Full-length transcriptome-referenced analysis reveals developmental and olfactory regulatory genes in Dermestes frischii

Full-length transcriptome-referenced analysis reveals developmental and olfactory regulatory genes in Dermestes frischii | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Full-length transcriptome-referenced analysis reveals developmental and olfactory regulatory genes in Dermestes frischii Gengwang Hu, Liangliang Li, Yifei Li, Shipeng Shao, Ruonan Zhang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4206363/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Dermestes frischii Kugelann, 1792 is a storage pest worldwide, and is important for estimating the postmortem interval in forensic entomology. However, because of the lack of transcriptome and genome resources, population genetics and biological control studies on D. frischii have been hindered. Here, single-molecule real-time sequencing and next-generation sequencing were combined to generate the full-length transcriptome of the five developmental stages of D. frischii , namely egg, young larva, mature larva, pupa, and adult. A total of 41665 full-length non-chimeric sequences and 59385 non-redundant transcripts were generated, of which 42756 were annotated in public databases. By comparing the transcripts from adjacent developmental stages, 24376, 11802, 20726, and 13262 differentially expressed genes were identified, respectively. Using the weighted gene co-expression network analysis, gene co-expression modules related to the five developmental stages were constructed and screened, and the genes in these modules subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The expression patterns of the DEGs related to olfaction and insect hormone biosynthesis were also explored. Transcription of most odorant binding proteins was up-regulated in the adult stage, suggesting they are important for foraging in adults. Many genes encoding for the ecdysone-inducible protein were up-regulated in the pupal stage. The results of the qRT-PCR were consistent with the RNA-seq results. This is the first full-length transcriptome sequencing of dermestids, and the data obtained here is vital for understanding the stage-specific development and olfactory system of D. frischii , providing valuable resources for storage pest and forensic research. Dermestes frischii developmental stage RNA-seq SMRT sequencing Transcriptome WGCNA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Introduction Dermestes frischii belongs to Dermestidae of the Coleoptera, and is an important storage pest distributed worldwide (Wilches et al. 2016). The adults and larvae can be found in storage items such as processed meat, animal medicinal materials, and animal specimens, and can directly drill into the wooden structure of grain depots and other wooden products to cause serious harm, or spread over long distances with the transportation of stored products, making them important quarantine pests (Athanassiou et al. 2019). Beneficially, D. frischii is used by museums to remove soft tissues attached to animal bone specimens (Peacock 1993), and this species also has important value in forensic entomology for estimating the postmortem interval (PMI). Dermestes frischii is a holometabolous insect, and goes through four stages of development: egg, larva, pupa, and adult. Previous studies have systematically studied the growth and developmental patterns of this species using morphology, providing important indicators for pest control and PMI estimation (Martín-Vega et al. 2017; Lambiase et al. 2018; Hu et al. 2023). As a storage pest perspective, the differential expression of genes at different developmental stages is the molecular basis for insect growth and development. Therefore, identification and functional analysis of some key genes can help to develop new bioinspired strategies for pest control based on disruption of related metabolic pathways of these genes (Zhang et al. 2021, Gao et al. 2022). For example, the growth and development of insects are mainly controlled by ecdysone and juvenile hormone, and chitin metabolism plays an essential role in insect development but is absent in mammals, making them potential targets for pest control (Li et al. 2022). From a forensic perspective, the differentially expressed gene (DEG) data can be subjected to error analysis, which is a major advantage as evidence in court. The results of blind testing have shown that DEG data is highly repeatable for the estimation of PMI, and that it can improve the estimation accuracy using stages with insignificant morphological changes, such as the post-feeding stage and the pupal stage (Ren et al. 2022). However, at present, most forensically important species, including D. frischii , have not been screened for marker genes of PMI estimation. Compared with other necrophagous insects, such as the Dipteran Calliphoridae, D. frischii arrives at the carcass later, has a longer life cycle, can establish stable populations on the carcasses, and is extremely resistant to drought. This renders it as an indicator for PMI estimation for advanced decayed or even skeletonized carcasses (Magni et al. 2015). Therefore, it is necessary to explore the stage-specific gene expression patterns of D. frischii , to understand the complex molecular regulatory mechanisms behind its developmental phenotypic traits. The olfactory system of an insect is its primary form of communication with the external environment, and plays an important role in behaviors such as foraging, finding mates, and choosing oviposition sites (Altner and Prillinger 1980). The study of repellents based on the insect olfactory system of D. frischii can reduce the economic losses caused by this species (Campbell et al. 2002). From a forensic perspective, the study of the olfactory genes of D. frischii could reveal the molecular mechanism of their perception and response to volatile organic compounds released by carcasses, thereby contributing to the enhancement of PMI estimation. Reference-free transcriptome analysis does not rely on reference genome information, and can be applied to the study of species with no reference or low-quality reference genomes, and provides valuable information for the development of biological research and genomics. At present, the combination of single-molecule real-time (SMRT) sequencing and next-generation sequencing (NGS) is commonly used for reference-free transcriptome analysis. The SMRT sequencing greatly reduces the difficulty of analyzing reference-free transcriptome data, enhances the ability to obtain a complete genome and full-length transcriptome, and is more conducive to in-depth analysis and information mining. More comprehensive annotation information can be obtained by using NGS data to analyze the specificity of transcript expression (Van Dijk et al. 2018). Thus, NGS transcriptome sequencing combined with SMRT sequencing was used to conduct an in-depth analysis of the transcriptional profiles of the five developmental stages of D. frischii , which will provide a more complete insight into the stage-specific development and olfactory system of this species, and ultimately aid storage pest and forensic research. Material and methods Colony establishment and sample collection In July 2021, twenty pairs of D. frischii adults were collected from pig carcasses ( Sus scrofa domestica L.) in Shizuishan City, Ningxia, China (38° 98' N, 106° 52' E), and a population was established in the laboratory. Morphological identification was carried out according to the identification key of Zhang et al. (Zhang et al. 2004), and molecular verification of the identification was done based on the COI gene sequence from the leg muscle tissue of a single adult D. frischii . The gene sequence was uploaded to GenBank (accession number: OQ842293). Adults were placed in a 20×14×8 cm plastic box and raised in a microenvironment incubator set to 25°C, 70% relative humidity, and L12:D12 photoperiod. Dried lean pork was regularly provided as a food source, and corks were used as the pupation substrate. After a stable laboratory population was established, samples from each of the five developmental stages were collected, including eggs (E, the first day after oviposition), young larvae (YL, the first instar), mature larvae (ML, the last instar), pupae (P, the first day after pupation), and adults (A, the first day after eclosion, males and females were 1:1). Total RNA was extracted from three samples from each stage for transcriptome sequencing. Generation of the full-length reference transcriptome for D. frischii Firstly, total RNA extracted from the three samples from each developmental stage was mixed to generate a pool for cDNA library construction. Subsequently, the sequencing of the library on a PacBio RS II platform was performed by Biomarker Technologies Co. Ltd. (Beijing, China). For the PacBio long read processing, the raw subreads were analyzed following the ISO-Seq3 pipeline ( https://github.com/PacificBiosciences/IsoSeq ). The pipeline included three initial steps: generation of circular consensus sequencing (CCS) reads, classification of full-length (FL) reads, and clustering of full-length non-chimeric (FLNC) reads. Polished CCS subreads were generated using CCS v6.2.0 and FL transcripts were identified by the poly(A) tails and the 5' and 3' cDNA primers. Lima v2.1.0 (https://lima.how/) and ISO-Seq3 (https://github.com/PacificBiosciences/IsoSeq) were used to remove the primers and poly(A) tails, respectively. Iterative clustering for error correction was used to obtain high-quality FL consensus sequences. In addition, high-quality FL consensus sequences were classified with the criteria of a post-correction accuracy above 99%. Finally, by removing redundancy using the cd-hit software, Iso-Seq high-quality FL transcripts were obtained for further analysis. Illumina sequencing and data analysis Illumina sequencing was performed on all samples collected from the above five developmental stages, including E, YL, ML, P, and A. The preparation of the gene library and sequencing of the transcriptome was carried out at Biomarker Technologies Co. Ltd. (Beijing, China). The raw reads of all RNA-seq libraries were deposited in the NCBI SRA (BioProject number: PRJNA1067750). For RNA-seq data processing, raw data in fastq format were processed through in-house Perl scripts. Clean data were obtained by removing reads containing adapters, reads containing poly-Ns and low-quality reads from raw data. All the downstream analyses were based on clean data with high quality. Identification of differentially expressed genes (DEGs) In order to ascertain relationships between different samples, principal component analysis (PCA) and Pearson correlation coefficient analysis were performed using the corrplot and factoextra packages in R 3.5.2 (R Core Team 2013, Vienna, Austria). The expression levels of transcripts were quantified by values of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM). The DESeq2 package was used to find significant differences in gene expression and the threshold significance level was set at FDR<0.05 