Introduction
The diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), is recognised as one of the most destructive pests affecting cruciferous vegetables worldwide, and its larvae feed on foliage of economically significant crops including cabbage, broccoli, and cauliflower, causing substantial yield and quality losses (Li et al., Reference Li, Feng, Liu, You and Furlong2016; Wang et al., Reference Wang, Li, Shao, Li, Guan, Guo and Chen2025b). P. xylostella is a highly adaptable pest that is distributed across all continents except Antarctica, and has been widely recognised as a major pest of cruciferous crops throughout China since the 1970s (Yang et al., Reference Yang, Tian, Xu, Xie, Wang, Zhang, Wang and Wu2015; Zhou et al., Reference Zhou, Lei, Chen, You and You2022; Hou et al., Reference Hou, Zhang, Zhu and Chen2024a). The conservative estimates show that annual global economic costs for P. xylostella management range between $4 and $5 billion, with chemical insecticides remaining the primary control strategy despite decades of implementation (Jaleel et al., Reference Jaleel, Saeed, Saeed, Naqqash, Sial, Aine, Yanyuan, Rui, He and Lu2019; Shehzad et al., Reference Shehzad, Bodlah, Siddiqui, Bodlah, Fareen and Islam2023). However, extensive use of chemical insecticides has driven the evolution of high levels of resistance in P. xylostella to nearly all major insecticide classes, including carbamates, chlorpyrifos, diamides (e.g. chlorantraniliprole and flubendiamide), organophosphates, and pyrethroids (Liu et al., Reference Liu, Tian, Li, Ni, Sun, Yin, Li, Zhang and Li2024; Wang et al., Reference Wang, Chen, Shi, Wu, Yang and Wang2025a; Wang et al., Reference Wang, Wang, Ravi, Zaraei, Alkubaisi, Wen, Zeng, El-gamal and Xu2025c). Additionally, the rapid reproductive capacity and overlapping generations of P. xylostella populations exacerbate control challenges, and thus it was urgent to explore new alternative prevention strategies for P. xylostella. Fortunately, recent advances suggest that double-stranded RNA (dsRNA) can act as a novel selective insecticide via RNA interference (RNAi) by targeting essential developmental genes, providing a potential alternative to conventional chemical control methods (Das and Nanda, Reference Das and Nanda2024; Liu et al., Reference Liu, Wang, Zhou, Zhang, Ge, Moussian, Yan, Gao and Wang2025).
Neuropeptides are the largest and most diverse class of signalling molecules in multicellular organisms, critically involved in regulating numerous physiological processes in insects (Schoofs et al., Reference Schoofs, De Loof and Van Hiel2017). These small proteins, derived from larger precursors mainly expressed in the central nervous and digestive systems, act as neurohormones, neurotransmitters and neuromodulators (Hou et al., Reference Hou, Jiang, Yang, Wang and Kang2015). Neuropeptides are processed through enzymatic cleavages and modifications to generate biologically active mature peptides of varying lengths, which are then secreted into the extracellular space to exert their effects by binding to and activating specific G-protein-coupled receptors on target cells (Hou et al., Reference Hou, Wang, Sun and Wang2023). Previous studies have demonstrated that neuropeptides regulate a wide range of insect behaviours and physiological processes, including feeding and foraging, growth and development, metabolism, reproduction and courtship (Schoofs et al., Reference Schoofs, De Loof and Van Hiel2017; Lu et al., Reference Lu, Pang, Wen, Smagghe, Liu and Gui2025; Pang et al., Reference Pang, Li, Lu, Smagghe, Liu and Gui2025). Neuropeptides are considered promising targets for RNAi-based and eco-friendly pest control strategies due to their key roles in vital insect physiological processes (Gäde and Goldsworthy, Reference Gäde and Goldsworthy2003; Yeoh et al., Reference Yeoh, Pandit, Zandawala, Nässel, Davies and Dow2017; Luo et al., Reference Luo, Lin, Linghu, Gong, Luo, Liu, Jin, Smagghe, Liu and Gui2024). Additionally, the rapid advancement of gene sequencing technologies has enabled the comprehensive identification and annotation of neuropeptides across the genomes and transcriptomes of numerous insect species, including Spodoptera litura (Yang et al., Reference Yang, Wang, Deng, Xiao, Ma, Huang and Lu2024), Mythimna separata (Li et al., Reference Li, Li, Yang, Li, Wu and Xu2023), Galleria mellonella (Luo et al., Reference Luo, Lin, Linghu, Gong, Luo, Liu, Jin, Smagghe, Liu and Gui2024), migratory locust (Hou et al., Reference Hou, Jiang, Yang, Wang and Kang2015), Antheraea pernyi (Wang et al., Reference Wang, Wang and Nie2023), Coccinella septempunctata (Han et al., Reference Han, Chen, Liu, Zhang, Guo, Liu, Wang, Shen and Zhang2024), Chrysoperla carnea (An et al., Reference An, Dou, Dang, Guo, Gao and Li2023), Tribolium castaneum (Li et al., Reference Li, Predel, Neupert, Hauser, Tanaka, Cazzamali, Williamson, Arakane, Verleyen, Schoofs, Schachtner, Grimmelikhuijzen and Park2008), Bemisia tabaci (Li et al., Reference Li, Shi, Lin, Yang and Liu2021), Spodoptera exigua (Llopis-Giménez et al., Reference Llopis-Giménez, Han, Kim, Ros and Herrero2019), Bombyx mori (Roller et al., Reference Roller, Yamanaka, Watanabe, Daubnerová, Žitňan, Kataoka and Tanaka2008), Pteromalus puparum (Xu et al., Reference Xu, Teng, Gu, Qi, Guo, Xiao, Wang, Fang, Wang, Song, Stanley and Ye2020), and Apis mellifera (Hummon et al., Reference Hummon, Richmond, Verleyen, Baggerman, Huybrechts, Ewing, Vierstraete, Rodriguez-zas, Schoofs and Robinson2006). Collectively, these studies have offered valuable insights into the diversity and functional roles of neuropeptides in these species.
