain and VNC (Figure 4D); CNMa, OK-B, and TR showed prominence in gut, while NPF1a, OK-A, and sNPF were also weakly expressed in gut tissue (Figure 4C,D). Multi-tissue expression: CRF-DH44 was ubiquitously expressed in the brain, VNC, and MT (Figure 4D).

3.4. G Protein-Coupled Receptors for Neuropeptides

We identified 31 putative neuropeptide G protein-coupled receptors (GPCRs) in the CNS transcriptome of H. vigintioctopunctata, and all have complete ORFs encoding proteins ranging from 249 to 1393 amino acids (aa) (Table S5). Structural prediction indicated that 30 GPCRs possess ≥ 6 transmembrane domains (TMDs), while the insulin receptor retained a single TMD (Table S5). Among these GPCRs, 25 receptors belong to Family A, 4 belong to Family B, and 2 belong to leucine-rich repeat-containing GPCRs (LGRs).
Ligand specificity of the 25 Family A receptors was inferred through BLASTP and phylogenetic analyses, resolving them into 19 functional groups (Table S5): Allatostatin A-like (HvpA1), AKH (HvpA2), Allatotropin (AT) (HvpA3, HvpA4), CAPA/periviscerokinin (PVK) (HvpA5), CCH1 (HvpA6), CCH2 (HvpA7), CCAP (HvpA8, HvpA9), ecdysis triggering hormone (ETH) (HvpA10, HvpA11), FMRF (HvpA12), insulin (HvpA13, HvpA14), MS (HvpA15), NPF (HvpA16), pyrokinin (PK) (HvpA17, HvpA18), RY (HvpA19), sex peptide/allatostatin B (SP/AstB) (HvpA20), TK/ITPL (HvpA21, HvpA22), trissin (TR) (HvpA23), SK (Hvp24), and orphan receptors (HvpA25-NPFF). The phylogenetic analysis reflected clear orthologous relationships within Family A neuropeptide GPCRs (Figure 5A).
For Family B GPCRs, the phylogenetic tree revealed that four receptors were classified into four branches (Figure 5B). HvpB1 clustered with B. mori and Drosophila DH31 receptors. HvpB2 grouped with H. axyridis and Drosophila DH44 receptors (Figure 5B). HvpB3 clustered with B. mori and Drosophila pigment dispersing factor (PDF) receptors. HvpB4 (orphan receptor) exhibited orthology to those of H. axyridis and T. madens PTH-like receptors (Figure 5B). Here, two LGRs were identified in H. vigintioctopunctata, and they were GPA2/GPA5 receptor (HvpLGR1) and bursicon receptor (HvpLGR2) (Table S5). The phylogenetic tree showed that HvpLGR1 is closely related to the receptor for glycoprotein hormones in D. melanogaster (Dme_LGR1) and HvpLGR2 clustered with bursicon receptors from other insects (Figure 5C).