and a log2 ratio of more than one. Gene annotation and functional enrichment analysis The gene functions were annotated based on the following databases: NR (NCBI non-redundant protein sequences); Pfam (Protein family); KOG/COG/eggNOG (Clusters of Orthologous Groups of proteins); Swiss-Prot (a manually annotated and reviewed protein sequence database); KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology). In order to understand the involvement of different regulatory pathways or biological processes, respectively, GO and KEGG enrichment analysis of the DEGs were implemented using the clusterProfiler R package (Yu et al. 2012), and GO terms and KEGG pathways with a p value<0.05 were considered significantly enriched. Weighted gene co-expression network analysis The co-expression network was constructed in the R package weighted gene co-expression network analysis (WGCNA) (Langfelder and Horvath 2008). Network construction and detection were completed using an unsigned TOM Type. Soft thresholding power was selected to make the entire network fit the scale-free topology with a minModuleSize of 30, and a mergeCutHeight of 0.25. Correlation analysis between each module and the different developmental stages was also performed to explore modules that were highly related to the developmental stages of D. frischii . The association between module eigengenes and differentiation traits was measured according to Spearman's correlation coefficient. Also, target genes involved in each module were used for GO and KEGG analyses. RNA-Seq data validation by real-time quantitative PCR To evaluate the relative expression levels of the RNA-Seq results, six pairs of primers were designed using Primer Premier 5.0 (Premier Biosoft Intl., California, USA), for the following genes: cytoplasmic polyadenylation element-binding protein 1 ( CPEB1 ), cell division cycle protein 20 homolog ( CDC20 ), lipase 1-like isoform X2 ( LIX2 ), farnesol dehydrogenase ( FDL ), ADP, ATP carrier protein ( AACP ), and cytochrome c-2 ( CYT C2 ) (Table S1). Samples from the five developmental stages of D. frischii were collected again, and total RNA was extracted from each sample using the RNA Simple Total RNA Kit (TIANGEN Biotech, Beijing, China) following the recommended protocol. Then 1 µg RNA was reverse-transcribed into cDNA using HiScript III RT SuperMix (Vazyme Biotech Co., Ltd., Nanjing, China). The 20 µL qRT-PCR reactions contained 10 µL SYBR Green qPCR mixture (Vazyme Biotech Co., Ltd., Nanjing, China), 1 µL cDNA, 0.4 µL forward and reverse primers and 8.2 µL ddH2O. The reactions were performed on a LightCycler 96 Real-Time PCR System (Roche Inc., Branchburg, NJ, USA) with the following conditions: 95°C for 30s; 95°C for 5s, 60°C for 30s for 40 cycles, and a final step of 95°C for 10s. The relative expressions of the targeted genes were normalized with the endogenous reference gene, ribosomal protein L18 (RPL18) (Table S1). The expression levels of the target genes were calculated using the 2 -ΔΔCt method (Livak and Schmittgen 2001). Results Quality of transcriptomic data Through the PacBio sequencing, we obtained 41.16G data and of the 482586 polished CCS, 86.32% were FLNC. Subsequently, 98456 consensus isoforms, 98431 high-quality isoforms, and 59385 non-redundant transcript sequences were obtained (Table S2). Meanwhile, the Illumina data was of high quality, and the information for each sample is provided in Table S3. Our results showed that 48996 open reading frames were predicted for D. frischii, where the complete open reading frames were 26665. In most organisms, simple sequence repeats are an important molecular marker and 13212 simple sequence repeats were detected in D. frischii . Four computational approaches (CPC/CNCI/CPAT/Pfam) were combined to sort lncRNAs and a total of 15082 lncRNAs were predicted. In addition, 42756 transcripts were annotated using NR, Pfam, KOG/COG/eggNOG, Swiss-Prot, GO, and the KEGG databases. Transcriptome comparison between different developmental stages and sample status validation Principal components analysis of transcriptomic differences between the developmental stages showed five groupings, corresponding to the five stages. Some samples from the YL and ML groups clustered together, and some samples from the P and A groups were clustered together. However, samples from E were more distinct from the other four developmental stages (Fig. 1A). The Pearson correlation coefficient is used as an evaluation of biological repeat correlation. A coefficient of determination, r 2 , that is closer to 1, suggests a stronger correlation between two duplicate samples. The Pearson's correlation coefficient analyses of all the samples verified the consistency of the biological replicates collected at each stage (Fig. 1B). DEG expression analysis Among the 59385 unigenes, DEGs were analyzed in biologically relevant comparisons, namely YL vs. E, ML vs. YL, P vs. ML, and A vs. P (Table S4). The DEGs from the different libraries were plotted in four volcano plots, showing significance on the y-axis and fold change on the x-axis. Fold change (log2FC≥1) and an adjusted p-value (≤0.05) were used as thresholds for significance testing (Fig. 2A). The number of transcripts differentially expressed in the different developmental stages is illustrated as a bar chart (Fig. 2B). A Venn diagram was also created to demonstrate the relationship between the DEGs in all comparison groups (Fig. 2C). Ultimately, 24376, 11802, 20726 and 13262 DEGs were identified for the comparison group YL vs. E, ML vs. YL, P vs. ML and A vs. P, respectively. Data suggests that the egg-young larvae and mature larvae-pupae transitions caused more complex transcriptional changes compared to the other transitions. In addition, all DEGs were mapped to GO terms (Fig. 3; Table S5) and KEGG pathways (Table S6) to explore the biological functions in which they may be involved. We found that the common biological process enriched by up-regulated genes in the YL vs. E group and down-regulated genes in the ML vs. YL group is "cuticle development", and the common biological process of up-regulated genes in the ML vs. YL group and down-regulated genes in the P vs. ML group includes "response to oxygen-containing compound" and "chitin metabolic process". The common GO terms of up-regulated genes in the P vs. ML group and down-regulated genes in the A vs. P group were mainly related to "microtubules and cilium activities", and "cell adhesion". The down-regulated genes in the YL vs. E group were significantly enriched in biological processes such as "DNA replication", and the GO terms enriched in the A vs. P group were mainly related to "energy metabolism activities". Gene co-expression network analysis In the constructed scale-free weighted gene co-expression network, the remaining 36987 genes after filtering were divided into 15 modules, with each color representing a module (Fig. 4A). The WGCNA identified 5 modules significantly associated with the five developmental stages (Pearson correlation r>0.8, p<0.05). To be specific, the turquoise, red, blue, brown and black modules were positively correlated with the egg, young larva, mature larva, pupa and adult stages, respectively (Fig. 4B). Subsequently, we calculated the gene significance and module membership values of all the genes in each module and found that gene significance and module membership were highly positively correlated in the first four modules (cor=0.78~0.95), but not the black module (cor=0.49) (Fig. 5A-E). Functional analysis of five-specific modules ClusterProfiler was used to examine the biological function of the five-specific modules by GO (Table S7) and KEGG pathway analysis (Table S8). The turquoise module was highly associated with the egg stage, where the top 10 biological processes in the GO analysis focused on mitotic regulation, mRNA processing and modification, and microtubule organization (Fig. 6A). The most enriched pathways were "MAPK signaling pathway", "mRNA surveillance pathway", "Hippo signaling pathway", "Wnt signaling pathway" and "RNA polymerase" (Fig. 7A). The red module was correlated with young larvae. The most enriched GO terms in the BP categories were "chitin-based cuticle development", "cuticle development" and "body morphogensis" (Fig. 6B). According to the KEGG plot, the most enriched pathways were "fatty acid degradation", "oxidative phosphorylation", and "phototransduction" (Fig. 7B), which implied that cuticle development, energy metabolism, and phototransduction are important for the young larvae of D. frischii . The blue module was mainly associated with the mature larvae. The top 10 enriched GO terms under the BP category were associated with biosynthesis and metabolism of substances, especially amino acids, amides, and peptides (Fig. 6C). The KEGG pathway results showed that, except for the calcium signaling pathway and apelin signaling pathway, the other pathways were closely related to the biosynthesis and metabolism of amino acids as well as glucose metabolism (Fig. 7C). The brown module was highly correlated with the pupal stage. The significantly enriched biological processes mainly focused on cell adhesion, morphogenesis, and axon development (Fig. 6D). According to the KEGG barplot, the most enriched pathways were "Hippo signaling pathway", "ECM-receptor interaction" and "axon guidance" (Fig. 7D). During the development, many of the pupal tissues and structures of D. frischii are broken down and reorganized into the structures and tissues of the adult and in response to metamorphosis, it requires the involvement of multiple biological functions, such as signal transduction and cell adhesion. The black module was mainly associated with the adult stage. The top five enriched GO terms were mitochondrial related, including "mitochondrial electron transport", "ATP synthesis", "tricarboxylic acid cycle (TCA)", "mitochondrial ATP synthesis coupled proton transport" and "mitochondrial electron transport, ubiquinol to cytochrome c" (Fig. 6E). Consistently, the KEGG significantly enriched pathways were "oxidative phosphorylation", "propanoate metabolism", "TCA cycle", and "amino acid degradation" (Fig. 7E). These results showed that adults need a boost in metabolism to complete activities such as migration, feeding, and mating. Expression of olfaction-related DEGs In order to further explore the relationship between the olfactory system and the developmental stages of D. frischii , the expression profiles of olfaction-related genes were examined in more detail. Communication between individual insects and between insects and their environment relies mainly on the senses of smell and taste, which involves several proteins, including odorant binding protein ( OBP ), chemosensory protein, odorant receptor, olfactory receptor, and odorant-degrading enzymes. According to the DEGs and their annotation information, 11 olfactory receptors, three odorant receptors, 16 OBPs , and 19 chemosensory proteins were found (Fig. 8). The results showed that most olfactory-related genes were down-regulated during egg development. However, the expression of two genes encoding