Energy homeostasis in insects requires precise regulation to balance fluctuating energy demands and food availability, a process primarily governed by the adipokinetic hormone (AKH) (Arrese and Soulages, Reference Arrese and Soulages2010; Gáliková et al., Reference Gáliková, Diesner, Klepsatel, Hehlert, Xu, Bickmeyer, Predel and Kühnlein2015; Sanchez et al., Reference Sanchez, Munoz, Chen and Giurfa2021). AKH is a short neuropeptide typically composed of 8–10 amino acid residues, first identified in the locust Locusta migratoria, and to date more than 60 variants have been annotated across insect species, highlighting its widespread distribution in arthropod neuroendocrine systems (Mayer and Candy, Reference Mayer and Candy1969; Lu et al., Reference Lu, Zhang, Chen, Li, Li, Cheng, Zhou, You and Zhou2018). AKH is synthesised in the corpora cardiaca and released into the haemolymph to regulate energy-demanding processes through receptor-mediated signalling (Hou et al., Reference Hou, Chen, Jiang, Wei, Gui, Wang and Smagghe2017). Upon binding to its G protein-coupled receptor (AKHR), the AKH pathway mobilises energy reserves, including lipids and carbohydrates, enabling metabolic adaptation during physiological challenges such as flight or starvation (Hou et al., Reference Hou, Chen, Jiang, Wei, Gui, Wang and Smagghe2017; Hughson, Reference Hughson2022; Xiao et al., Reference Xiao, Jin, Tan, Tang and Huang2023). Furthermore, AKHs have been shown to be involved in feeding behaviour, foraging activity, oxidative stress, reproduction, and midgut proteolytic activity in insects (Lee and Park, Reference Lee and Park2004; Bednářová et al., Reference Bednářová, Kodrík and Krishnan2013; Lu et al., Reference Lu, Zhang, Chen, Li, Li, Cheng, Zhou, You and Zhou2018; Sanchez et al., Reference Sanchez, Munoz, Chen and Giurfa2021). Moreover, the AKH has been found to be a taxon-specific neuropeptide exclusive to arthropods with no vertebrate homologs (Shen et al., Reference Shen, Liu, Chen, Li, Mao, Wang and Zhang2024; Kong et al., Reference Kong, Xie, Cao, Li, Zhang, Zou, Xia and Xin2025). Therefore, it emerges as a potential target for precision pest control through employing an RNAi strategy, enabling species-specific management while preserving non-target organisms.
However, the sequence information of neuropeptides and the potential roles of AKH in P. xylostella remain poorly understood. We hypothesise that PxAKH is a key regulator of lipid mobilisation and feeding behaviour in P. xylostella, and that its functional characterisation could provide insights into species-specific pest control strategies. To test this hypothesis, we established a research framework as follows. Firstly, we performed a comprehensive annotation of neuropeptide precursor genes using the available genomic data of P. xylostella. Secondly, we aimed to identify PxAKH genes in P. xylostella and analyse their spatial and temporal expression patterns using qPCR. Thirdly, we investigated how starvation affects lipid metabolism, food consumption, and the expression of PxAKH1 and PxAKH2 in P. xylostella. Fourthly, we employed RNAi gene silencing technology (dsPxAKH1 injection) to elucidate the crucial roles of PxAKH1 in lipid mobilisation and food intake in P. xylostella larvae. Overall, this study aims to provide a conceptual understanding of the roles of PxAKH in regulating nutrient metabolism and feeding behaviour in P. xylostella and to evaluate PxAKH1 as a potential target for RNAi-based pest management.
Materials and methods
Insects
The laboratory colony of P. xylostella used in this study was maintained for successive generations in artificial climate chambers at Yangzhou University. In brief, P. xylostella were reared under standardised laboratory conditions, with temperature maintained at 27 ± 1 °C, relative humidity at 70 ± 5%, a 16 : 8 h light–dark photoperiod, and no exposure to pesticides.
The P. xylostella larvae were fed on fresh leaves of cabbage, Brassica oleracea, and upon adult emergence, the moths were transferred to rearing cages and supplied with a 10% honey solution prepared by diluting pure honey in distilled water as a food source. To induce oviposition, transparent plastic films (5 cm × 5 cm) were immersed in cabbage juice for 30 seconds, removed, blotted dry with filter paper to remove surface moisture, and then placed inside the rearing cages.
Genome-wide identification of neuropeptides
In this study, the P. xylostella neuropeptide precursors were identified by performing sequence alignments using the NCBI online tool BLAST (Basic Local Alignment Search Tool). The whole-genome sequence of P. xylostella (NCBI Assembly: GCA_000330985.13) obtained from the GenBank database was compared against the conserved neuropeptide gene sequences from other different insects. These reference sequences were sourced from published studies on B. mori, D. melanogaster, S. litura, Bactrocera dorsalis, Nilaparvata lugens, and M. separata (Roller et al., Reference Roller, Yamanaka, Watanabe, Daubnerová, Žitňan, Kataoka and Tanaka2008; Nässel and ÅME, Reference Nässel and Åme2010; Tanaka et al., Reference Tanaka, Suetsugu, Yamamoto, Noda and Shinoda2014; Gui et al., Reference Gui, Jiang, Smagghe and Wang2017; Li et al., Reference Li, Li, Yang, Li, Wu and Xu2023; Yang et al., Reference Yang, Wang, Deng, Xiao, Ma, Huang and Lu2024). In addition, the detailed neuropeptide gene information of P. xylostella was obtained via the NCBI Gene database, including Gene Name, GenBank Accession Number (GenBank acc. no.), and Genomic Location. The GeneMark genome online tool was used to analyse gene features such as Start Site, Stop Site, Strand Direction, and Open Reading Frame (ORF).
Gene amplification of PxAKH1 and PxAKH2
According to the manufacturer’s instructions, total RNA was extracted from fourth-instar P. xylostella larvae by using the RNA-easy Isolation Reagent (Nanjing Novozan Biotechnology Co., Ltd., China). RNA concentration was determined by absorbance at 260 nm using a NanoDrop spectrophotometer (Shimadzu, Kyoto, Japan), while RNA purity was assessed by OD260/280 and OD260/230 ratios, and integrity was confirmed by 1% agarose gel electrophoresis. Then, cDNA synthesis was performed using the HiScript II First-Strand cDNA Synthesis Kit with gDNA wiper (Nanjing Novozan Biotechnology Co., Ltd., China). Subsequently, the complete coding sequences of PxAKH1 and PxAKH2 were amplified by PCR using specific primers designed from P. xylostella genome information (Table S1).