4. Discussion

Neuropeptides play critical roles in insect physiology by regulating processes like feeding, reproduction, and behavior through intercellular communication [20,26]. Their essential functions throughout insect life cycles make neuropeptide signaling pathways promising targets for developing selective insecticides with improved environmental profiles. In this study, 58 neuropeptide precursors and 31 neuropeptide receptors were identified by RNA sequencing in the pest 28-spotted ladybird H. vigintioctopunctata.
Conventional transcriptome sequencing typically yields 6 Gb per sample. To obtain more complete neuropeptide precursor sequences, we generated 11.78 Gb of raw data. The clean reads Q20 (%) are 98.95% > 90%, and clean reads Q30 (%) are 96.62% > 80%, which indicate the transcriptome results are of good quality. Compared to the NR database, the proportion of H. vigintioctopunctata transcripts annotated to different species was higher in Coccinella septempunctata and Harmonia axyridis than other Coleoptera insects (Figure 1C); this may be because they all belong to the ladybird family of insects.
In total, 58 identified neuropeptide precursors were identified in H. vigintioctopunctata, compared to 64 in T. castaneum [20,26], 57 in Grapholita molesta [18], 50 in Tenebrio molitor [21], 17 in Coccinella septempunctata [22], and 36 in Apis mellifera [44]. Interestingly, Calcitonin, and CRF-DH47 had not been identified in H. vigintioctopunctata; this may be because their expression levels are very low at the transcript level. Additionally, ACP, Elevenin, and AstC are absent in Coccinellidae species. ACP is also absent in Igelater, Photinus, Aquatica, and Leptinotarsa; Elevenin is also absent in Pogonus and Leptinotarsa. Crz and AstA were not identified in any Coleoptera databases so far. However, the absence of specific families in Coccinellidae (ACP, Elevenin, AstC) and Coleoptera-wide gaps (Crz and AstA) highlights lineage-specific gene loss or divergence, which may be linked to ecological adaptations.
ILPs play important roles in growth and development, reproduction, stress resistance, and lifespan [10]. ILPs have been found in numerous insect species, and the number of ILPs is significantly different, such as only one in Locusta migratoria [45], but the silkworm has 50 insulin-like peptides [28]. Here, in H. vigintioctopunctata, six genes encoding ILP were identified (Table S5). These insulin genes display significant sequence diversity, akin to what has been observed in coleopteran species [26]. ILPs possess some conserved residues (six cysteines, one leucine, and one tyrosine) within the A- and B-chains that are indispensable for tertiary structure formation [46,47] (Figure S32).
The PBAN/Pyrokinin neuropeptide family, with an FXPRLamide motif in the C-terminus, stimulates pheromone biosynthesis, hindgut muscle contraction, diapause, and cuticle melanization [48]. The precursor gene of PBAN encodes five neuropeptides including DH, α-SGNP, β-SGNP, γ-SGNP, and PBAN, in moths such as B. mori and Helicoverpa assulta [49,50]. However, two neuropeptides encoded by the PBAN precursor gene were predicted in H. vigintioctopunctata (Table S4). This possibly indicates the pleiotropic nature of the PBAN/Pyrokin family of peptides across different insects.
The presence of a single AKH precursor (AKH2) in H. vigintioctopunctata contrasts with the three AKH paralogs (AKH1, AKH2, AKH3) reported in other insects, such as Tribolium castaneum, Locusta migratoria and Grapholita molesta [18,20,51]. This discrepancy raises intriguing questions about the evolutionary dynamics and functional specialization of AKH genes across insect lineages [51,52,53]; they typically exhibit functional diversification through gene duplication in different species. The absence of AKH1 and AKH3 may reflect functional redundancy or reduced selective pressure for specialized paralogs in H. vigintioctopunctata’s ecological niche that need further studies to verify.
The expansion of conservation to alternative splicing patterns is a significant finding. The fact that several neuropeptide genes, including Ast CC, CRF-DH 37, OK, NPLP1, and NPF1 have consistent transcript variants in H. vigintioctopunctata implies that alternative splicing can generate distinct neuropeptide sequences. Phylogenetic clustering of H. vigintioctopunctata neuropeptides with orthologs from diverse insects (e.g., Tribolium, Drosophila) suggests sequence-level similarities.