chemosensory proteins ( MS_transcript_28421 and MS_transcript_27306 ) was highly up-regulated in the egg stage and subsequently down-regulated in the larval stages. Specifically, four olfactory receptors were mainly up-regulated in the P and A stages, while the other seven olfactory receptor 142-like genes were mainly up-regulated in the YL and ML stages, and then down-regulated thereafter. The expression patterns of the two odorant receptor 4-like isoform X1 were also similar to those of the seven olfactory receptors. Interestingly, the expression of almost all OBP genes was up-regulated in the A stage. It is hypothesized that OBPs play an important role for the adults of D. frischii in the discovery of carcasses. We also found that most chemosensory protein genes were up-regulated in the YL and ML stages. However, four chemosensory protein genes were up-regulated in the A stage ( MS_transcript_88355 , MS_transcript_28982 , MS_transcript_48953 , and MS_transcript_72021 ) and the expression of chemosensory protein CSP8 was up-regulated in the YL and P stages. Expression of DEGs in insect hormone biosynthesis Juvenile hormone ( JH ) and molt hormone synergistically regulate insect metamorphosis and development. In juvenile hormone biosynthesis, a total of 49 DEGs were significantly (p<0.05) enriched in D. frischii (Fig. 9). These DEGs were mainly down-regulated in the E stage, especially farnesyl diphosphate synthase ( FPPS ), NADP + -dependent farnesol dehydrogenase ( FOHSDR ) and juvenile hormone diol kinase ( JHDK ). The DEGs encoding for FOHSDR were mainly up-regulated from the P to the A stage, while the DEGs encoding aldehyde dehydrogenases ( ALDH ) were mainly up-regulated from the YL to the ML stage. In addition, the expression of the methyl farnesoate epoxidase ( CYP15A1 ) gene was up-regulated in the E and P stages. These three genes encode for key enzymes involved in the biosynthesis of JH III , which is the principal component of juvenile hormone. It is hypothesized that these enzymes play a key role in the precise regulation of juvenile hormone concentrations at different developmental stages of D. frischii . For the degradation process of JH III , the genes encoding juvenile hormone esterase ( JHE ) were up-regulated from the ML to the A stage, and the genes encoding juvenile hormone epoxide hydrolase ( JHEH ) were up-regulated in the ML stage. Both JHE and JHEH are key enzymes responsible or degrading juvenile hormone. This suggests that JHE and JHEH control changes in the concentration of the juvenile hormones by degrading them during the development of D. frischii from mature larvae to adults. In addition, the expression of the JHDK gene was significantly down-regulated in the E stage, and then maintained at a constant expression level, suggesting it promoted the growth and development of eggs by inhibiting the degradation of juvenile hormone. Molting hormone (20-hydroxyecdysone, 20E ) is a typical steroid hormone regulating molting, metamorphosis, and reproduction. Only six DEGs were found in the molting hormone biosynthesis pathway (Fig. 10). The gene encoding for CYP307A1 was up-regulated in the E and P stages, while the gene encoding for CYP315A1 was up-regulated from the ML to the P stage and the gene encoding for CYP314A1 was up-regulated in the P stage. In addition, the enzyme CYP18A1 , which is responsible for the degradation of 20E , was highly expressed in the YL and ML stages. These genes strictly control the change of the 20E titer in D. frischii by regulating the 20E biosynthesis process. Ecdysone receptor ( EcR ) and ultraspiracle ( USP ) are members of the nuclear receptor superfamily and are molecular targets of 20E . When 20E induces the formation of transcription factors such as HR3 , E75 , E78 , Brc , and others by interacting with the heterodimer formed by EcR and USP , it initiates an internal regulatory cascade response that causes insect molting and metamorphosis. Results showed that the gene encoding EcR was up-regulated from the YL to the P stage, while the gene encoding USP was up-regulated from the E to the ML stage. Interestingly, results showed that a large number of DEGs encoding ecdysone-inducible protein (e.g., E74 , E75 , E78 , and E93 ) was markedly up-regulated during the P stage. This suggests that the 20E biosynthesis pathway is active in the P stage, which led to a change in the 20E concentration and the activation of a large number of ecdysone-inducible proteins, thereby regulating the expression of different downstream target genes, thus controlling the eclosion process of D. frischii . Validation of gene expression levels In order to validate the results of RNA-seq, six genes related to cell division, cell cycle regulation, energy metabolism, and JH biosynthesis were selected for qRT-PCR analysis (Fig. 11). The results showed agreement between the RNA-Seq analysis and qRT-PCR results, indicating that the transcriptome analysis was accurate and reliable. Discussion Important biological functions at different developmental stages Through PacBio Iso-Seq and Illumina RNA-seq sequencing, this work, for the first time, provides the full-length transcriptome information and the complete transcriptome data of the five developmental stages of D. frischii , to determine the molecular mechanisms underlying its metamorphic development. DEGs were identified using the p-value and the fold change thresholds of the comparisons of the developmental stages. Then, GO and KEGG analyses were performed to better understand the DEGs relevant to the biological processes and pathways. Meanwhile, in order to further understand the biological functions involved in the development of D. frischii , a weighted gene co-expression network was also constructed. The resulting WGCNA allowed for the identification of stage-specific modules (gene clusters) during development, as did the enrichment analysis in the GO and KEGG annotation (Fig. 12). In the turquoise module, which was positively correlated with the egg stage, 46, 47, and 100 genes were enriched to the biological processes of "mitotic spindle organization", "microtubule cytoskeleton organization involved in mitosis" and "cell division", respectively. Among the down-regulated DEGs in the YL vs. E comparison group, 72, 93, and 87 genes were enriched to the biological processes of "DNA replication", "DNA integration" and "cell division", respectively. After the female D. frischii lays eggs, the continuous development of the embryo involves numerous cell cycles. The cell cycle process can be divided into two phases, interphase and mitosis, where interphase can be further divided into pre-DNA synthesis, DNA synthesis and post-DNA synthesis. The cytoskeleton comprising microtubules, microfilaments, and intermediate fibers is the internal structure that supports and maintains the morphology and structure of the cell. Microtubules are fibrous structures composed of tubulin proteins that form a network to support and guide the movement of materials inside the cell. Thus, cell division, DNA replication, and microtubule cytoskeleton organization are important biological processes for the developing egg, which ensures the normal morphogenesis and development of the embryo. In addition, the common enrichment pathways between the turquoise module and the down-regulated genes of the YL vs. E comparison group included the "MAPK signaling pathway", the "mRNA surveillance pathway", the "Wnt signaling pathway", and the "Hippo signaling pathway". The MAPK signaling pathway plays a pivotal role in the development of metazoans, by controlling cell proliferation and cell differentiation through receptor tyrosine kinases (Baril et al. 2014). The Hippo signaling pathway is a highly conserved kinase cascade that affects organ size by regulating cell proliferation, survival, and differentiation (Manno 2021). The mRNA surveillance pathway mainly monitors and regulates the translation of mRNA to ensure correct protein synthesis, which is important for accurate gene expression and the normal function of cells. The results from this work showed that these enrichment pathways play a key regulatory role in the growth and development of the egg stage of D. frischii . After hatching from the egg, D. frischii enters the young larvae stage, which was significantly positively correlated with the red module. The GO analysis identified 21 genes enriched in the "chitin-based cuticle development" biological process and among the up-regulated DEGs in the YL vs. E comparison group, a large number of chitin-related genes involved in the biological processes of "chitin-based cuticle development", "chitin metabolic process", "cuticle development" and "chitin catabolic development" was found. Chitin is abundantly present in the insect cuticle and is important for the formation of the larval epidermis during the early stages of development. Especially for D. frischii , a multi-instar species with 4-11 molts, frequent molting means a significant demand for chitin synthesis. Chitin can provide physical support, prevent desiccation, protect from physical and chemical damages, and defend against pathogens. On the other hand, chitin is also an important signaling molecule in insects, and is involved in regulating the growth and development of larvae. In addition, the top three enriched pathways involved in the red module were "fatty acid degradation", "oxidative phosphorylation" and "phototransduction". These three pathways were included in the top five pathways enriched for the down-regulated DEGs in the ML vs. YL comparison group, suggesting that oxidative phosphorylation and fatty acid degradation serve energy production in early larval metabolism. Compared to the egg stage, the young larvae require a large amount of energy to support morphogenesis and organ development. At the same time, transition to the larval stage involves autonomous activities, such as feeding and developing vision through phototransduction to continuously perceive a large amount of external environmental information or stimuli to survive. Some pathways enriched in the red module, such as "drug metabolism-cytochrome P450 ", and "metabolism of xenobiotics by cytochrome P450 ", suggest that cytochrome P450 may help protect the larvae from damage by other organisms. The blue module was closely associated with the mature larval stage. The biological functions of the mature larvae focused on metabolism-related processes, especially the biosynthesis and metabolism of amino acids, which are necessary for growth and development. During the development from young larvae to mature adults, the body size of D. frischii increases after each molt and the focus of the biological functions shifts from chitin synthesis and energy metabolism to amino acid synthesis, to prepare for the increased energy consumption in the post-feeding stage, metamorphosis and growth during the pupal stage. Results also showed that immune-related GO terms were dominant in the up-regulated DEGs of the ML vs. YL comparison group and the down-regulated DEGs of the P vs. ML comparison group. These include "leukocyte mediated immunity", "neutrophil