Sequence and phylogenetic analysis
Protein sequences of PxAKH1 and PxAKH2 from different insect species were aligned using the DNAMAN 5.2.2 software (Lynnon Biosoft, Quebec, Canada). According to the multiple alignment results of these sequences, a distance matrix was generated using MEGA 6.0, and a neighbour-joining phylogenetic tree was constructed with the Poisson correction model and pairwise gap deletion (Tamura et al., Reference Tamura, Stecher, Peterson, Filipski and Kumar2013). Eventually, the reliability of the phylogenetic branches was assessed using 1,000 bootstrap replicates.
Temporal and spatial expression of PxAKH1 and PxAKH2
The developmental stage samples included 1–2-day-old eggs, first- to fourth-instar larvae, prepupae, 3-day-old pupae, and 5-day-old adults. For the tissue-specific analysis, the head, midgut (Gut), Malpighian tubules (MT), epidermis (EP), and fat body (FB) were individually dissected from fourth-instar larvae in ice-cold PBS (pH 7.4). As described above, total RNA was extracted and then used for cDNA synthesis. The qPCR analysis was conducted on a Bio-Rad CFX 96 system using 10 μl reaction volumes containing 1 μl cDNA template (approximately 200 ng/μl), 5 μl GoTaq qPCR Master Mix (Promega), 0.5 μl each of forward and reverse primers (10 μM), and 3 μl nuclease-free water. The qPCR thermal cycling involved an initial denaturation at 95 °C for 2 minutes, 40 cycles of 95 °C for 15 seconds and 60 °C for 30 seconds, followed by a melting curve analysis from 60 to 95 °C to confirm the specificity of amplification. All primer sequences are presented in Table S1, and RPL32 (GenBank accession no. AB180441) was selected as the reference gene because of its consistent expression in P. xylostella (Zhou et al., Reference Zhou, Guo, Kang, Qin, Gong, Sun, Guo, Zhu, Bai, Zhang, Zhou and Zhang2020; An et al., Reference An, Dou, Dang, Guo, Gao and Li2023; Hou et al., Reference Hou, Zhu, Fang and Chen2024b). The qPCR assays were carried out with four biological replicates, and the relative expressions of PxAKH1 and PxAKH2 were determined using the 2-ΔΔCt method (Livak and Schmittgen, Reference Livak and Schmittgen2001). For the expression analyses, the egg stage and Malpighian tubules were used as the reference groups for developmental and tissue-specific comparisons of PxAKH1 and PxAKH2, respectively.
Effect of starvation on lipid mobilisation, food intake and PxAKH transcripts
For the starvation experiment, newly moulted fourth-instar larvae of uniform size were allocated to five treatment groups, with each group comprising four replicates, each consisting of 20 P. xylostella larvae. Larvae in the control group were continuously fed for 24 h, while the experimental groups were subjected to starvation for 3, 6, 12, and 24 h, respectively. For the feeding assays, starved third-instar P. xylostella larvae were placed in each well of a 6-well plate containing freshly excised cabbage leaf discs (12 mm diameter) prepared with a sterile cork borer and aseptically arranged in a triangular pattern on 2% agar to prevent overlap (n = 4 replicates per treatment group). Feeding activity was monitored at 6-hour intervals under controlled conditions (27 ± 1 °C, 70% RH, 16 : 8 L : D photoperiod). In this study, the grid method was utilised to quantify the consumed leaf area. A transparent 12 × 12 mm reference grid (1 mm2 resolution) was overlaid on digital images of consumed leaf discs using ImageJ (v1.53), and the eaten area was calculated by summing fully covered grid units and fractional coverage (≥75% = 1 unit, 50–74% = 0.5 unit, 25–49% = 0.25 unit, < 25% = 0 unit), with the total multiplied by 1 mm2 to obtain the leaf area consumed. Additionally, the effects of starvation on the transcripts of PxAKH1 and PxAKH2 were determined in P. xylostella. As described above, total RNA was isolated and used for cDNA synthesis, followed by qPCR performed on a Bio-Rad CFX 96 system with a 10 μl reaction volume. The relative expression levels of PxAKH1 and PxAKH2 were calculated using the 2-ΔΔCt method after P. xylostella larvae were starved for different durations (0, 6, 12, and 24 h). For qPCR analysis of PxAKH1/PxAKH2 transcripts after starvation, four biological replicates were performed, each consisting of three randomly selected larvae pooled from a group of 20 individuals.
Effect of RNAi on lipid mobilisation and food intake
In this study, according to the qPCR analysis, PxAKH1 was selected for further functional analysis by employing the RNAi approach. The T7 promoter-tailed primers listed in Table S1 were employed for PCR amplification of the PxAKH1 mRNA target region. According to the manufacturer’s instructions, dsPxAKH1 was then synthesised using the Transcript Aid T7 High Yield Transcription Kit (Thermo Scientific, Lithuania), and its quality was confirmed by 1% agarose gel electrophoresis. With regard to the RNAi experiments, fourth-instar P. xylostella larvae were injected with 3.0 μg (approximately 300 nl) of either dsPxAKH1 or dsGFP control using a Nanoject II Auto-Nanoliter Injector. Afterwards, qPCR was used to assess RNAi efficiency at 12, 24, and 48 h after injection, with four biological replicates performed, each consisting of 10 individually dsRNA-injected larvae, from which three larvae were randomly selected and pooled for RNA extraction and RNAi efficiency assessment.
According to the previously reported method (Grönke et al., Reference Grönke, Beller, Fellert, Ramakrishnan, Jäckle and Kühnlein2003; Hou et al., Reference Hou, Chen, Jiang, Wei, Gui, Wang and Smagghe2017), the lipid mobilisation of P. xylostella was determined after RNAi. Namely, the triacylglycerol (TAG) content was detected by using an enzymatic assay, and insects were homogenised in cold PBS using a Potter–Elvehjem homogeniser. After centrifugation at 3,000 g for 10 minutes at 4 °C to remove impurities, the supernatants of the homogenates were collected and used for TAG determination with the Triglycerides Kit (Doles Reagentes, Goiânia, Brazil). Meanwhile, the lipid droplets in the fat body of P. xylostella larvae were visualised using Nile red staining. Specifically, fat bodies from control (dsGFP) and treated (dsPxAKH1) larvae were dissected in chilled PBS (pH 7.4) and fixed in 4% formalin on glass slides for 3 h at room temperature. The fat bodies were washed with PBS and then stained by immersion in a Nile red solution (1 mg/ml Nile red stock in acetone diluted 1: 100 in PBS) for 30 minutes. Following a final PBS wash, the stained fat bodies were imaged using a Zeiss LSM510 fluorescence microscope (Zeiss, Jena, Germany) with excitation at 488 nm and emission collected between 570 and 640 nm. For each larval specimen, five random microscopic fields were captured, and all subsequent image observation and data analysis were performed in a blinded manner, with researchers unaware of the treatment group assignments throughout the analytical process to minimise subjective bias. Additionally, food intake in P. xylostella was evaluated following RNAi-mediated silencing of PxAKH1 using the methods described above. For all RNAi-based functional experiments, including TAG quantification, Nile red staining, and post-RNAi feeding assays, four biological replicates were performed, each consisting of 10 individually dsRNA-injected larvae, from which three larvae were randomly selected and pooled per replicate to prepare homogenates or isolate fat bodies for downstream biochemical and cytological analyses
Data analysis
All data are presented as mean ± standard error (SE), and differences in PxAKH1 and PxAKH2 expression following starvation, RNAi efficiency of PxAKH1, as well as TAG content and food intake after dsPxAKH1 injection were analysed using Student’s t-test (P < 0.05). Statistical analyses were performed with SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). A sample size of n = 4 biological replicates per group was used, which is consistent with common practice in similar experimental systems and sufficient to detect the observed effect sizes.
