Neuropeptides are pivotal in regulating essential physiological processes such as development, reproduction, circadian rhythm, and feeding in insects. They are predominantly synthesized and released from neurosecretory cells within the central nervous system (CNS) (Nässel and Winther, 2010; Schoofs et al., 2017; Abou El Asrar et al., 2020; Toprak, 2020). Most of the known neuropeptides are oligopeptides or small protein molecules composed of several to tens of amino acids (Schoofs et al., 2017; Abou El Asrar et al., 2020). Biologically active neuropeptides undergo intricate post-translational processing from their precursor forms. Acting as neuromodulators or neurohormones, neuropeptides transmit signals by binding to specific membrane receptors. Most neuropeptide receptors belong to the G protein-coupled receptor family, initiating intracellular cascade reactions upon coupling with G proteins in the cell membrane, ultimately eliciting cellular responses (Audsley and Down, 2015; Rosenbaum et al., 2009). Some neuropeptide receptors are not GPCRs, for example, insulin-like receptor is tyrosine kinase receptors (TKR) (Choi and Bai, 2023). Recently, high-throughput sequencing techniques (transcriptome and genome analysis) and bioinformatics technology has greatly promoted the prediction and identification of insect neuropeptides and their receptors (Tanaka et al., 2014; Wang et al., 2018; Li FF. et al., 2020). Over the past 2 decades, substantial progress has been made in identifying novel neuropeptides and homologous receptors across various insect species of significance in human health, agriculture, and ecological environments. Examples include the discovery of 35 neuropeptides and peptide hormones in Anopheles gambiae (Riehle et al., 2002), 37 neuropeptides in Bombyx mori (Roller et al., 2008), 36 neuropeptides in Apis mellifera (Hummon et al., 2006), and 41 genes encoding neuropeptides and protein hormones in Tribolium castaneum (Li et al., 2008).

The lady beetle, C. septempunctata, is a natural predator of agricultural pests, and it is economically important for controlling aphids, whiteflies, mites, thrips, and lepidopteran pests (Chen et al., 2023). This beneficial insect has been extensively mass-cultured and deployed in greenhouses and farmlands across Europe, Asia, and North America (Yang et al., 2014). In Northern China, C. septempunctata adults enter winter diapause in response to short photoperiods and low temperatures, and our previous work identified the specific conditions that elicit diapause (Wang et al., 2013). Diapause creates challenges and opportunities for biological control. While diapause may interrupt rearing activities and reduce field effectiveness (as diapausing beetles do not feed on pests), regulating diapause can enhance the long-term storage and timely deployment of C. septempunctata. Mastering the mechanism of insect diapause and regulating the diapause of natural enemy insects can promote the shelf-life and effective release of mass-produced predatory ladybeetles. Given the pivotal role of neuropeptide signaling systems in physiological behavioral processes, as well as the high specificity and low target interference of neuropeptide receptors, insect neuropeptides are considered ideal targets for cultivating specific insect regulators. Thus, elucidating the structure and function of neuropeptides and their receptors involved in C. septempunctata diapause is crucial.

In this study, we employed transcriptome sequencing and bioinformatics analysis to identify genes encoding neuropeptide precursors and their receptors in C. septempunctata. Additionally, we conducted structural analysis and predicted the mature peptides of the identified neuropeptide precursors. Neuropeptides were classified into subclasses through phylogenetic analysis. Using qRT-PCR, we examined the expression profiles of partial neuropeptides precursor in five different tissues (head, thorax, fat body, ovary and midgut) of female adults at diapause-inducing/non-diapause inducing condition. The sequences of these identified genes serve as a foundational resource for further exploring the roles of neuropeptides precursor in diapause regulation in C. septempunctata.

Transcriptome sequencing is fundamental to dentification of genes, and identification of neuropeptides and their receptors is the first and foremost step of deep function depth studies in physiological processes. However, detailed information on neuropeptide types and expression in C. septempunctata remains scarce. Therefore, to address this problem, we performed mRNA sequencing analysis of the head in C. septempunctata.