activation involved in immune response", and "neutrophil activation". The GO terms related to cellular stress, such as "cellular response to oxygen-containing compound", and "cellular response to chemical stimulus" were also enriched for this phase. Since mature larvae usually live in the carcass where many microorganisms breed, a heightened immune response and increased expression of stress-related genes will ensure their survival in this harsh environment. The brown module was highly associated with the pupal stage, and the top five GO terms enriched for the BP category and the down-regulated DEGs of the A vs. P comparison group were identical, focusing on cell adhesion and morphogenesis. Cell adhesion is the process in which cells attach to neighboring cells through the interaction of specialized molecules on the cell surface and it is speculated that during metamorphosis, D. frischii undergoes extensive cell rearrangement and tissue remodeling through cell adhesion. However, in the up-regulated DEGs of the P vs. ML comparison group, the top GO terms were cilium and microtubule related. Researchers have found that cilium is an important microtubule-based organelle that influences the cell cycle and regulates various cell life activities, such as stem cell maintenance, differentiation, and asymmetric division (Zheng et al. 2016; Patel and Tsiokas 2021). Therefore, cilium dynamics are closely related to cellular processes, such as the cell cycle and differentiation, and it is speculated that the process of complete metamorphosis is mainly mediated by cilium and microtubule activity. The black module was closely associated with the adult stage. The enriched GO terms and KEGG pathways in both the black module and the up-regulated DEGs of the A vs. P comparison group were significantly related to energy metabolism, including "ATP synthesis coupled proton transport", and "TCA cycle". This suggests an increased energy consumption in adult D. frischii . Expression characteristics of olfactory-related genes Insect ecology, to a strong degree, depends on the chemosensory modalities of smell and taste (Walker et al. 2022) and the olfactory system plays an important role in guiding behaviors, including feeding, mating, and oviposition (Al-Jalely et al. 2021). For example, with D. frischii , male adults are first attracted by the odor emitted by decaying carcasses or stored materials. After confirming through olfaction that the environment is suitable for offspring development, male adults will release sex hormones to attract females to mate. Eventually, females, attracted to the pheromones, will mate and lay their eggs on carcasses or storage rich in organic matter. However, little is known about the molecular mechanism of olfactory reception in insects. In Pachyrhinus yasumatsui (Kono and Morimoto 1960)(Coleoptera: Curculionidae), 113 genes were identified to be involved in chemosensory functions (Hong et al. 2023). In Chilo sacchariphagus (Bojer, 1856)(Lepidoptera: Crambidae), a key pest of sugarcane, 72 candidate chemosensory genes were identified from different tissues and genders (Liu et al. 2021). Here, a total of 78 candidate genes were associated with the sense of smell, including 32 olfactory receptors, four odorant receptors, 19 OBPs , and 23 chemosensory proteins. Among them, 49 olfactory genes were differentially expressed during the developmental process of D. frischii . Most olfactory-related genes maintained low expression levels during the egg stage. Subsequently, these genes showed divergent expression patterns as development progressed. Notably, almost all the OBPs were up-regulated at the adult stage. Insect OBPs are a class of small soluble proteins that facilitate the binding and transport of small molecules and can be found in various tissues. In the Japanese pine sawyer Monochamus alternatus Hope, 1842 (Coleoptera: Cerambycidae), an apparent gradient expression pattern of OBP19 was detected. The binding protein was highly and specifically expressed in the antennae and played an essential role in the detection of camphene (Li et al. 2022). Throughout the development of Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae), the transcription of OBP27 steadily increased and the transcripts of this gene were abundant in the fat body and male reproductive organs (Han et al. 2023). Thus, data suggests that the up-regulated expression levels of OBPs in the adult stage ensure a faster detection of the corpse or breeding site. Investigating these olfactory-related genes will help reveal the molecular mechanism of odorant recognition in D. frischii and may aid in the development of novel control strategies for this species. Expression characteristics of insect hormone biosynthesis related DEGs Two major hormones, JH and 20E , regulate insect growth and development according to their precisely coordinated titers, which are controlled by both biosynthesis and degradation pathways (Zhang et al. 2017). The JHs are a family of sesquiterpenoid molecules that are secreted into the hemolymph, and play a key role in preventing larval precocious metamorphosis, maintaining the larval state, controlling adult sexual development, and promoting insect egg maturation (Cai et al. 2022). Enzymes involved in the biosynthesis of JH include FPPS , FOHSDR , ALDH , juvenile hormone acid methyltransferase ( JHAMT ), and CYP15A1 , where JHAMT is a rate-limiting enzyme of JH biosynthesis in insects. It transfers the methyl group of S-adenosyl methionine to either the carboxyl group of JH acids or the farnesoic acid to produce JH (Zhang et al. 2022). In this study, among the 42756 annotated transcripts, we did not find transcripts encoding for JHAMT . Another key enzyme-encoding gene, CYP15A1 , was reported to encode for the cytochrome P450 enzyme responsible for the epoxidation of methyl farnesoate to JH (Marchal et al. 2011). The strong up-regulation of two transcripts encoding for CYP15A1 found here might contribute to JH accumulation in promoting egg maturation. In multicellular organisms, most of the developmental transitions are driven by steroid hormones. Steroid hormone 20E is secreted by prothoracic glands and transported to the target organs via the hemolymph (Lee et al. 2022). Previous studies showed that 20E is actually regulated by six P450 genes (five P450 genes belonging to the Halloween family and a CYP18A1 gene) in model insects (Liu et al. 2020). In Manduca sexta (Linnaeus, 1763) (Lepidoptera: Sphingidae), CYP306A1 , CYP302A1 , and CYP315A1 , which mediate the final hydroxylation in the biosynthesis of ecdysone, were selectively expressed in the prothoracic glands, and changes in their expression correlate with the haemolymph ecdysteroid titer during the fifth (final) larval instar. Transcript levels of CYP314A1 , the 20-hydroxylase, which converts ecdysone into the more active 20E , closely parallels the enzyme activity measured in vitro (Rewitz et al. 2006). CYP18A1 has been shown to play a key role in insect steroid hormone inactivation through 26-hydroxylation (Li et al. 2014). In Bombyx mori (Linnaeus, 1758) (Lepidoptera: Bombycidae), overexpression of CYP18A1 resulted in developmental arrest during the final instar larval stage. Also, the 20E titers in the transgenic B. mori expressing CYP18A1 were lower compared to the levels in the control group (Li et al. 2014). In this study, the expression of CYP315A1 and CYP314A1 were up-regulated and the expression of CYP18A1 was down-regulated in the pupal stage, suggesting that these genes were transcriptionally regulated to support the high 20E biosynthesis activity that produces the ecdysteroid pulses triggering the pupation of D. frischii . A heterodimeric complex of two nuclear receptors, EcR and USP , transduces 20E signaling to modulate insect growth and development (Yu et al. 2023). In Apis mellifera Linnaeus (1758) (Hymenoptera: Apidae), 20E induced EcR expression and RNAi knockdown of the EcR gene lead to a delay in larval transition to the pupal stage. In the presence of 20E , EcR can bind to USP and increase the expression of 20E -inducible genes for pupal-adult development (Song et al. 2023). These early ecdysone-inducible genes include E74 , E75 , E93 , and HR3 . For example, E74 is a key transcription factor induced by 20E , which plays a role in many physiological events during insect growth and development, including vitellogenesis, organ remodeling, and new tissue formation, as well as programmed cell death and metamorphosis (Zhang et al. 2022). More specifically, E74 is encoded within the 74EF early puff and consists of two overlapping transcription units, E74A and E74B . In Drosophila , mutations in E74A and E74B are lethal during prepupal and pupal development, which is consistent with the critical role their gene products play in metamorphosis (Fletcher et al. 1995). In Sitobion avenae (Fabricius, 1775) (Hemiptera: Aphididae), as a major component of the insect ecdysone signaling pathway, the expression of ecdysone-inducible E75 was low in the adult stage, but high in the pseudo embryo and nymphal stages (Zheng et al. 2023). In this study, we speculate that the strong up-regulation of these ecdysone-inducible genes ( E74 , E75 , E78 , and E93 ) is because of a rapidly increasing 20E pulse, leading to undergoing extensive metamorphic remodeling in the pupae. In summary, D. frischii exhibits unique and important biological functions at all developmental stages. In the egg stage, the biological functions of D. frischii mainly focus on cell division, DNA replication, and microtubule-related processes. In the young larvae, the biological functions of D. frischii mainly include cuticle development, energy metabolism, and phototransduction. In the mature larval stage, the main biological functions are transformed into amino acid synthesis and metabolism, immunity, and stress. In the pupal stage, the biological functions of D. frischii were mainly focused on cilium assembly and movement, cell adhesion, and morphogenesis. In the adult stage, its main biological function is mainly related to energy metabolism. Transcription of most odorant binding proteins was up-regulated in the adult stage, and many genes encoding for the ecdysone-inducible protein were up-regulated in the pupal stage. Declarations Acknowledgment This work was supported by the National Natural Science Foundation of China [grant number 31872258, 32070508, and 82002007] and Priority Academic Program Development of Jiangsu Higher Education. Funding This work was supported by the National Natural Science Foundation of China [grant number 31872258, 32070508, and 82002007] and Priority Academic Program Development of Jiangsu Higher Education. Competing Interests The authors declare no conflict of interest. Authors’ contributions GH and ZG conceived experiments and wrote manuscript. YW (Yu Wang) designed experiments. LL, YL, SS, RZ, YG (Yundi Gao), YG (Yi Guo) and YW (Yinghui Wang) conducted experiments. GH and ZG analyzed data. All authors read and approved the manuscript. 