Results
Genome-wide annotation of neuropeptides in P. xylostella
In this study, 45 neuropeptide genes belonging to 33 gene families were identified in the genome of the diamondback moth by utilising homologous genes in different insect species. As shown in Table 1, we found that two neuropeptide genes, allatotropin and trissin, contained at least two transcripts, while others such as adipokinetic hormone, allatostatin, CCHamide, insulin-like peptide, and neuropeptide F exhibited two or more isoforms. Here, each neuropeptide gene of P. xylostella could be mapped to specific chromosomes, and all annotated neuropeptide genes consisted of the complete open reading frames (ORFs), encoding the specific neuropeptide precursors ranging from 42 to 508 amino acids in length.
Genomic identification of neuropeptide precursors in P. xylostella
Table 1 Long description
Genomic records for 38 neuropeptide precursor transcripts in P. xylostella list each peptide name, transcript variant, nucleotide and protein GenBank accessions, chromosomal location, genomic start and stop, strand, ORF length in base pairs, and predicted protein length in amino acids. Entries are distributed across many chromosomes, including multiple loci on chromosomes 2, 6, 7, 12, 14, 23, and 24. Most genes have a single transcript variant, but some show multiple variants at the same locus, including allatotropin with X1 and X2, diuretic hormone 44 with two variants labeled DH1 and DH45, and trissin with X1 and X2. ORF lengths range from 129 bp for ion transport peptide to 1527 bp for natalisin, with corresponding protein lengths from 42 aa to 508 aa. Several peptides are compact, such as AKH1 and AKH2 at 222 and 210 bp, while longer precursors include leucokinin at 996 bp and proctolin at 804 bp. Strand orientation varies by gene, with both plus and minus strand annotations across the genome. Coordinates indicate genomic span rather than coding length, so large start to stop intervals can reflect introns and do not directly match ORF size.
The cDNA sequence of PxAKH in P. xylostella
Here, two PxAKH genes, designated as PxAKH1 (fig. 1A) and PxAKH2 (fig. 1B), were identified in P. xylostella via homology comparison with AKH genes from B. mori and S. frugiperda. The PxAKH1 gene has an open reading frame (ORF) of 222 bp, encoding a protein of 73 amino acids, including a 20-amino-acid signal peptide (MHKISFLFVLLACLVIVSEA). Based on predicted signal peptide cleavage sites, a 10-amino-acid putative mature peptide (QLTFTSSWGG) is inferred. Meanwhile, the ORF of the PxAKH2 gene consisted of 210 bp, encoding a 69-amino-acid protein with a 20-amino-acid signal peptide (MFRAFFVFFCVAAMLVLADA). An 8-amino-acid putative mature peptide (QLTFTPNW) is predicted following conserved protease cleavage. Phylogenetic analysis using the Neighbour-Joining method indicated that both PxAKH1 and PxAKH2 sequences exhibit high conservation with AKH sequences from other lepidopteran insects (fig. 2).
Amino acid alignment of adipokinetic hormone (PxAKH1) (A) and (PxAKH2) (B) sequences from Plutella xylostella. The bioactive mature peptide of AKH was highlighted in a sequence logo, where the height of each amino acid symbol reflects its conservation level. Grapholitha molesta (GmAKH1, QMS54688.1; GmAKH2, QMS54689.1), Loxostege sticticalis (LsAKH1, KAL0818720.1), Spodoptera exigua (SeAKH1, KAF9407176.1; SeAKH2, AXY04230.1), Ostrinia furnacalis (OfAKH1, XP_028164252.1; OfAKH2, XP_028164252.1), Agrotis ipsilon (AiAKH1, C0HL91.1; AiAKH2, C0HL91.1), Mythimna separata (MsAKH1, ALX27200.1; MsAKH2, KAJ8709409.1), Manduca sexta (MasAKH1, KAG6451321.1), Papilio xuthus (PaxAKH1, KPI97662.1; PaxAKH2, KPI97662.1), Bombyx mori (BmAKH1, NP_001104825.1; BmAKH1, ABY81279.1), Spodoptera frugiperda (SfAKH1, KAF9794929.1; SfAKH2, KAF9794929.1), Helicoverpa armigera (HaAKH1, AGH25544.1; HaAKH2, AGH25545.1), Cydia pomonella (CpAKH2, QDK59876.1) and Galleria mellonella (GamAKH2, WLY76811.1).
Figure 1 Long description
Textual illustration showing amino acid alignment of adipokinetic hormone sequences. Panel A displays PxAKH1 and Panel B shows PxAKH2. Each panel contains sequences from various species, with conserved residues shaded dark. The mature peptide sequences are highlighted in color: ′QLTFTSSWGG′ for PxAKH1 and ′QLTFTPNW′ for PxAKH2. These sequences are also enlarged below each panel, emphasizing conservation and key differences across species.
Phylogenetic analysis of adipokinetic hormone (AKH) in different insect species. AKH1 and AKH2 were represented as red and green, respectively, and the solid dot showed AKH from Plutella xylostella. The evolutionary relationship was established by using the Neighbour-Joining method with a bootstrap value of 1000.