The spatial expression profiling of neuropeptide precursor genes in H. vigintioctopunctata larvae reveals distinct tissue-specific patterns. The predominant expression of most neuropeptide precursors in the brain and VNC aligns with their canonical functional properties as neurohormones or neuromodulators in central and peripheral nervous systems. For instance, the high expression of AKH, EH, PTTH, and ILPs in the brain may correlate with their involvement in critical processes such as energy mobilization [52], ecdysis [54], developmental timing [55], and insulin-like signaling [56]. Similarly, the enrichment of CAPA, CCAP, and ETH in the VNC suggests their potential roles in myotropic effects on heart muscles [57] and ecdysis-related behaviors [58,59].
The co-expression of 22 precursors in both the brain and VNC (e.g., AT, CRF-DH37, NPF1b) implies their dual roles in integrating systemic and local signaling. For example, neuropeptides like CRF-DH44 (expressed across brain, VNC, and Malpighian tubules) have multiple roles, including regulation of body-fluid secretion, internal nutrient sensing, and CO2-dependent response in Drosophila [60]. The tissue-restricted expression of CNMa, OK B, and TR in the gut highlights their potential involvement in digestive or peristaltic regulation, while ADF-b1/4/5 in Malpighian tubules may contribute to controlling water balance as in Tenebrio molitor [61]. Notably, the expression of NPF1a and sNPF in the gut aligns with their known roles in appetite regulation and gut motility in other insects [9]. Overall, the spatial divergence in neuropeptide expression underscores the modularity of peptidergic signaling, enabling precise regulation of tissue-specific processes while maintaining systemic coordination. Future studies could validate these patterns via in situ hybridization or RNAi to clarify functional hierarchies and cross-talk among these neuropeptides in this species.
The identification of 31 putative neuropeptide GPCRs in the central nervous system (CNS) transcriptome of H. vigintioctopunctata. Notably, the majority of these receptors belong to Family A (25 out of 31), which aligns with observations in other insects. Phylogenetic analysis and BLASTP-based ligand predictions classified these receptors into 19 distinct groups, including receptors for allatostatins, AKH, CCAP, ETH, and others, underscoring their potential roles in regulating critical processes such as growth, metabolism, ecdysis, and reproduction. Family B GPCRs, though fewer in number, exhibited clear orthology to receptors for diuretic hormones (DH31, DH44) and pigment-dispersing factor (PDF), suggesting they may be involved in osmoregulation, stress responses, and circadian rhythm modulation. These functional inferences are provisional pending ligand-binding and knockdown experiments.
The identification of neuropeptides and their receptor sequences in the pest H. vigintioctopunctata provides molecular insights for advancing pest management strategies. Specifically, neuropeptides and their receptors play pivotal regulatory roles in insect growth, development, and behavior, making them promising targets for pest control. For instance, analogs or inhibitors of neuropeptides and their receptors can suppress pest development and increase mortality. CAPA/CAP2b neuropeptides, known to modulate water and ion balance by regulating physiological metabolism, have been shown to induce higher mortality in stink bugs and aphids when applied as analogs [62,63]. Similarly, biostable multi-Aib analogs of tachykinin-related peptides (TRPs), multifunctional neuropeptides widespread in arthropods, exhibit potent oral aphicidal activity in the pea aphid Acyrthosiphon pisum [64]. Disruption of insect diapause, a survival strategy to evade adverse seasons, has been achieved using agonists and antagonists of diapause hormone in Helicoverpa zea [65]. In recent years, RNA interference (RNAi)-based biopesticides have gained significant attention due to their high specificity and low ecological risk. A notable example is Calantha, a sprayable RNAi formulation developed by GreenLight Biosciences to target the proteasome subunit beta type-5 (PSMB5) gene of the Colorado potato beetle (Leptinotarsa decemlineata) [66]. RNAi targeting neuropeptides and their receptors has similarly demonstrated potential for pest control. For example, silencing bursicon or its receptor disrupted cuticle tanning and wing expansion, causing lethal developmental defects in Aphis citricidus and Henosepilachna vigintioctomaculata [67,68]. In Tribolium castaneum, RNAi-mediated knockdown of adipokinetic hormones (AKHs) and their receptor significantly reduced locomotor activity [69]. Similarly, silencing CCH1 neuropeptide and its receptor in aphids led to reduced feeding and reproductive capacity [70]. These cases underscore the broad applicability of neuropeptide-targeted RNAi in disrupting critical physiological and behavioral processes in pests.