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Supplementary Files Supplementaryflies.zip Supplementary Information Table S1 Primers used for quantitative Real-Time PCR in this study Table S2-S3 Statistical summary of the PacBio Iso-Seq and Illumina RNA-seq sequencing Table S4 Differentially expressed genes in different comparison groups Table S5 GO enrichment analysis of DEGs in different comparison groups Table S6 KEGG enrichment pathway analysis of DEGs in different comparison groups Table S7 GO enrichment analysis of genes in different stages-specfic modules Table S8 KEGG enriched pathway analysis of genes in different stages-specific modules Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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(A) Volcano Plot representation of DEGs across developmental stages. The fold-change and p-value are shown for the comparisons across developmental stages: YL vs. E; ML vs. YL; P vs. ML; A vs. P. Up-regulated genes are shown in red and down-regulated genes are shown in green. (B) Bar graph of up- and down-regulated genes from pairwise comparisons. (C) Venn diagram shows the relationship between DEGs among all comparison\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4206363/v1/dc7ab05b72d6b0da525e5129.png"},{"id":54356410,"identity":"a886776e-0131-4dba-a1df-1a50b0dab2bb","added_by":"auto","created_at":"2024-04-09 09:59:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":168388,"visible":true,"origin":"","legend":"\u003cp\u003eBiological process enrichment analysis of up-regulated and down-regulated DEGs. GO terms of up-regulated (A) and down-regulated (B) DEGs identified in the comparison group YL vs. E; GO terms of up-regulated (C) and down-regulated (D) DEGs identified in the comparison group ML vs. YL; GO terms of up-regulated (E) and down-regulated (F) DEGs identified in the comparison group P vs. ML; GO terms of up-regulated (G) and down-regulated (H) DEGs identified in the comparison group A vs. P\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4206363/v1/b109796bf185e491b25e7004.png"},{"id":54356408,"identity":"eff170ec-d161-4cef-83ac-3dc6a64533f9","added_by":"auto","created_at":"2024-04-09 09:59:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":112186,"visible":true,"origin":"","legend":"\u003cp\u003eScale-free weighted gene co-expression network construction analysis for \u003cem\u003eDermestes frischii\u003c/em\u003e. (A) All genes of the network were divided into 15 modules, and each color represents a module; (B) Heatmap showing the correlation between modules and developmental stages. Each row corresponds to a module and each column corresponds to a developmental stage. A high degree of positive correlation (close to 1) is indicated by dark red, and a high degree of negative correlation (close to -1) is indicated by dark blue\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4206363/v1/b91c351d9e7add5e01cb326b.png"},{"id":54356409,"identity":"7c11e845-5644-4f87-8315-da4330e2a7a6","added_by":"auto","created_at":"2024-04-09 09:59:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":178516,"visible":true,"origin":"","legend":"\u003cp\u003eThe relevance of members in the modules and stages of the scale-free weighted gene co-expression network construction analysis for \u003cem\u003eDermestes frischii\u003c/em\u003e. (A-E) Gene-trait significance vs. module membership scatterplots for \u003cem\u003eDermestes frischii\u003c/em\u003e stage-associated modules, along with correlation and p values. The stage-associated modules are black, turquoise, blue, brown, and red, respectively\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4206363/v1/e94dd75a45a9881284f31ed3.png"},{"id":54356413,"identity":"0adbde19-b90e-4f97-8f67-f846d03ec3bc","added_by":"auto","created_at":"2024-04-09 09:59:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":168091,"visible":true,"origin":"","legend":"\u003cp\u003eGO enrichment analysis of \u003cem\u003eDermestes frischii\u003c/em\u003e genes in stage-specific modules. (A) Enriched biological processes in egg-related module (MEturquoise); (B) Enriched biological processes in young larvae-related module (MEred); (C) Enriched biological processes in mature larvae-related module (MEblue); (D) Enriched biological processes in pupae-related module (MEbrown); (E) Enriched biological processes in adult-related module (MEblack)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4206363/v1/7d124eec2e06bd2da7e1d403.png"},{"id":54357216,"identity":"243c5625-5408-494d-b325-2a60ea23e0fc","added_by":"auto","created_at":"2024-04-09 10:07:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":173133,"visible":true,"origin":"","legend":"\u003cp\u003eEnriched KEGG pathways in stage-specific modules \u003cem\u003eDermestes frischii\u003c/em\u003e. (A) Enriched pathways in egg-related module (MEturquoise); (B) Enriched pathways in young larvae-related module (MEred); (C) Enriched pathways in mature larvae-related module (MEblue); (D) Enriched pathways in pupae-related module (MEbrown); (E) Enriched pathways in adult-related module (MEblack)\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4206363/v1/e16c0ec3bfc1a613b529af8d.png"},{"id":54356412,"identity":"60bf6a31-eb86-40d0-a04e-b37baa1d1f79","added_by":"auto","created_at":"2024-04-09 09:59:35","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":179906,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of olfaction-related DEGs in the different developmental stages of \u003cem\u003eDermestes frischii\u003c/em\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4206363/v1/d3b12f0f3e1045f7ce2f3120.png"},{"id":54356419,"identity":"96afbc11-fe5d-4b92-8d4b-e57c18cc6c3a","added_by":"auto","created_at":"2024-04-09 09:59:36","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":141100,"visible":true,"origin":"","legend":"\u003cp\u003eExpression patterns of DEGs related to the juvenile hormone biosynthesis pathway of \u003cem\u003eDermestes frischii\u003c/em\u003e. FPPP, farnesyl diphosphate synthase; FOHSDR, NADP+-dependent farnesol dehydrogenase; ALDH, aldehyde dehydrogenase (NAD+); JHAMT, juvenile hormone III synthase; CYP15A1, methyl farnesoate epoxidase; JHE, juvenile hormone esterase; JHEH, juvenile hormone epoxide hydrolase; JHDK, juvenile hormone diol kinase\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4206363/v1/2cbf1e93fb00ee07a4b2fb70.png"},{"id":54356416,"identity":"5ca38528-8e54-4669-becf-145bf8e8e313","added_by":"auto","created_at":"2024-04-09 09:59:35","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":121968,"visible":true,"origin":"","legend":"\u003cp\u003eExpression patterns of DEGs related to the molting hormone biosynthesis pathway of \u003cem\u003eDermestes frischii\u003c/em\u003e. NVD, cholesterol 7-desaturase; CYP307A1, cytochrome P450 family 307 subfamily A; CYP306A1, ecdysteroid 25-hydroxylase; CYP302A1, ecdysteroid 22-hydroxylase; CYP315A1, ecdysteroid 2-hydroxylase; CYP314A1, ecdysone 20-monooxygenase; EO, ecdysone oxidase; CYP18A1, 26-hydroxylase; EcR, ecdysone receptor; USP, ultraspiracle; E74, ecdysone-induced protein 74EF; E75, ecdysone-inducible protein E75; E78, ecdysone-induced protein 78C; E93, ecdysone-induced protein 93F\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-4206363/v1/998095386c73111d42b7a1f1.png"},{"id":54356418,"identity":"17a13a6a-6b54-4c42-89b1-69c1a80bfb22","added_by":"auto","created_at":"2024-04-09 09:59:35","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":78698,"visible":true,"origin":"","legend":"\u003cp\u003eExpression level validation of six DEGs of \u003cem\u003eD. frischii\u003c/em\u003e using quantitative real-time PCR (qRT-PCR). Expression levels of cytoplasmic polyadenylation element-binding protein 1 (CPEB1), cell division cycle protein 20 homolog (CDC20), lipase 1-like isoform X2 (LIX2), farnesol dehydrogenase (FDL), ADP, ATP carrier protein (AACP), cytochrome c-2 (CYT C2) in the different developmental stages. Error bars represent the means ± standard deviations from three replicates\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-4206363/v1/c9c615189f5fb3c7c8d01f94.png"},{"id":54356417,"identity":"ac2ba3f5-9c68-42b7-b281-b39a0b66325e","added_by":"auto","created_at":"2024-04-09 09:59:35","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":131673,"visible":true,"origin":"","legend":"\u003cp\u003eGO and KEGG pathway analysis of the main biological functions of \u003cem\u003eDermestes frischii\u003c/em\u003ein its five developmental stages\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-4206363/v1/75ab731b70cc8f9faaa03006.png"},{"id":57625945,"identity":"4f75f4fa-e3c2-48c5-973e-588d2138dae6","added_by":"auto","created_at":"2024-06-03 14:01:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2128819,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4206363/v1/c3bd4eeb-98cb-4c10-945f-193479ef3c5c.pdf"},{"id":54356414,"identity":"9c9c0681-cc64-4be9-b7a9-97177d08031a","added_by":"auto","created_at":"2024-04-09 09:59:35","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14249508,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S1\u003c/strong\u003e Primers used for quantitative Real-Time PCR in this study\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S2-S3 \u003c/strong\u003eStatistical summary of the PacBio Iso-Seq and Illumina RNA-seq sequencing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S4\u003c/strong\u003e Differentially expressed genes in different comparison groups\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S5\u003c/strong\u003e GO enrichment analysis of DEGs in different comparison groups\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S6\u003c/strong\u003e KEGG enrichment pathway analysis of DEGs in different comparison groups\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S7\u003c/strong\u003e GO enrichment analysis of genes in different stages-specfic modules\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S8\u003c/strong\u003e KEGG enriched pathway analysis of genes in different stages-specific modules\u003c/p\u003e","description":"","filename":"Supplementaryflies.zip","url":"https://assets-eu.researchsquare.com/files/rs-4206363/v1/2569f4533fabcfdad003f692.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Full-length transcriptome-referenced analysis reveals developmental and olfactory regulatory genes in Dermestes frischii","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eDermestes frischii\u003c/em\u003e belongs to Dermestidae of the Coleoptera, and is an important storage pest distributed worldwide\u0026nbsp;(Wilches et al. 2016). The adults and larvae can be found in storage items such as processed meat, animal medicinal materials, and animal specimens, and can directly drill into the wooden structure of grain depots and other wooden products to cause serious harm, or spread over long distances with the transportation of stored products, making them important quarantine pests\u0026nbsp;(Athanassiou et al. 2019). Beneficially, \u003cem\u003eD. frischii\u003c/em\u003e is used by museums to remove soft tissues attached to animal bone specimens\u0026nbsp;(Peacock 1993), and this species also has important value in forensic entomology for estimating the postmortem interval (PMI).