Figure 2 Long description
The phylogenetic tree illustrates the evolutionary relationships of adipokinetic hormone genes AKH1 and AKH2 across different insect species. The tree is divided into two main sections, with AKH1 represented in red and AKH2 in green. Each section is organized vertically, showing the hierarchical clustering of species based on genetic similarity. The red section begins with Spodoptera frugiperda AKH1 at the top, followed by Agrotis ipsilon AKH1 and continues downwards through various species, including Plutella xylostella AKH1, which is marked with a solid red dot. The green section starts with Spodoptera frugiperda AKH2, followed by Helicoverpa armigera AKH2 and includes Plutella xylostella AKH2, marked with a solid green dot. Bootstrap values are displayed at each node, indicating the confidence level of the branching, with values such as 96, 99 and 100. The tree branches are labeled with insect orders, including Lepidoptera, Hymenoptera, Hemiptera, Diptera, Coleoptera and Orthoptera, showing the taxonomic classification of each species. The branches are primarily diagonal, indicating the evolutionary divergence from common ancestors, with nodes representing speciation events. The tree visually demonstrates the genetic conservation and divergence of AKH genes among the listed insect species.
Development- and tissue-specific transcript profiling of PxAKH in P. xylostella
The results of qPCR analysis showed that both PxAKH1 (fig. 3A) and PxAKH2 (fig. 3B) were highly expressed in the third- and fourth-instar larval stages of P. xylostella. Additionally, the results of qPCR suggested that both PxAKH subtypes could be detected in various tissues, with relatively high expression in the fat body and head tissues for the PxAKH1 gene (fig. 3C), and fat body and gut tissues for the PxAKH2 gene (fig. 3D).
Temporal (A and C) and spatial (B and D) distribution of adipokinetic hormone (PxAKH1 and PxAKH2) transcripts were detected by qPCR in Plutella xylostella. Abbreviations used on the X-axis (A and C): E (eggs), L1 (first-instar larvae), L2 (second-instar larvae), L3 (third-instar larvae), L4 (fourth-instar larvae), PP (prepupa), P (pupae), and A (Adults). Abbreviations used on the X-axis (B and D): Gut (midgut), FB (fat body), EP (epidermis), and MT (Malpighian tubules). Four replicates were conducted (the red dots on the bar chart represent data points from different replicates), and the data are presented as mean ± SE.
Figure 3 Long description
PxAKH1 across developmental stages. The horizontal axis label is Different developmental stages with categories E, L1, L2, L3, L4, PP, P, A. The vertical axis label is Relative expression level. The vertical axis range is 0.0 to 4.0. The tallest bar is at L3, a little above 3.0. The next tallest bar is at L4, just below 3.0. P is around 2.0. L2 is around 1.5. PP is around 1.2. L1 is around 1.0. E is around 0.8. A is around 0.5. Each bar has a vertical error bar and multiple overlaid circular points. PxAKH1 across tissues. The horizontal axis label is Different tissues with categories Head, Gut, FB, EP, MT. The vertical axis label is Relative expression level. The vertical axis range is 0.0 to 15.0. The tallest bar is at Head, around 12 to 13. FB is around 2.0. Gut is around 1.0. EP is around 1.0. MT is around 1.0. Each bar has a vertical error bar and multiple overlaid circular points. PxAKH2 across developmental stages. The horizontal axis label is Different developmental stages with categories E, L1, L2, L3, L4, PP, P, A. The vertical axis label is Relative expression level. The vertical axis range is 0.0 to 6.0. The tallest bar is at L3, around 4.5 to 5.0. L4 is around 3.0. L1 is around 2.0. L2 is around 2.0. PP is around 2.0. P is around 2.0. A is around 2.0. E is around 1.0. Each bar has a vertical error bar and multiple overlaid circular points. PxAKH2 across tissues. The horizontal axis label is Different tissues with categories Head, Gut, FB, EP, MT. The vertical axis label is Relative expression level. The vertical axis range is 0.0 to 15.0. The tallest bar is at FB, around 10 to 11. Gut is around 8 to 9. Head is around 1.0. EP is around 1.0. MT is around 1.0. Each bar has a vertical error bar and multiple overlaid circular points. Across developmental stages, both PxAKH1 and PxAKH2 have the tallest bar at L3. Across tissues, PxAKH1 has the tallest bar at Head, while PxAKH2 has the tallest bar at FB, with Gut also high.
Changes in food intake and PxAKH transcripts after starvation
After subjecting larvae to different durations of starvation, measurements were conducted to assess the changes in food intake (fig. 4) and two AKH transcripts (fig. 5A and B) of P. xylostella. In this study, food intake analysis showed that larvae starved for 3 h resumed feeding rapidly after nutrient restoration, with leaf areas of 26.46 ± 2.86 mm2 at 6 h, 149.46 ± 7.76 mm2 at 12 h, and 223.50 ± 19.19 mm2 at 24 h. A similar trend was observed in larvae starved for 6 h, with feeding areas of 22.00 ± 1.63 mm2, 94.46 ± 4.49 mm2, and 122.46 ± 18.37 mm2 at 6, 12, and 24 h, respectively. Notably, both 3 h- and 6 h-starved larvae exhibited significantly enhanced feeding within 24 h compared with the control group (CK: 103.46 ± 2.86 mm2 at 24 h) (fig. 4). In contrast, larvae starved for 12 h showed reduced feeding ability immediately after nutrient restoration, with minimal feeding within the first 6 h (5.00 ± 2.45 mm2), but gradually recovered to 61.00 ± 4.08 mm2 at 12 h and 97.46 ± 2.04 mm2 at 24 h, approaching control levels. However, larvae starved for 24 h failed to recover normal feeding, showing consistently low feeding areas of 1.33 ± 0.47 mm2, 3.00 ± 1.63 mm2, and 44.50 ± 11.84 mm2 at 6, 12, and 24 h, respectively, which remained far below the control level (fig. 4).
The effect of starvation treatment on food intake with different durations in Plutella xylostella. (A) The observation of P. xylostella larvae feeding performance by using the leaf disc method. (B) The feeding amount of P. xylostella larvae under starvation treatment with different durations. Four replicates were conducted (the dots of different shapes on the line chart represent data points from different replicates), and the data are presented as mean ± SE.