5. Conclusions

In general, our study describes a combined strategy of CNS transcriptome of H. vigintioctopunctata whose screening resulted in the discovery and identification of its neuropeptide and GPCR genes. These results provide a basis for further pharmacological studies to design mimetic analogs of peptides or antagonists and agonists of receptors for control strategy on this solanaceous crop pest. Future functional studies, including ligand-receptor binding assays, tissue-specific localization, RNA interference, and CRISPR-Cas9 gene editing, will be critical to validate these predictions and unravel the precise roles of these receptors in H. vigintioctopunctata physiology. The sequence identification of these neuropeptides and their receptors provides a foundational resource for exploring insect neuroendocrinology and developing targeted strategies for pest management.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/insects16060624/s1. Table S1: Summary of RNA-Seq data in Henosepilachna vigintioctopunctata central nervous system transcriptome; Table S2: Summary of assembled unigenes in H. vigintioctopunctata central nervous system transcriptome; Table S3: Sequences for putative transcriptions of annotated neuropeptide precursors genes deduced from H. vigintioctopunctata central nervous system transcriptome; Table S4: Overview of the presence of neuropeptide precursors in H. vigintioctopunctata and other insects; Table S5: G protein-coupled receptors for neuropeptide identified in H. vigintioctopunctata; Table S6: Primers in this study; Table S7: GenBank accession numbers used for the multiple sequence alignment; Figure S1: Functional annotation of unigenes from transcriptome of H. vigintioctopunctata CNS; Figure S2: GO annotations analysis of unigenes from transcriptome of H. vigintioctopunctata CNS; Figure S3 The standard curves, melting curves, amplification plots of the primers for Quantitative Real-Time PCR (qRT-PCR) of 58 neuropeptide genes; Figure S4–S53: Sequence alignment of the neuropeptide precursors.

Author Contributions

Conceptualization, X.L. (Xiaoming Liu), Q.L. and S.A.; methodology, Z.W. and S.Y.; software, Y.Z. and A.L.; validation, Y.Z. and C.S.; data curation, Q.L. and X.L. (Xiaoguang Liu); writing—original draft preparation, X.L. (Xiaoming Liu), Q.L., Z.W., A.L., S.Y., Y.Z., C.S., X.L. (Xiaoguang Liu), M.D. and S.A.; writing—review and editing, X.L. (Xiaoming Liu), M.D., Y.Z., C.S. and S.A.; project administration, X.L. (Xiaoming Liu), M.D., Y.Z. and S.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by Henan Agricultural Research System (HARS-22-09-G3) and the High-level Talents–Top Talents Project of Henan Agricultural University (30501417).

Data Availability Statement

The research data are deposited in the article and Supplementary Materials, and the identified sequences have been uploaded to NCBI (GenBank Accession Numbers are listed in Table 1 and Table S5). Further inquiries can be directed to the corresponding author.