Based on transcriptome analysis, we identified 17 neuropeptides precursor and 12 receptor transcripts encoding 9 neuropeptide receptors from C. septempunctata in this study. The confirmation of these receptor information still requires a lot of work, such as pharmacological characterization was determined with all possible neuropeptides of similar motifs. Most neuropeptide receptors belong to the G protein-coupled receptor family. However, we have noticed that insulin-like receptor isoform XI and insulin-like receptor are not GPCRs but tyrosine kinase receptors (TKR), which includes catalytic domain of insulin receptor-like protein tyrosine kinases, furin-like cysteine rich region, receptor l domain and fibronectin type domain (Choi and Bai, 2023). For some neuropeptide signaling systems, C. septempunctata seems to lack both receptor and neuropeptide precursor, such as SK, Crz, Trissin, Ast and Elevenin. For others, the neuropeptide precursor was not found or is lacking, but its presumed receptor is present (e.g., PDF, ILP, CNMa, etc.). There are several factors that may account for the difference in the number of identified genes of specific functions which has been discussed (Wang et al., 2015; Huang et al., 2021; Li X. et al., 2020). Firstly, the head used as the sequenced samples did not cover complete the individual and all stages of life cycle of C. septempunctata and some genes may not have been expressed in these measured samples. Secondly, some genes are expressed at low levels in head tissue making it challenging to detect their expression quantitatively or they may not be expressed at all in the sampled conditions. Thirdly, due to the lack of strong sequence conservation among some neuropeptide precursors or neuropeptide receptors, their clear orthologs could not be found in C. septempunctata based on homology searches. Lastly, some neuropeptides may indeed be absent in C. septempunctata, a phenomenon observed in various insects. For instance, sex peptide was reported only in the Drosophila genus, while neuroparsin is missing in Drosophila melanogaster and other Drosophila species (Veenstra, 2010).

Generally, neuropeptide precursor undergoes alternative splicing, regulated cleavages and post-translational to produce functionally active mature neuropeptides (Agrawal et al., 2019; Christie, 2017). Multiple sequence alignment of NPF precursors between the C. septempunctata and other insects revealed a conserved C-terminal sequence-RPRYamide. The invertebrate NPF is considered to be a superfamily of NPY in which an amidated tyrosine (Y) residue is replaced with an amidated phenylalanine (F) residue at its C-terminal end (Cui and Zhao, 2020). In insects, most NPFs are consisted of 36–40 amino acids and the sequence of amino acids at C-terminal end is relatively conservative with RPRFamide, which is important to maintain functional activity of NPFs. According to the different C-terminus, insect NPFs can be divided into the following type: the general structure of NPFs is RPRFa, which accounts for more than 80% of known insect NPFs (except drosophila melanogaster NPF: RVRFa (Brown et al., 1999)); Additionally, a unique structure, RGRYa (often GRxRYa or Gx1x2RYa, NPF2 or NPY), has been identified exclusively in Lepidoptera insects with some species exhibiting both RPRFa and RGRYa structures (Wang et al., 2021; Li et al., 2023); Furthermore, a distinct structure known as KARYa has been found only in Hymenoptera insects, such as A. mellifera NPF (Hummon et al., 2006).

NPFs are widely distributed in brain neurons of larvae and adults, as well as the foregut, midgut, and hindgut in most insects (Wang et al., 2021; Li et al., 2023; Johard et al., 2008; Liu et al., 2013; Nässel et al., 2008; Yue et al., 2016). NPF peptides are a group of multifunctional peptides and exert multi-actions in insects. The differential distribution of NPF across various tissues and cells suggests a close relationship between its location and function. For instance, NPF expression in the midgut correlates with feeding behavior, while its expression in the brain (clock neurons, mushroom body) relates to daily circadian rhythm and sleep (Cui and Zhao, 2020). In our study, we observed predominant expression of NPF in the head and midgut, with lower levels in the thorax and fat body of female C. septempunctata adults. Interestingly, throughout the entire stage of diapause induction, NPF expression levels were consistently lower compared to females under non-diapause conditions. In our previous research, we noted that female C. septempunctata exhibit suppressed ovaries and accumulate lipids during prolonged diapause periods, often accompanied by reduced or no feeding (Li FF. et al., 2020; Chen et al., 2023). Given the multifaceted roles of NPF in insects and its expression profile in C. septempunctata, we plan to conduct further investigations into the functions of NPF genes during diapause.