Figure 4 Long description
A showing a grid of circular leaf discs arranged in 5 rows and 4 columns. Column labels at the top read 0h, 6h, 12h, 24h. Row labels on the left read CK, Starved 3 h, Starved 6 h, Starved 12 h, Starved 24 h. B showing a line graph with a legend listing CK, Starved 3 h, Starved 6 h, Starved 12 h, Starved 24 h. The x-axis label reads Feeding time after starvation. The x-axis tick labels read 6h, 12h, 24h. The y-axis label reads Feeding amount (mm superscript 2). The y-axis range is 0.0 to 300.0 with tick labels 0.0, 100.0, 200.0, 300.0. Plotted values by series: CK: at 6h about 40; at 12h about 40; at 24h about 100. Starved 3 h: at 6h about 30; at 12h about 150; at 24h about 220. Starved 6 h: at 6h about 20; at 12h about 100; at 24h about 120. Starved 12 h: at 6h about 5; at 12h about 60; at 24h about 100. Starved 24 h: at 6h about 1; at 12h about 3; at 24h about 40.
The effect of starvation treatment on expression levels of (A) PxAKH1 and (B) PxAKH2 with different durations in Plutella xylostella. (C) The knockdown efficiency of PxAKH1 after the injection of dsRNA into fourth-instar larvae of Plutella xylostella at 12, 24, and 48 h. Four replicates were conducted (the red dots on the bar chart represent data points from different replicates), and the data are presented as mean ± SE. Significant differences between the two treatments were analysed by an independent samples t-test (*P < 0.05, **P < 0.01).
Figure 5 Long description
Three bar graphs compare expression and transcript levels. The first graph, PxAKH1, shows relative expression levels with vertical bars. Categories are CK, Starved 3 h, 6 h, 12 h and 24 h. PxAKH1 expression peaks at Starved 3 h, about 3.5 and decreases significantly by Starved 24 h, about 0.5. The second graph, PxAKH2, shows expression levels with similar categories. PxAKH2 remains stable initially but drops significantly at Starved 12 h and 24 h, around 0.5. The third graph, dsPxAKH1, shows transcript levels at 12, 24 and 48 hours. Each time point compares dsGFP and dsPxAKH1. dsPxAKH1 shows reduced levels compared to dsGFP, with significant differences at 12 and 24 hours. Bars are vertical, with different fills indicating treatments. Red dots represent individual data points. Starvation induces early PxAKH1 expression, reduces PxAKH2 at later times and dsPxAKH1 lowers transcript levels. Significant differences are marked with asterisks.
This study further found that starvation had differential effects on the transcript levels of the two PxAKH genes in P. xylostella larvae (fig. 5A and B). Specifically, the qPCR analysis showed that the expression level of the PxAKH1 gene exhibited a trend of initial increase followed by decrease under starvation stress conditions. Namely, the PxAKH1 transcript levels were significantly elevated at the 3 and 6 h time points, and the expression levels were significantly lower compared to the control group at the 12 and 24 h time points. Conversely, the PxAKH2 gene demonstrated a significant down-regulation trend across all time points of starvation stress.
Effect of RNAi on lipid mobilisation and food intake
After the injection with dsPxAKH1, the target gene was suppressed by 73.03% within 12 h compared to the control, with silencing efficiencies of 57.87% and 35.58% at 24 and 48 h, respectively (fig. 5C). Following RNAi-mediated knockdown of PxAKH1, the TAG content in P. xylostella larvae was significantly higher than in the control group injected with dsGFP at 24 h (fig. 6A). In addition, Nile red staining revealed that the content and density of lipid droplets were higher in dsPxAKH1-injected larvae than in the control group, indicating lipid accumulation in P. xylostella larvae (fig. 6B). Importantly, we also observed that the food intake of P. xylostella larvae injected with dsPxAKH1 was significantly lower than that of the control larvae injected with dsGFP (fig. 7).
The lipid content changes of Plutella xylostella larvae that treated with dsPxAKH1. The effect of dsPxAKH1 injection on triacylglyceride levels (A) and fat droplets (B) in P. xylostella larvae. Four replicates were conducted (the red dots on the bar chart represent data points from different replicates), and the data are presented as mean ± SE. Significant differences between the two treatments were analysed with an independent samples t-test (*P < 0.05). Scale bars = 20 μm.
Figure 6 Long description
Bar chart with two vertical bars and overlaid dot markers. Text above the plot reads 24h. The x-axis has two category labels: dsGFP and dsPxAKH1. The y-axis label reads Triacylglyceride (microgram). The y-axis runs from 0.0 to 30.0 with labeled ticks at 0.0, 10.0, 20.0 and 30.0. The dsGFP bar reaches about 24.0. Four dot markers are plotted near the top of the dsGFP bar at about 24.0. The dsPxAKH1 bar reaches about 26.0. Four dot markers are plotted near the top of the dsPxAKH1 bar at about 26.0. A bracket connects the two bars with an asterisk above the bracket. Two micrographs placed side by side. The left micrograph has the label dsGFP above it. The right micrograph has the label dsPxAKH1 above it. Each micrograph contains many small, round bright spots distributed across the field. A small scale bar is visible near the lower right corner of each micrograph.
The effect of dsPxAKH1 injection on food intake in Plutella xylostella. (A) The observation of P. xylostella larvae feeding performance by using the leaf disc method after the PxAKH1 gene was knocked down. (B) The feeding amount of larvae after injecting with dsPxAKH1 in P. xylostella. Four replicates were conducted (the dots of different shapes on the line chart represent data points from different replicates), and the data are presented as mean ± SE. Significant differences between the two treatments were analysed by an independent samples t-test (*P < 0.05, **P < 0.01).
Figure 7 Long description
Two horizontal rows of leaf disc photos arranged in five columns. Column headings read 0h, 6h, 12h, 18h, 24h. Row labels at the left read dsGFP for the first row and dsAKH1 for the second row. Each photo shows three circular leaf discs. Across time from 0h to 24h, the discs show increasing missing areas. At 24h, the dsGFP row shows larger missing areas than the dsAKH1 row. A line graph with plotted points for two groups labeled dsGFP and dsAKH1. The x-axis label reads Time. The x-axis tick labels read 6h, 12h, 18h, 24h. The y-axis label reads Feeding amount left parenthesis mm superscript 2 right parenthesis. The y-axis tick labels read 0.0, 50.0, 100.0, 150.0, 200.0, 250.0. Data points are shown at each time for both groups, with multiple points per time. At 6h, dsGFP points are near 0.0 to about 25.0 and dsAKH1 points are near 0.0 to about 10.0. At 12h, dsGFP points are about 60.0 to 90.0 and dsAKH1 points are about 20.0 to 40.0. At 18h, dsGFP points are about 110.0 to 140.0 and dsAKH1 points are about 60.0 to 90.0. At 24h, dsGFP points are about 210.0 to 230.0 and dsAKH1 points are about 100.0 to 150.0. A single asterisk appears above the dsGFP series near 6h, 12h and 18h and a double asterisk appears above the dsGFP series near 24h.