Acknowledgments

We sincerely thank Ji-Ping Liu for collecting the materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Insects 16 00624 g001
Figure 1. Transcriptomics analysis of H. vigintioctopunctata central nervous system (CNS). (A) Anatomy of the CNS from H. vigintioctopunctata. (B) A Venn diagram across NR, Swiss-Prot, Pfam, and KEGG databases. (C) Comparative analysis against the NR database revealed taxonomic distribution patterns of H. vigintioctopunctata transcripts.
Figure 1. Transcriptomics analysis of H. vigintioctopunctata central nervous system (CNS). (A) Anatomy of the CNS from H. vigintioctopunctata. (B) A Venn diagram across NR, Swiss-Prot, Pfam, and KEGG databases. (C) Comparative analysis against the NR database revealed taxonomic distribution patterns of H. vigintioctopunctata transcripts.
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Figure 2. Distribution of transcriptomic unigenes in the KEGG pathways. (A) KEGG pathway analysis assigned unigenes to six functional groups: Environmental Information Processing, Human Diseases, Organismal Systems, Cellular Processes, Genetic Information Processing, and Metabolism. (B) The KEGG pathways in the pathway ‘Nervous system’. (C) The KEGG pathways in the pathway ‘Endocrine system’.
Figure 2. Distribution of transcriptomic unigenes in the KEGG pathways. (A) KEGG pathway analysis assigned unigenes to six functional groups: Environmental Information Processing, Human Diseases, Organismal Systems, Cellular Processes, Genetic Information Processing, and Metabolism. (B) The KEGG pathways in the pathway ‘Nervous system’. (C) The KEGG pathways in the pathway ‘Endocrine system’.
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Insects 16 00624 g003
Figure 3. Phylogenetic tree of neuropeptide precursors. The Neighbor-Joining method is used to construct the phylogenetic tree by MEGA11 based on the deduced amino acid sequences of neuropeptide precursors. The different color dots at each node indicate the percentage of bootstrap value after 1000 replications. The GenBank accession numbers is listed in Table S7.
Figure 3. Phylogenetic tree of neuropeptide precursors. The Neighbor-Joining method is used to construct the phylogenetic tree by MEGA11 based on the deduced amino acid sequences of neuropeptide precursors. The different color dots at each node indicate the percentage of bootstrap value after 1000 replications. The GenBank accession numbers is listed in Table S7.
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Insects 16 00624 g004
Figure 4. Spatial expression of neuropeptide precursors in H. vigintioctopunctata. (A) Brain-enriched precursors. (B) VNC-enriched precursors. (C) Brain–VNC co-expressed precursors. (D) Non-neural tissue expression. Br: Brain; VNC: ventral nerve cord (VNC); MT: Malpighian tubes. Data are mean ± standard error (mean ± SE). Different letters indicate significant differences among groups (p < 0.05).
Figure 4. Spatial expression of neuropeptide precursors in H. vigintioctopunctata. (A) Brain-enriched precursors. (B) VNC-enriched precursors. (C) Brain–VNC co-expressed precursors. (D) Non-neural tissue expression. Br: Brain; VNC: ventral nerve cord (VNC); MT: Malpighian tubes. Data are mean ± standard error (mean ± SE). Different letters indicate significant differences among groups (p < 0.05).
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Insects 16 00624 g005
Figure 5. Phylogenetic tree of the Family A neuropeptide G protein-coupled receptors (GPCRs) (A), Family B neuropeptide GPCRs (B), and leucine-rich repeat-containing GPCRs (C). The Neighbor-Joining method is used to construct the phylogenetic tree by MEGA11 based on the deduced amino acid sequences of transmembrane domains 1–7 of GPCRs. The different-colored dots at each node indicate the percentage of bootstrap value after 1000 replications. The GenBank accession numbers are listed in Table S7.
Figure 5. Phylogenetic tree of the Family A neuropeptide G protein-coupled receptors (GPCRs) (A), Family B neuropeptide GPCRs (B), and leucine-rich repeat-containing GPCRs (C). The Neighbor-Joining method is used to construct the phylogenetic tree by MEGA11 based on the deduced amino acid sequences of transmembrane domains 1–7 of GPCRs. The different-colored dots at each node indicate the percentage of bootstrap value after 1000 replications. The GenBank accession numbers are listed in Table S7.
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Table 1. Neuropeptide precursors identified from Henosepilachna vigintioctopunctata.
Table 1. Neuropeptide precursors identified from Henosepilachna vigintioctopunctata.