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDermestes frischii\u003c/em\u003e is a holometabolous insect, and goes through four stages of development: egg, larva, pupa, and adult. Previous studies have systematically studied the growth and developmental patterns of this species using morphology, providing important indicators for pest control and PMI estimation\u0026nbsp;(Martín-Vega et al. 2017; Lambiase et al. 2018; Hu et al. 2023). As a storage pest perspective, the differential expression of genes at different developmental stages is the molecular basis for insect growth and development. Therefore, identification and functional analysis of some key genes can help to develop new bioinspired strategies for pest control based on disruption of related metabolic pathways of these genes\u0026nbsp;(Zhang et al. 2021, Gao et al. 2022). For example, the growth and development of insects are mainly controlled by ecdysone and juvenile hormone, and chitin metabolism plays an essential role in insect development but is absent in mammals, making them potential targets for pest control\u0026nbsp;(Li et al. 2022).\u003c/p\u003e\n\u003cp\u003eFrom a forensic perspective, the differentially expressed gene (DEG) data can be subjected to error analysis, which is a major advantage as evidence in court. The results of blind testing have shown that DEG data is highly repeatable for the estimation of PMI, and that it can improve the estimation accuracy using stages with insignificant morphological changes, such as the post-feeding stage and the pupal stage\u0026nbsp;(Ren et al. 2022). However, at present, most forensically important species, including \u003cem\u003eD. frischii\u003c/em\u003e, have not been screened for marker genes of PMI estimation. Compared with other\u0026nbsp;necrophagous\u0026nbsp;insects, such as the Dipteran Calliphoridae, \u003cem\u003eD. frischii\u003c/em\u003e arrives at the carcass later, has a longer life cycle, can establish stable populations on the carcasses, and is extremely resistant to drought. This renders it as an indicator for PMI estimation for advanced decayed or even skeletonized carcasses\u0026nbsp;(Magni et al. 2015). Therefore, it is necessary to explore the stage-specific gene expression patterns of \u003cem\u003eD. frischii\u003c/em\u003e, to understand the complex molecular regulatory mechanisms behind its developmental phenotypic traits.\u003c/p\u003e\n\u003cp\u003eThe olfactory system of an insect is its primary form of communication with the external environment, and plays an important role in behaviors such as foraging, finding mates, and choosing oviposition sites\u0026nbsp;(Altner and Prillinger 1980). The study of repellents based on the insect olfactory system of \u003cem\u003eD. frischii\u003c/em\u003e can reduce the economic losses caused by this species\u0026nbsp;(Campbell et al. 2002). From a forensic perspective, the study of the olfactory genes of \u003cem\u003eD. frischii\u003c/em\u003e could reveal the molecular mechanism of their perception and response to volatile organic compounds released by carcasses, thereby contributing to the enhancement of PMI estimation.\u003c/p\u003e\n\u003cp\u003eReference-free transcriptome analysis does not rely on reference genome information, and can be applied to the study of species with no reference or low-quality reference genomes, and provides valuable information for the development of biological research and genomics. At present, the combination of single-molecule real-time (SMRT) sequencing and next-generation sequencing (NGS) is commonly used for reference-free transcriptome analysis. The SMRT sequencing greatly reduces the difficulty of analyzing reference-free transcriptome data, enhances the ability to obtain a complete genome and full-length transcriptome, and is more conducive to in-depth analysis and information mining. More comprehensive annotation information can be obtained by using NGS data to analyze the specificity of transcript expression\u0026nbsp;(Van Dijk et al. 2018). Thus, NGS transcriptome sequencing combined with SMRT sequencing was used to conduct an in-depth analysis of the transcriptional profiles of the five developmental stages of \u003cem\u003eD. frischii\u003c/em\u003e, which will provide a more complete insight into the stage-specific development and olfactory system of this species, and ultimately aid storage pest and forensic research.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003eColony establishment and sample collection\u003c/p\u003e\n\u003cp\u003eIn July 2021, twenty pairs of \u003cem\u003eD. frischii\u003c/em\u003e adults were collected from pig carcasses (\u003cem\u003eSus scrofa domestica\u003c/em\u003e L.) in Shizuishan City, Ningxia, China (38° 98' N, 106° 52' E), and a population was established in the laboratory. Morphological identification was carried out according to the identification key of Zhang et al.\u0026nbsp;(Zhang et al. 2004), and molecular verification of the identification was done based on the COI gene sequence from the leg muscle tissue of a single adult \u003cem\u003eD. frischii\u003c/em\u003e. The gene sequence was uploaded to GenBank (accession number: OQ842293). Adults were placed in a 20×14×8 cm plastic box and raised in a microenvironment incubator set to 25°C, 70% relative humidity, and L12:D12 photoperiod. Dried lean pork was regularly provided as a food source, and corks were used as the pupation substrate.\u003c/p\u003e\n\u003cp\u003eAfter a stable laboratory population was established, samples from each of the five developmental stages were collected, including eggs (E, the first day after oviposition), young larvae (YL, the first instar), mature larvae (ML, the last instar), pupae (P, the first day after pupation), and adults (A, the first day after eclosion, males and females were 1:1). Total RNA was extracted from three samples from each stage for transcriptome sequencing.\u003c/p\u003e\n\u003cp\u003eGeneration of the full-length reference transcriptome for D. frischii\u003c/p\u003e\n\u003cp\u003eFirstly, total RNA extracted from the three samples from each developmental stage was mixed to generate a\u0026nbsp;pool\u0026nbsp;for cDNA library construction. Subsequently, the sequencing of the library on a PacBio RS II platform was performed by Biomarker Technologies Co. Ltd. (Beijing, China).\u003c/p\u003e\n\u003cp\u003eFor the PacBio long read processing, the raw subreads were analyzed following the ISO-Seq3 pipeline\u0026nbsp;(\u003ca href=\"https://github.com/PacificBiosciences/IsoSeq\" target=\"_blank\"\u003ehttps://github.com/PacificBiosciences/IsoSeq\u003c/a\u003e). The pipeline included three initial steps: generation of circular consensus sequencing (CCS) reads, classification of full-length (FL) reads, and clustering of full-length non-chimeric (FLNC) reads. Polished CCS subreads were generated using CCS v6.2.0 and FL transcripts were identified by the poly(A) tails and the 5' and 3' cDNA primers. Lima v2.1.0 (https://lima.how/) and ISO-Seq3 (https://github.com/PacificBiosciences/IsoSeq) were used to remove the primers and poly(A) tails, respectively. Iterative clustering for error correction was used to obtain high-quality FL consensus sequences. In addition, high-quality FL consensus sequences were classified with the criteria of a post-correction accuracy above 99%. Finally, by removing redundancy using the cd-hit software, Iso-Seq high-quality FL transcripts were obtained for further analysis.\u003c/p\u003e\n\u003cp\u003eIllumina sequencing and data analysis\u003c/p\u003e\n\u003cp\u003eIllumina sequencing was performed on all samples collected from the above five developmental stages, including E, YL, ML, P, and A. The preparation of the gene library and sequencing of the transcriptome was carried out at Biomarker Technologies Co. Ltd. (Beijing, China).\u0026nbsp;The raw reads of all RNA-seq libraries were deposited in the NCBI SRA (BioProject number: PRJNA1067750).\u003c/p\u003e\n\u003cp\u003eFor RNA-seq data processing, raw data in fastq format were processed through in-house Perl scripts. Clean data were obtained by removing reads containing adapters, reads containing poly-Ns and low-quality reads from raw data. All the downstream analyses were based on clean data with high quality.\u003c/p\u003e\n\u003cp\u003eIdentification of differentially expressed genes (DEGs)\u003c/p\u003e\n\u003cp\u003eIn order to ascertain relationships between different samples, principal component analysis (PCA) and Pearson correlation coefficient analysis were performed using the corrplot and factoextra packages in R 3.5.2 (R Core Team 2013, Vienna, Austria).\u003c/p\u003e\n\u003cp\u003eThe expression levels of transcripts were quantified by values of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM). The DESeq2 package was used to find significant differences in gene expression and the threshold significance level was set at FDR\u0026lt;0.05 and a log2 ratio of more than one.\u003c/p\u003e\n\u003cp\u003eGene annotation and functional enrichment analysis\u003c/p\u003e\n\u003cp\u003eThe gene functions were annotated based on the following databases: NR (NCBI non-redundant protein sequences); Pfam (Protein family); KOG/COG/eggNOG (Clusters of Orthologous Groups of proteins); Swiss-Prot (a manually annotated and reviewed protein sequence database); KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology).\u003c/p\u003e\n\u003cp\u003eIn order to understand the involvement of different regulatory pathways or biological processes, respectively, GO and KEGG enrichment analysis of the DEGs were implemented using the clusterProfiler R package\u0026nbsp;(Yu et al. 2012), and GO terms and KEGG pathways with a p value\u0026lt;0.05 were considered significantly enriched.\u003c/p\u003e\n\u003cp\u003eWeighted gene co-expression network analysis\u003c/p\u003e\n\u003cp\u003eThe co-expression network was constructed in the R package weighted gene co-expression network analysis (WGCNA)\u0026nbsp;(Langfelder and Horvath 2008). Network construction and detection were completed using an unsigned TOM Type. Soft thresholding power was selected to make the entire network fit the scale-free topology with a minModuleSize of 30, and a mergeCutHeight of 0.25. Correlation analysis between each module and the different developmental stages was also performed to explore modules that were highly related to the developmental stages of \u003cem\u003eD. frischii\u003c/em\u003e. The association between module eigengenes and differentiation traits was measured according to Spearman's correlation coefficient. Also, target genes involved in each module were used for GO and KEGG analyses.\u003c/p\u003e\n\u003cp\u003eRNA-Seq data validation by real-time quantitative PCR\u003c/p\u003e\n\u003cp\u003eTo evaluate the relative expression levels of the\u0026nbsp;RNA-Seq results,\u0026nbsp;six pairs of primers were designed using Primer Premier 5.0 (Premier Biosoft Intl., California, USA), for the following genes: cytoplasmic polyadenylation element-binding protein 1 (\u003cem\u003eCPEB1\u003c/em\u003e), cell division cycle protein 20 homolog (\u003cem\u003eCDC20\u003c/em\u003e), lipase 1-like isoform X2 (\u003cem\u003eLIX2\u003c/em\u003e), farnesol dehydrogenase (\u003cem\u003eFDL\u003c/em\u003e), ADP, ATP carrier protein (\u003cem\u003eAACP\u003c/em\u003e), and cytochrome c-2 (\u003cem\u003eCYT C2\u003c/em\u003e) (Table S1).