The prothoracicotropic hormone (PTTH) is a brain neurosecretory peptide of insects that stimulates the prothoracic glands (PGs) to secrete ecdysteroid, thereby inducing molting and metamorphosis (Mizoguchi, 2001). It is widely accepted that PTTH is released at specific times during insect development, such as larval molting, larval–pupal transformation and adult development. In addition, Some studies indicate that JH regulates the brain to produce and/or release PTTH. In lepidopterans, JH blocked the PTTH secretion activity of the brain–corpora cardiaca–corpora allata complex (Br–CC–CA) of JHA-treated larvae, thereby indirectly inhibiting the secretion of ecdysteroid that induces the larval–pupal transformation (Rountree and Bollenbacher, 1986; Watson et al., 1987). Our previous research found that the absence of the juvenile hormone (JH) which resulted from the upregulation of two JH degradation genes (CsJHE, CsJHEH), is a critical factor during reproductive diapause in C. septempunctata (Li et al., 2022). It is unknown whether JH regulates the PTTH of C.septenunctata females. Further in-depth research is needed to determine whether PTTH is involved in regulating the reproductive diapause of C. septempunctata females. Pupal diapause of lepidoptera insects is regulated by the major developmental hormones prothoracicotropic hormone (PTTH) and ecdysone, such as Scrobipalpa ocellatella (Ahmadi et al., 2021), Pieris napi (Süess et al., 2022) and Helicoverpa armigera (Wei et al., 2005). There is currently no research on the regulation of diapause by PTTH in coleoptera insects.

Adipokinetic hormone (AKH) produced by the corpora cardiaca is involved in innutrient and energy metabolism, maintaining lipid, carbohydrate levels and female reproduction in insect (Wei et al., 2005; Lee and Park, 2004; Bharucha et al., 2008). In D. melanogaster, decreased AKH reduces trehalose content in hemolymph, glucose homeostasis and low energy metabolism (Lee and Park, 2004; Kim and Rulifson, 2004; Isabel et al., 2005). In B. mori, injection of AKH maturation peptide increases carbohydrate content and lipid levels in hemolymph (Sehadova et al., 2020). Knockdown AKHR suppresses the mobilization of triacylglycerol and trehalose, reduces vitellogenin expression in the fat body, and delays oocyte development in migratory locusts (Zheng et al., 2020). In Bactrocera dorsalis, AKHR silencing causes triglyceride (TG) accumulation and affects courtship activity and fecundity (Hou et al., 2017). In our previous research, CsAKH and CsAKHR are involved in the regulation of lipid accumulation and ovarian development during diapause in C. septempunctata (Shen et al., 2024).

In addition, for the identified neuropeptides and neuropeptide receptors, we conducted evolutionary tree analysis and multiple sequence alignment. Amino acid sequence alignment results showed that CAPA receptor and SP receptor in C. septempunctata respectively had a much longer sequence than their closely related species (Supplementary Figures S8 and S9); SIFamide receptor in C. septempunctata had a shorter sequence than its closely related species (Supplementary Figure S3). In addition, Supplementary Figure S2 showed that tachykinin receptor in Harmonia axyridis had a longer sequence than its closely related species. We infered that this may be caused by alternative splicing. Due to selection pressure or optimization of gene expression, the alternative splicing of precursor mRNA leads to additional exon insertion or intron retention, resulting in the production of various mature mRNA molecules (Koch, 2017). These different mRNA have distinct functional or structural features. So, genes of certain species may have shorter or longer mRNA sequences than homologous genes of other species.

In conclusion, our study employed transcriptome sequencing to identify a comprehensive panel of 17 neuropeptides precursor and 9 neuropeptide receptors genes from the adult head of C. septempunctata. This initial exploration provides a foundational understanding of these genes’ functions. We conducted phylogenetic analyses with sequences of other insect species and examined their gene expression profiles in different tissues including the head, thorax, fat body, ovary and midgut in female. These findings suggest their potential roles in the development and diapause of C. septempunctata. Overall, our research contributes valuable insights into neuropeptides precursor and neuropeptide receptor genes in insect diapause, advancing our understanding and potentially facilitating the mass production and storage of C. septempunctata.