Discussion
It is widely known that insect neuropeptides play essential roles in regulating development, physiology, and behaviour, making them promising targets for RNAi-based neurological insecticides (Veenstra, Reference Veenstra2019; Luo et al., Reference Luo, Lin, Linghu, Gong, Luo, Liu, Jin, Smagghe, Liu and Gui2024). Considering the important roles of neuropeptides in pest management, genes encoding these peptides have been identified and annotated in available genome and transcriptome datasets from diverse insect species, and more than 50 neuropeptide families have been reported to date (Roller et al., Reference Roller, Yamanaka, Watanabe, Daubnerová, Žitňan, Kataoka and Tanaka2008; Li et al., Reference Li, Shi, Lin, Yang and Liu2021; Yan et al., Reference Yan, Aniruddha, Nachman, Olivier, Davies, Dow and Guy2023). In this study, 45 neuropeptide precursor genes were annotated in the P. xylostella genome through orthologous comparisons and classified into 33 neuropeptide gene families. Besides, the comprehensive identification and annotation of neuropeptides revealed remarkable structural and functional diversity in P. xylostella, providing a foundation for future functional studies in this important pest (Hou et al., Reference Hou, Jiang, Yang, Wang and Kang2015; Yang et al., Reference Yang, Wang, Deng, Xiao, Ma, Huang and Lu2024).
AKH is an insect-specific neuropeptide that plays a key role in energy metabolism in arthropods by promoting the mobilisation and release of lipids, carbohydrates, and amino acids (Alves-Bezerra et al., Reference Alves-Bezerra, De Paula, Medina, Silva-oliveira, Medeiros, Gäde and Gondim2016; Jiang et al., Reference Jiang, Marco, Scheich, He, Wang, Gaede and McMahon2023). Previous studies have reported that AKH isoforms exhibited inter-specific variation among insects, such as L. migratoria possessing four AKH isoforms, B. mori has three subtypes, Manduca sexta and Glossina morsitans exhibiting two isoforms, while only a single AKH has been identified in B. tabaci, N. lugens, and D. melanogaster (Lu et al., Reference Lu, Zhang, Chen, Li, Li, Cheng, Zhou, You and Zhou2018; Gäde et al., Reference Gäde, Šimek and Marco2019; Christie, Reference Christie2020; Li et al., Reference Li, Shi, Lin, Yang and Liu2021). In the current study, two PxAKH isoforms (PxAKH1 and PxAKH2) were identified in P. xylostella. The subsequent sequence analysis revealed notable evolutionary conservation of AKH proteins across different insect orders and families, especially exhibiting high similarity to orthologues in other lepidopteran insects. Meanwhile, structural variation among AKH isoforms has been shown to drive functional divergence, as demonstrated in L. migratoria, where AKH1 primarily regulates lipid metabolism during prolonged flight by mobilising unsaturated fatty acids, AKH2 is associated with carbohydrate metabolism in the initial flight phase, and AKH3 modulates nutrient metabolism during resting states (Tomcala et al., Reference Tomcala, Bartu, Simek and Kodrik2010; Zheng et al., Reference Zheng, Chen, Liu, Song and Zhou2024).
In this study, to elucidate the tissue-specific and developmental stage-dependent expression profiles of PxAKH1 and PxAKH2, qPCR was applied to systematically analyse their transcriptional patterns across various tissues and life stages of P. xylostella. The results suggested that the expression levels of the two PxAKH isoforms were both elevated in the third- and fourth-instar larval stages of P. xylostella. We found that this increase coincides with the feeding frenzy stage characterised by rapid larval growth during these developmental stages. Similarly, previous studies in some other insects such as Helicoverpa armigera and G. morsitans have reported the specific high expression of AKH genes during the final instar larvae, followed by a decrease in transcript levels during the pupal stage (Caers et al., Reference Caers, Janssen, Van Rompay, Broeckx, Van Den Abbeele, Gäde, Schoofs and Beets2016; Xiao et al., Reference Xiao, Gao, Tang, Xiao, Hu, Xie, Zaghloul and Huang2025). In P. xylostella, the last two instar larvae consume large amounts of food to accumulate energy reserves and nutrients for subsequent activities, indicating the observed upregulation in PxAKH expression is closely involved in regulating larval feeding behaviour and energy metabolism (Li et al., Reference Li, Li, Wang, Liu, Dong, Wang and Zhao2025). Meanwhile, AKH expression showed a decreasing trend from the third to the fourth instar, likely reflecting stage-specific physiological demands. The third instar is a rapid growth-initiating stage with highly active energy metabolism, whereas the fourth instar represents a phase of intensive feeding and energy storage in preparation for pupation, resulting in a shift in metabolic regulation. Spatial expression analysis showed that both PxAKH1 and PxAKH2 were predominantly expressed in the fat body of fourth-instar P. xylostella larvae. The insect fat body is a key organ for lipid storage and energy metabolism, making it a primary site of AKH gene expression (Azeez et al., Reference Azeez, Meintjes and Chamunorwa2014; Rovenko et al., Reference Rovenko, Perkhulyn, Gospodaryov, Sanz, Lushchak and Lushchak2015). As reported in previous studies, the AKH signalling system was involved in regulating mobilise and release of energy substrates from this core metabolic tissue to support normal insect development (Alves-Bezerra et al., Reference Alves-Bezerra, De Paula, Medina, Silva-oliveira, Medeiros, Gäde and Gondim2016; Hou et al., Reference Hou, Chen, Jiang, Wei, Gui, Wang and Smagghe2017). Being consistent with other worms (Sehadova et al., Reference Sehadova, Takasu, Zaloudikova, Lin and Zurovec2020), we also found the specific high expression of the PxAKH1 gene in the head of P. xylostella larvae. The head, as the sensory and neural centre of P. xylostella, shows highly expressed PxAKH1, which may be involved in modulating neural signalling to influence larval behaviours and physiological responses, such as feeding behaviour and stimulus responses (Zemanová et al., Reference Zemanová, Stašková and Kodrík2016). Similarly, the high expression of PxAKH2 in the gut of P. xylostella larvae suggests a potential role in nutrient absorption and digestive processes, possibly through AKH-mediated stimulation of digestive enzyme activity in this tissue (Bodláková et al., Reference Bodláková, Jedlicka and Kodrík2017; Li et al., Reference Li, Li, Yang, Li, Wu and Xu2023). However, these proposed functions remain hypothetical and require further experimental validation.