\u003c/p\u003e\n\u003cp\u003eSamples from the five developmental stages of\u0026nbsp;\u003cem\u003eD. frischii\u003c/em\u003e were collected again,\u0026nbsp;and total RNA was extracted from\u0026nbsp;each sample\u0026nbsp;using the RNA Simple Total RNA Kit (TIANGEN Biotech, Beijing, China) following the recommended protocol.\u0026nbsp;Then 1 µg RNA was reverse-transcribed into cDNA using HiScript III RT SuperMix (Vazyme Biotech Co., Ltd., Nanjing, China).\u0026nbsp;The\u0026nbsp;20 µL\u0026nbsp;qRT-PCR reactions contained\u0026nbsp;10 µL SYBR Green qPCR mixture (Vazyme Biotech Co., Ltd., Nanjing, China), 1 µL cDNA, 0.4 µL forward and reverse primers and 8.2 µL ddH2O. The reactions were performed on a LightCycler 96\u0026nbsp;Real-Time\u0026nbsp;PCR System (Roche Inc., Branchburg, NJ, USA) with the following conditions: 95°C\u0026nbsp;for 30s; 95°C\u0026nbsp;for 5s, 60°C\u0026nbsp;for 30s for 40 cycles, and a final step of 95°C\u0026nbsp;for 10s. The relative expressions of the targeted genes were normalized with the endogenous reference gene, ribosomal protein L18 (RPL18) (Table S1).\u0026nbsp;The expression levels of the target genes were calculated using the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method\u0026nbsp;(Livak and Schmittgen 2001).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eQuality of transcriptomic data\u003c/p\u003e\n\u003cp\u003eThrough the PacBio sequencing, we obtained 41.16G data and of the 482586 polished CCS, 86.32% were FLNC. Subsequently, 98456 consensus isoforms, 98431 high-quality isoforms, and 59385 non-redundant transcript sequences were obtained (Table S2). Meanwhile, the Illumina data was of high quality, and the information for each sample is provided in Table S3.\u003c/p\u003e\n\u003cp\u003eOur results showed that 48996 open reading frames were predicted for \u003cem\u003eD. frischii,\u0026nbsp;\u003c/em\u003ewhere the complete open reading frames were 26665. In most organisms, simple sequence repeats are an important molecular marker and 13212 simple sequence repeats were detected in \u003cem\u003eD. frischii\u003c/em\u003e. Four computational approaches (CPC/CNCI/CPAT/Pfam) were combined to sort lncRNAs and a total of 15082 lncRNAs were predicted. In addition, 42756 transcripts were annotated using NR, Pfam, KOG/COG/eggNOG, Swiss-Prot, GO, and the KEGG databases.\u003c/p\u003e\n\u003cp\u003eTranscriptome comparison between different developmental stages and sample status validation\u003c/p\u003e\n\u003cp\u003ePrincipal components analysis of transcriptomic differences between the developmental stages showed five groupings, corresponding to the five stages. Some samples from the YL and ML groups clustered together, and some samples from the P and A groups were clustered together. However, samples from E were more distinct from the other four developmental stages (Fig. 1A).\u003c/p\u003e\n\u003cp\u003eThe Pearson correlation coefficient is used as an evaluation of biological repeat correlation. A coefficient of determination, r\u003csup\u003e2\u003c/sup\u003e, that is closer to 1, suggests a stronger correlation between two duplicate samples. The Pearson\u0026apos;s correlation coefficient analyses of all the samples verified the consistency of the biological replicates collected at each stage (Fig. 1B).\u003c/p\u003e\n\u003cp\u003eDEG expression analysis\u003c/p\u003e\n\u003cp\u003eAmong the 59385 unigenes, DEGs were analyzed in biologically relevant comparisons, namely YL vs. E, ML vs. YL, P vs. ML, and A vs. P (Table S4). The DEGs from the different libraries were plotted in four volcano plots, showing significance on the y-axis and fold change on the x-axis. Fold change (log2FC\u0026ge;1) and an adjusted p-value (\u0026le;0.05) were used as thresholds for significance testing (Fig. 2A). The number of transcripts differentially expressed in the different developmental stages is illustrated as a bar chart (Fig. 2B). A Venn diagram was also created to demonstrate the relationship between the DEGs in all comparison groups (Fig. 2C). Ultimately, 24376, 11802, 20726 and 13262 DEGs were identified for the comparison group YL vs. E, ML vs. YL, P vs. ML and A vs. P, respectively. Data suggests that the egg-young larvae and mature larvae-pupae transitions caused more complex transcriptional changes compared to the other transitions.\u003c/p\u003e\n\u003cp\u003eIn addition, all DEGs were mapped to GO terms (Fig. 3; Table S5) and KEGG pathways (Table S6) to explore the biological functions in which they may be involved. We found that the common biological process enriched by up-regulated genes in the YL vs. E group and down-regulated genes in the ML vs. YL group is \u0026quot;cuticle development\u0026quot;, and the common biological process of up-regulated genes in the ML vs. YL group and down-regulated genes in the P vs. ML group includes \u0026quot;response to oxygen-containing compound\u0026quot; and \u0026quot;chitin metabolic process\u0026quot;. The common GO terms of up-regulated genes in the P vs. ML group and down-regulated genes in the A vs. P group were mainly related to \u0026quot;microtubules and cilium activities\u0026quot;, and \u0026quot;cell adhesion\u0026quot;. The down-regulated genes in the YL vs. E group were significantly enriched in biological processes such as \u0026quot;DNA replication\u0026quot;, and the GO terms enriched in the A vs. P group were mainly related to \u0026quot;energy metabolism activities\u0026quot;.\u003c/p\u003e\n\u003cp\u003eGene co-expression network analysis\u003c/p\u003e\n\u003cp\u003eIn the constructed scale-free weighted gene co-expression network, the remaining 36987 genes after filtering were divided into 15 modules, with each color representing a module (Fig. 4A). The WGCNA identified 5 modules significantly associated with the five developmental stages (Pearson correlation r\u0026gt;0.8, p\u0026lt;0.05). To be specific, the turquoise, red, blue, brown and black modules were positively correlated with the egg, young larva, mature larva, pupa and adult stages, respectively (Fig. 4B). Subsequently, we calculated the gene significance and module membership values of all the genes in each module and found that gene significance and module membership were highly positively correlated in the first four modules (cor=0.78~0.95), but not the black module (cor=0.49) (Fig. 5A-E).\u003c/p\u003e\n\u003cp\u003eFunctional analysis of five-specific modules\u003c/p\u003e\n\u003cp\u003eClusterProfiler\u0026nbsp;was used to examine the biological function of the five-specific modules by GO (Table S7) and KEGG pathway analysis (Table S8). The turquoise module was highly associated with the egg stage, where the top 10 biological processes in the GO analysis focused on mitotic\u0026nbsp;regulation, mRNA processing and modification, and microtubule organization (Fig. 6A). The most enriched pathways were \u0026quot;MAPK signaling pathway\u0026quot;, \u0026quot;mRNA surveillance pathway\u0026quot;, \u0026quot;Hippo signaling pathway\u0026quot;, \u0026quot;Wnt signaling pathway\u0026quot; and \u0026quot;RNA polymerase\u0026quot; (Fig. 7A).\u003c/p\u003e\n\u003cp\u003eThe red module was correlated with young larvae. The most enriched GO terms in the BP categories were \u0026quot;chitin-based cuticle development\u0026quot;, \u0026quot;cuticle development\u0026quot; and \u0026quot;body morphogensis\u0026quot; (Fig. 6B). According to the KEGG plot, the most enriched pathways were \u0026quot;fatty acid degradation\u0026quot;, \u0026quot;oxidative phosphorylation\u0026quot;, and \u0026quot;phototransduction\u0026quot; (Fig. 7B), which implied that cuticle development, energy metabolism, and phototransduction are important for the young larvae of \u003cem\u003eD. frischii\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe blue module was mainly associated with the mature larvae. The top 10 enriched GO terms under the BP category were associated with biosynthesis and metabolism of substances, especially amino acids, amides, and peptides (Fig. 6C). The KEGG pathway results showed that, except for the calcium signaling pathway and apelin signaling pathway, the other pathways were closely related to the biosynthesis and metabolism of amino acids as well as glucose metabolism (Fig. 7C).\u003c/p\u003e\n\u003cp\u003eThe brown module was highly correlated with the pupal stage. The significantly enriched biological processes mainly focused on cell adhesion, morphogenesis, and axon development (Fig. 6D). According to the KEGG barplot, the most enriched pathways were \u0026quot;Hippo signaling pathway\u0026quot;, \u0026quot;ECM-receptor interaction\u0026quot; and \u0026quot;axon guidance\u0026quot; (Fig. 7D). During the development, many of the pupal tissues and structures of \u003cem\u003eD. frischii\u0026nbsp;\u003c/em\u003eare broken down and reorganized into the structures and tissues of the adult and in response to metamorphosis, it requires the involvement of multiple biological functions, such as signal transduction and cell adhesion.\u003c/p\u003e\n\u003cp\u003eThe black module was mainly associated with the adult stage. The top five enriched GO terms were mitochondrial related, including \u0026quot;mitochondrial electron transport\u0026quot;, \u0026quot;ATP synthesis\u0026quot;, \u0026quot;tricarboxylic acid cycle (TCA)\u0026quot;, \u0026quot;mitochondrial ATP synthesis coupled proton transport\u0026quot; and \u0026quot;mitochondrial electron transport, ubiquinol to cytochrome c\u0026quot; (Fig. 6E). Consistently, the KEGG significantly enriched pathways were \u0026quot;oxidative phosphorylation\u0026quot;, \u0026quot;propanoate metabolism\u0026quot;, \u0026quot;TCA cycle\u0026quot;, and \u0026quot;amino acid degradation\u0026quot; (Fig. 7E). These results showed that adults need a boost in metabolism to complete activities such as migration, feeding, and mating.