ClassificationFamilyNeuropeptide PrecursorsGenBank
Accession No.		Transcripts Per Million (TPM)Precursor size (aa)Signal Peptide (aa)Homology Search with Known Protein (Blastp)
SpeciesE-ValueIdentity (%)Accession No.
NeuropeptidesAdipokinetic hormone/
Hipertrehalosemic hormone/
Red pigment-concentrating		Adipokinetic hormone 2 (AKH2)PV645140116.4572, complete20Tribolium castaneum8.00 × 101858.90%NP_001107818.1
NeuropeptidesAgatoxin-like peptide aAgatoxin-like (ALP)/LQDVamidePV64514114.86106, complete23Carabus violaceus1.00 × 104266.67%XP_968442.2
NeuropeptidesAllatostatinAllatostatin B (AstB)/Prothoracicostatic peptide (PTSP)/myoinhibitory peptide (MIP)PV645142541.37192, complete29Tribolium castaneum2.00 × 105048.45%RZC34805.1
NeuropeptidesAllatostatinAllatostatin CC (AstCC) X1PV645143188.64137, complete23Zophobas atratus1.00 × 105367.72%UXO98062.1
NeuropeptidesAllatostatinAllatostatin CC (AstCC) X2PV6451444.2140, complete26Zophobas atratus2.00 × 105367.72%UXO98062.1
NeuropeptidesAllatostatinAllatostatin CCC (AstCCC)PV645145215.35104, complete23Coccinella septempunctata9.00 × 103664%XP_044759342 [26]
NeuropeptidesAllatotropin/OrexinAllatotropin (AT)PV645146189.2099, complete24Tribolium castaneum1.00 × 101340.95%NP_001137204.1
Osmoregulatory neuropeptidesAntidiuretic hormoneAntidiuretic factor b-1 (ADF-b1)PV64514714.64129, complete22Tribolium castaneum1.00 × 102138.00%EFA07530.1 [20]
Osmoregulatory neuropeptidesAntidiuretic hormoneAntidiuretic factor b-4 (ADF-b4)PV645148146.37159, complete18Tribolium castaneum5.00 × 103357.00%EFA07533.2 [20]
Osmoregulatory neuropeptidesAntidiuretic hormoneAntidiuretic factor b-5 (ADF-b5)PV645149197.04138, complete18Tribolium castaneum2.00 × 103048.00%EFA07534.1 [20]
Osmoregulatory neuropeptidesVasopressin/oxytocinArginine-vasopressin-like (AVPL)PV645150388.95152, complete21Tribolium castaneum1.00 × 105561.22%NP_001078831.1
Other putative neuropeptide genesNABaratin (NVP-like)PV645151684.9314, complete21Tribolium castaneum1.00 × 108051.36%EFA09163.1 [20]
Protein hormones (polypeptides)Cystine knotBursicon alpha (Bur α)PP430623544.05169, complete29Tribolium castaneum4.00 × 109492.00%NP_001107779.1
Protein hormones (polypeptides)Cystine knotBursicon beta (Bur β)/partner of bursiconPP430624385.62142, complete26Tribolium castaneum3.00 × 107080.15%NP_001107780.1
NeuropeptidesPyrokinin/Periviscerokinin/
Pheromone biosynthesis
activating neuropeptide		Capability (CAPA)/Periviscerokinin (PVK)/Cardioacceleratory peptide 2b (CAP2b)PV645153598.23144, complete19Zophobas atratus3.00 × 100937.07%UXO98070.1
NeuropeptidesCrustacean cardioactive peptideCrustacean cardioactive peptide (CCAP)PV645160423.61142, complete19Diorhabda sublineata3.00 × 104962.24%XP_056637896.1
NeuropeptidesCCHamideCCHamide 1 (CCH1)PV645154173.87161, complete33Tenebrio molitor6.00 × 102337.09%UXO98161.1
NeuropeptidesCCHamideCCHamide 2 (CCH2)PV645155260.93118, complete27Tenebrio molitor3.00 × 102741.23%UXO98162.1
NeuropeptidesCNMamideCNMamide (CNMa)PV6451568.72142, complete15Tenebrio molitor5.00 × 101234.04%UXO98163.1
NeuropeptidesDiuretic hormoneCalcitonin-like diuretic hormone 31 (CL-DH31)PV64515278.7969, complete30Tribolium castaneum3.00 × 100761.54%EEZ99367.2
Osmoregulatory neuropeptidesCorticotropin-releasing hormone binding protein/Diuretic hormone-relatedCorticotropin-releasing factor-like diuretic hormone 37 X1 (CRF-DH 37 X1)PV645157156.13136, complete18Tribolium castaneum1.00 × 101234.31%NP_001164096.1 [26]
Osmoregulatory neuropeptidesCorticotropin-releasing hormone binding protein/Diuretic hormone-relatedCorticotropin-releasing factor-like diuretic hormone 37 X2 (CRF-DH 37 X2)PV64515892.62155, complete18Tribolium castaneum2.00 × 103345.57%XP_015835155.1 [26]
Osmoregulatory neuropeptidesCorticotropin-releasing hormone binding protein/Diuretic hormone-relatedCorticotropin-releasing factor-like diuretic hormone 44 (CRF-DH 44)PV645159135.5349, complete22Grapholitha molesta1.00 × 107046.00%MN639889
Protein hormones (polypeptides)Eclosion hormoneEclosion hormone (EH)PV64516216.6381, complete25Halyomorpha halys6.00 × 102866.22%XP_024214295.1
Protein hormones (polypeptides)Ecdysis-triggering hormoneEcdysis-triggering hormone (ETH)PV64516126.51155, complete22Colaphellus bowringi1.00 × 101443.44%UDO48204.1
NeuropeptidesFMRFamide-related peptideFMRFamide (FMRF)PV645163469.43207, complete17Harmonia axyridis1.00 × 106252.11%XP_045478717.1
Protein hormones (polypeptides)Glycoprotein hormoneGlycoprotein hormone alpha 2 (GPA2)PV645164187.93122, complete16Tribolium castaneum4.00 × 106777.87%NP_001164244.1
Protein hormones (polypeptides)Glycoprotein hormoneGlycoprotein hormone beta 5 (GPB5)PV645165144.95154, complete20Tribolium castaneum1.00 × 106564.47%NP_001280517.1