Insect behavioural processes are strongly influenced by physiological states such as food availability and nutritional status, primarily regulated through hormone pathways activated by feeding or starvation (Lebreton et al., Reference Lebreton, Mansourian, Bigarreau and Dekker2016). Here, the larvae subjected to short-term starvation (3 or 6 h) exhibited stronger compensatory feeding behaviour, with their food intake significantly higher than that of the control group in P. xylostella. Consistently, some studies have reported compensatory feeding in insects following starvation periods (Yu et al., Reference Yu, Huang, Ye, Zhang, Wu, Cheng, Jia and Wang2016; Zhao et al., Reference Zhao, Yin, Wang, Shen, Dong and Yan2025). This phenomenon was characterised by significantly reducing fat content that rapidly recovers upon resuming feeding, accompanied by a notable increase in food consumption. These findings demonstrate an adaptive strategy for energy replenishment in food-limited environments. However, when P. xylostella larvae were subjected to prolonged starvation, particularly for 24 hours, and then allowed to resume feeding, their food intake was significantly reduced, suggesting that extended starvation stress may cause physiological damage and impair both feeding capacity (Lee, Reference Lee2017). Moreover, the present study showed that PxAKH1 and PxAKH2 of P. xylostella exhibited distinct dynamic expression patterns under starvation stress, and importantly, we found that PxAKH1 was significantly induced after treatment with starvation for 3 and 6 h, respectively. Numerous studies have shown that the AKH signalling system is essential for lipid mobilisation, with its components’ expression strongly influenced by nutritional status, and that upregulated AKH genes participate in regulating the adaptive response to starvation (Grönke et al., Reference Grönke, Müller, Hirsch, Fellert, Andreou, Haase, Jäckle and Kühnlein2007; Huang et al., Reference Huang, Shi, Deng, Jia, Ding, Hou and Chen2024). According to these previous results, we speculated that the higher abundance of PxAKH1 could trigger the utilisation of stored lipid reserves, which is critical for modulating lipid homeostasis and sustaining energy supply under starvation conditions to meet the energy demands of P. xylostella (Hou et al., Reference Hou, Chen, Jiang, Wei, Gui, Wang and Smagghe2017; Gáliková and Klepsatel, Reference Gáliková and Klepsatel2023). In this study, injection of dsPxAKH1 was used to further investigate the critical functions of the PxAKH1 gene in P. xylostella. As the AKH signalling system is a central regulator of energy homeostasis in insects (Dong et al., Reference Dong, Chen, Xu, Li, Wang, Pan, Zhang, Li, Wang and Li2022), it is not surprising that the dsPxAKH1 injection exerted significant effects on lipid storage and mobilisation of P. xylostella. As expected, the RNAi-mediated silencing of PxAKH1 significantly elevated TAG levels and the density of lipid droplets relative to controls in P. xylostella. Being similar in N. lugens, NlAKH silencing would increase TAG levels, whereas NlAKH peptide injection led to a substantial reduction of TAG levels in the fat body (Lu et al., Reference Lu, Zhang, Chen, Li, Li, Cheng, Zhou, You and Zhou2018). Interestingly, some previous studies suggested that the AKH gene exhibited a strong correlation with food intake regulation in insects, and silencing AKH caused a negative impact on feeding behaviour due to an imbalance of energy metabolism (Huang et al., Reference Huang, Song, Su, Lai, Qin, Tian, Dong and Wang2020; Sanchez et al., Reference Sanchez, Munoz, Chen and Giurfa2021; Kong et al., Reference Kong, Xie, Cao, Li, Zhang, Zou, Xia and Xin2025). Importantly, our RNAi experiment indicated that PxAKH1 is essential for larval feeding, and the observed phenotypic changes may result from disrupted nutrient mobilisation and metabolic transport in P. xylostella following dsPxAKH1 injection (Sajwan et al., Reference Sajwan, Sidorov, Staskova, Zaloudikova, Takasu, Kodrik and Zurovec2015; Gáliková and Klepsatel, Reference Gáliková and Klepsatel2023).
Generally speaking, the current study identified a total of 45 neuropeptide precursors from the P. xylostella genome, consisting of two AKH subtypes (PxAKH1 and PxAKH2). The functional analysis showed the critical roles of PxAKH1 in starvation adaptation, lipid mobilisation, and food intake of P. xylostella. These findings demonstrated that knocking down PxAKH1 disrupted lipid metabolism and significantly suppressed larval food intake in P. xylostella, with the feeding area decreasing from 221.33 ± 9.10 mm2 in control larvae to 123.33 ± 18.80 mm2 in PxAKH1-silenced larvae. However, several limitations should be acknowledged. First, the relatively small sample size (n = 4 biological replicates per group) may limit the statistical power of some analyses and preclude formal verification of normality and homogeneity of variance assumptions. Second, the present study primarily focused on PxAKH1, while the functional characterisation of PxAKH2 remains unexplored, and its physiological role is still to be determined, particularly given its distinct expression pattern in the gut, which may suggest a different or complementary function. Third, the conclusions are mainly based on transcript level changes (qPCR data) without direct measurement of AKH peptide levels in the haemolymph. Fourth, all experiments were conducted under laboratory conditions, and no data under field conditions were included to assess the applicability of the findings for pest management. Finally, although a dsGFP control was included in RNAi experiments, a systematic assessment of potential off-target effects was not performed. Future studies should focus on the field evaluation of dsRNA-based RNAi strategies to assess their practical applicability under natural conditions, as well as the comprehensive functional characterisation of PxAKH2 to better understand its physiological roles. In addition, further experiments measuring AKH peptide levels in the haemolymph and assessing potential off-target effects of RNAi would strengthen the validation of PxAKH1 as an effective target. Together, these efforts will provide deeper insights into AKH-mediated regulatory mechanisms and facilitate the development of more effective and sustainable RNAi-based pest management strategies.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0007485326101254.
Financial support
This study was supported by the National Natural Sciences Foundation (32001906), the Jiangsu Agricultural Science and Technology Innovation Fund (CX(23)3017), and the Natural Science Foundation of Jiangsu Province, China (BK20190901).
Competing interests
The authors declare that they have no conflict of interest in this paper.