NeuropeptidesNAHansolinPV6451665.20121, complete20Tenebrio molitor4.00 × 101333.61%UXO98170.1
NeuropeptidesNAIDLSRF-like peptide (IDLSRF)PV645167260.25208, complete34Coccinella septempunctata4.00 × 1014495.19%XP_044765879.1
Protein hormones (polypeptides)Insulin/Insulin-like growth factor/RelaxinInsulin-like peptide 1 (ILP1)PV64516816.26123, complete21Harmonia axyridis1.00 × 104258.68%XP_045479779.1
Protein hormones (polypeptides)Insulin/Insulin-like growth factor/RelaxinInsulin-like peptide 2 (ILP2)PV645169122.58124, complete21Harmonia axyridis1.00 × 101838.64%XP_045479306.1
Protein hormones (polypeptides)Insulin/Insulin-like growth factor/RelaxinInsulin-like peptide 3 (ILP3)PV64517013.66131, complete20Coccinella septempunctata1.00 × 101336.36%XP_044748903.1
Protein hormones (polypeptides)Insulin/Insulin-like growth factor/RelaxinInsulin-like peptide 4 (ILP4)PV64517115.62109, complete21Camponotus floridanus5.00 × 100834.62%XP_025267327.1
Protein hormones (polypeptides)Insulin/Insulin-like growth factor/RelaxinInsulin-like peptide 5 (ILP5)PV6451726.54120, complete23Cryptolaemus montrouzieri3.00 × 100836.59%KAL3266362.1
Protein hormones (polypeptides)Insulin/Insulin-like growth factor/RelaxinInsulin-like peptide 7 (ILP7)/RelaxinPV64517379.70144, complete24Coccinella septempunctata8.00 × 108179.86%XP_044762237.1
Protein hormones (polypeptides)Crustacean hyperglycaemic hormone familyion transport peptide (ITP)PV64517448.91122, complete28Coccinella septempunctata3.00 × 107385.95%XP_044746445.1
Other putative neuropeptide genesITG-likeITG-like (ITG)PV645175850.08216, complete20Harmonia axyridis3.00 × 1013189.30%XP_045481008.1
NeuropeptidesMyosuppressinMyosuppressin (MS)PV6451782.0486, complete24Anoplophora glabripennis1.00 × 102657.89%XP_018573133.1
NeuropeptidesTachykinin-related peptidesNatalisin (NTL)PV64517911.50150, complete19Rhynchophorus ferrugineus4.00 × 102038.78%QGA72564.1
Protein hormones (polypeptides)Neuroparsin/Ovary ecdysteroidogenic hormoneNeuroparsin A (NPA)PV645180129.02101, complete25Harmonia axyridis7.00 × 103262.38%XP_045460883.1
NeuropeptidesNeuropeptide YNeuropeptide F 1a (NPF1a)PV6452116.2588, complete26Tenebrio molitor8.00 × 102157.95%UXO98177.1
NeuropeptidesNeuropeptide YNeuropeptide F 1b (NPF1b)PV64521223.98125, complete26Zophobas atratus4.00 × 103958.73%UXO98088.1
Other putative neuropeptide genesNeuropeptide-like precursorNeuropeptide-like precursor 1 X1 (NPLP1 X1)PV64520929.12371, complete24Coccinella septempunctata5.00 × 1014763.71%XP_044759118.1
Other putative neuropeptide genesNeuropeptide-like precursorNeuropeptide-like precursor 1 X2 (NPLP1 X2)PV6452104.74371, complete24Coccinella septempunctata2.00 × 1014364.36%XP_044759118.1
NeuropeptidesOrcokininOrcokinin A (OK A)PV64521317.87151, complete19Harmonia axyridis7.00 × 107773.33%XP_045477291.1
NeuropeptidesOrcokininOrcokinin B (OK B)PV6452148.59283, complete19Harmonia axyridis4.00 × 107552.63%XP_045477290.1
NeuropeptidesPigment-dispersing hormone/Pigment- dispersing factorPigment-dispersing factor (PDF)PV645215207.47105, complete27Photinus pyralis3.00 × 100737.50%XP_031349268.1
Protein hormones (polypeptides)NAProthoracicotropic hormone (PTTH)PV64521715.86176, complete18Tribolium madens2.00 × 101931.25%XP_044258306.1
NeuropeptidesProctolinProctolin (Pro)PV6452163543.7286, complete30Rhynchophorus ferrugineus4.00 × 101653.25%QGA72571.1
NeuropeptidesPyrokinin/Periviscerokinin/ Pheromone biosynthesis activating neuropeptidePyrokinin (PK)/phermone biosynthesis activating neuropeptide like (PBAN-like)PV645218467.84142, complete23Tribolium castaneum7.00 × 101338.32%XP_015835244.1
NeuropeptidesRFamide neuropeptideRFLamide (RF)PV64521959.54182, complete27Tenebrio molitor1.00 × 104243.46%UXO98182.1
NeuropeptidesLuqin/RyamideRyamide (RY)PV64522032.54120, complete23Coccinella septempunctata1.00 × 103863.64%XP_044747600.1
NeuropeptidesNeuropeptide Yshort neuropeptide F (sNPF)PV645221217.9299, complete26Harmonia axyridis5.00 × 104168.82%XP_045470701.1
NeuropeptidesFMRFamide related peptideSIFamide (SIF)PV645222130.0374, complete25Coccinella septempunctata1.00 × 102567.12%XP_044744559.1
NeuropeptidesGastrin/cholecystokininSulfakinin (SK)PV64522310.41104, complete27Coccinella septempunctata8.00 × 102549.51%XP_044752147.1
NeuropeptidesTachykinin-related peptidesTachykinin (TK)PV64522481.99276, complete22Coccinella septempunctata5.00 × 109761.25%XP_044759920.1
NeuropeptidesTrissinTrissin (TR)PV64522510.2486, complete21Anoplophora glabripennis2.00 × 101450.00%XP_018571856.1
NA: not applicable.
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MDPI and ACS Style

Lei, Q.; Wang, Z.; Yao, S.; Lin, A.; Zhang, Y.; Sun, C.; Liu, X.; Du, M.; Liu, X.; An, S. Transcriptome-Wide Identification of Neuropeptides and Neuropeptide Receptors in the Twenty-Eight-Spotted Ladybird Henosepilachna vigintioctopunctata. Insects 2025, 16, 624. https://doi.org/10.3390/insects16060624

AMA Style

Lei Q, Wang Z, Yao S, Lin A, Zhang Y, Sun C, Liu X, Du M, Liu X, An S. Transcriptome-Wide Identification of Neuropeptides and Neuropeptide Receptors in the Twenty-Eight-Spotted Ladybird Henosepilachna vigintioctopunctata. Insects. 2025; 16(6):624. https://doi.org/10.3390/insects16060624

Chicago/Turabian Style

Lei, Quanxing, Ziming Wang, Shuangyan Yao, Aili Lin, Yunhui Zhang, Chengxian Sun, Xiaoguang Liu, Mengfang Du, Xiaoming Liu, and Shiheng An. 2025. "Transcriptome-Wide Identification of Neuropeptides and Neuropeptide Receptors in the Twenty-Eight-Spotted Ladybird Henosepilachna vigintioctopunctata" Insects 16, no. 6: 624. https://doi.org/10.3390/insects16060624

APA Style

Lei, Q., Wang, Z., Yao, S., Lin, A., Zhang, Y., Sun, C., Liu, X., Du, M., Liu, X., & An, S. (2025). Transcriptome-Wide Identification of Neuropeptides and Neuropeptide Receptors in the Twenty-Eight-Spotted Ladybird Henosepilachna vigintioctopunctata. Insects, 16(6), 624. https://doi.org/10.3390/insects16060624

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