\u003c/p\u003e\n\u003cp\u003eExpression of olfaction-related DEGs\u003c/p\u003e\n\u003cp\u003eIn order to further explore the relationship between the olfactory system and the developmental stages of \u003cem\u003eD. frischii\u003c/em\u003e, the expression profiles of olfaction-related genes were examined in more detail. Communication between individual insects and between insects and their environment relies mainly on the senses of smell and taste, which involves several proteins, including odorant binding protein (\u003cem\u003eOBP\u003c/em\u003e), chemosensory protein, odorant receptor, olfactory receptor, and odorant-degrading enzymes. According to the DEGs and their annotation information, 11 olfactory receptors, three odorant receptors, 16 \u003cem\u003eOBPs\u003c/em\u003e, and 19 chemosensory proteins were found (Fig. 8). The results showed that most olfactory-related genes were down-regulated during egg development. However, the expression of two genes encoding chemosensory proteins (\u003cem\u003eMS_transcript_28421\u003c/em\u003e and \u003cem\u003eMS_transcript_27306\u003c/em\u003e) was highly up-regulated in the egg stage and subsequently down-regulated in the larval stages. Specifically, four olfactory receptors were mainly up-regulated in the P and A stages, while the other seven olfactory receptor 142-like genes were mainly up-regulated in the YL and ML stages, and then down-regulated thereafter. The expression patterns of the two odorant receptor 4-like isoform X1 were also similar to those of the seven olfactory receptors. Interestingly, the expression of almost all \u003cem\u003eOBP\u003c/em\u003e genes was up-regulated in the A stage. It is hypothesized that \u003cem\u003eOBPs\u003c/em\u003e play an important role for the adults of \u003cem\u003eD. frischii\u003c/em\u003e in the discovery of carcasses. We also found that most chemosensory protein genes were up-regulated in the YL and ML stages. However, four chemosensory protein genes were up-regulated in the A stage (\u003cem\u003eMS_transcript_88355\u003c/em\u003e, \u003cem\u003eMS_transcript_28982\u003c/em\u003e, \u003cem\u003eMS_transcript_48953\u003c/em\u003e, and \u003cem\u003eMS_transcript_72021\u003c/em\u003e) and the expression of chemosensory protein \u003cem\u003eCSP8\u003c/em\u003e was up-regulated in the YL and P stages.\u003c/p\u003e\n\u003cp\u003eExpression of DEGs in insect hormone biosynthesis\u003c/p\u003e\n\u003cp\u003eJuvenile hormone (\u003cem\u003eJH\u003c/em\u003e) and molt hormone synergistically regulate insect metamorphosis and development. In juvenile hormone biosynthesis, a total of 49 DEGs were significantly (p\u0026lt;0.05) enriched in \u003cem\u003eD. frischii\u0026nbsp;\u003c/em\u003e(Fig. 9). These DEGs were mainly down-regulated in the E stage, especially farnesyl diphosphate synthase (\u003cem\u003eFPPS\u003c/em\u003e), NADP\u003csup\u003e+\u003c/sup\u003e-dependent farnesol dehydrogenase (\u003cem\u003eFOHSDR\u003c/em\u003e) and juvenile hormone diol kinase (\u003cem\u003eJHDK\u003c/em\u003e). The DEGs encoding for \u003cem\u003eFOHSDR\u003c/em\u003e were mainly up-regulated from the P to the A stage, while the DEGs encoding aldehyde dehydrogenases (\u003cem\u003eALDH\u003c/em\u003e) were mainly up-regulated from the YL to the ML stage. In addition, the expression of the methyl farnesoate epoxidase (\u003cem\u003eCYP15A1\u003c/em\u003e) gene was up-regulated in the E and P stages. These three genes encode for key enzymes involved in the biosynthesis of \u003cem\u003eJH III\u003c/em\u003e, which is the principal component of juvenile hormone. It is hypothesized that these enzymes play a key role in the precise regulation of juvenile hormone concentrations at different developmental stages of \u003cem\u003eD. frischii\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eFor the degradation process of \u003cem\u003eJH III\u003c/em\u003e, the genes encoding juvenile hormone esterase (\u003cem\u003eJHE\u003c/em\u003e) were up-regulated from the ML to the A stage, and the genes encoding juvenile hormone epoxide hydrolase (\u003cem\u003eJHEH\u003c/em\u003e) were up-regulated in the ML stage. Both \u003cem\u003eJHE\u003c/em\u003e and \u003cem\u003eJHEH\u003c/em\u003e are key enzymes responsible or degrading juvenile hormone. This suggests that \u003cem\u003eJHE\u003c/em\u003e and \u003cem\u003eJHEH\u003c/em\u003e control changes in the concentration of the juvenile hormones by degrading them during the development of \u003cem\u003eD. frischii\u0026nbsp;\u003c/em\u003efrom mature larvae to adults. In addition, the expression of the\u003cem\u003e\u0026nbsp;JHDK\u003c/em\u003e gene was significantly down-regulated in the E stage, and then maintained at a constant expression level, suggesting it promoted the growth and development of eggs by inhibiting the degradation of juvenile hormone.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMolting hormone (20-hydroxyecdysone, \u003cem\u003e20E\u003c/em\u003e) is a typical steroid hormone regulating molting, metamorphosis, and reproduction. Only six DEGs were found in the molting hormone biosynthesis pathway (Fig. 10). The gene encoding for \u003cem\u003eCYP307A1\u003c/em\u003e was up-regulated in the E and P stages, while the gene encoding for \u003cem\u003eCYP315A1\u003c/em\u003e was up-regulated from the ML to the P stage and the gene encoding for \u003cem\u003eCYP314A1\u003c/em\u003e was up-regulated in the P stage. In addition, the enzyme \u003cem\u003eCYP18A1\u003c/em\u003e, which is responsible for the degradation of \u003cem\u003e20E\u003c/em\u003e, was highly expressed in the YL and ML stages. These genes strictly control the change of the \u003cem\u003e20E\u003c/em\u003e titer in \u003cem\u003eD. frischii\u0026nbsp;\u003c/em\u003eby regulating the \u003cem\u003e20E\u003c/em\u003e biosynthesis process.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEcdysone receptor (\u003cem\u003eEcR\u003c/em\u003e) and ultraspiracle (\u003cem\u003eUSP\u003c/em\u003e) are members of the nuclear receptor superfamily and are molecular targets of \u003cem\u003e20E\u003c/em\u003e. When \u003cem\u003e20E\u003c/em\u003e induces the formation of transcription factors such as \u003cem\u003eHR3\u003c/em\u003e,\u003cem\u003e\u0026nbsp;E75\u003c/em\u003e, \u003cem\u003eE78\u003c/em\u003e, \u003cem\u003eBrc\u003c/em\u003e, and others by interacting with the heterodimer formed by \u003cem\u003eEcR\u003c/em\u003e and \u003cem\u003eUSP\u003c/em\u003e, it initiates an internal regulatory cascade response that causes insect molting and metamorphosis. Results showed that the gene encoding \u003cem\u003eEcR\u003c/em\u003e was up-regulated from the YL to the P stage, while the gene encoding \u003cem\u003eUSP\u003c/em\u003e was up-regulated from the E to the ML stage. Interestingly, results showed that a large number of DEGs encoding ecdysone-inducible protein (e.g., \u003cem\u003eE74\u003c/em\u003e, \u003cem\u003eE75\u003c/em\u003e, \u003cem\u003eE78\u003c/em\u003e, and \u003cem\u003eE93\u003c/em\u003e) was markedly up-regulated during the P stage. This suggests that the \u003cem\u003e20E\u003c/em\u003e biosynthesis pathway is active in the P stage, which led to a change in the \u003cem\u003e20E\u003c/em\u003e concentration and the activation of a large number of ecdysone-inducible proteins, thereby regulating the expression of different downstream target genes, thus controlling the eclosion process of \u003cem\u003eD. frischii\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eValidation of gene expression levels\u003c/p\u003e\n\u003cp\u003eIn order to validate the results of RNA-seq, six genes related to cell division, cell cycle regulation, energy metabolism, and \u003cem\u003eJH\u003c/em\u003e biosynthesis were selected for qRT-PCR analysis (Fig. 11). The results showed agreement between the RNA-Seq analysis and qRT-PCR results, indicating that the transcriptome analysis was accurate and reliable.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eImportant biological functions at different developmental stages\u003c/p\u003e\n\u003cp\u003eThrough PacBio Iso-Seq and Illumina RNA-seq sequencing, this work, for the first time, provides the full-length transcriptome information and the complete transcriptome data of the five developmental stages of \u003cem\u003eD. frischii\u003c/em\u003e, to determine the molecular mechanisms underlying its metamorphic development. DEGs were identified using the p-value and the fold change thresholds of the comparisons of the developmental stages. Then, GO and KEGG analyses were performed to better understand the DEGs relevant to the biological processes and pathways. Meanwhile, in order to further understand the biological functions involved in the development of \u003cem\u003eD. frischii\u003c/em\u003e, a weighted gene co-expression network was also constructed. The resulting WGCNA allowed for the identification of stage-specific modules (gene clusters) during development, as did the enrichment analysis in the GO and KEGG annotation (Fig. 12).\u003c/p\u003e\n\u003cp\u003eIn the turquoise module, which was positively correlated with the egg stage, 46, 47, and 100 genes were enriched to the biological processes of \"mitotic spindle organization\", \"microtubule cytoskeleton organization involved in mitosis\" and \"cell division\", respectively. Among the down-regulated DEGs in the YL vs. E comparison group, 72, 93, and 87 genes were enriched to the biological processes of \"DNA replication\", \"DNA integration\" and \"cell division\", respectively. After the female \u003cem\u003eD. frischii\u0026nbsp;\u003c/em\u003elays eggs, the continuous development of the embryo involves numerous cell cycles. The cell cycle process can be divided into two phases, interphase and mitosis, where interphase can be further divided into pre-DNA synthesis, DNA synthesis and post-DNA synthesis. The cytoskeleton comprising microtubules, microfilaments, and intermediate fibers is the internal structure that supports and maintains the morphology and structure of the cell. Microtubules are fibrous structures composed of tubulin proteins that form a network to support and guide the movement of materials inside the cell. Thus, cell division, DNA replication, and microtubule cytoskeleton organization are important biological processes for the developing egg, which ensures the normal morphogenesis and development of the embryo. In addition, the common enrichment pathways between the turquoise module and the down-regulated genes of the YL vs. E comparison group included the \"MAPK signaling pathway\", the \"mRNA surveillance pathway\", the \"Wnt signaling pathway\", and the \"Hippo signaling pathway\". The MAPK signaling pathway plays a pivotal role in the development of metazoans, by controlling cell proliferation and cell differentiation through receptor tyrosine kinases\u0026nbsp;(Baril et al. 2014). The Hippo signaling pathway is a highly conserved kinase cascade that affects organ size by regulating cell proliferation, survival, and differentiation\u0026nbsp;(Manno 2021). The mRNA surveillance pathway mainly monitors and regulates the translation of mRNA to ensure correct protein synthesis, which is important for accurate gene expression and the normal function of cells. The results from this work showed that these enrichment pathways play a key regulatory role in the growth and development of the egg stage of \u003cem\u003eD. frischii\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eAfter hatching from the egg, \u003cem\u003eD. frischii\u0026nbsp;\u003c/em\u003eenters the young larvae stage, which was significantly positively correlated with the red module. The GO analysis identified 21 genes enriched in the \"chitin-based cuticle development\" biological process and among the up-regulated DEGs in the YL vs. E comparison group, a large number of chitin-related genes involved in the biological processes of \"chitin-based cuticle development\", \"chitin metabolic process\", \"cuticle development\" and \"chitin catabolic development\" was found. Chitin is abundantly present in the insect cuticle and is important for the formation of the larval epidermis during the early stages of development. Especially for \u003cem\u003eD. frischii\u003c/em\u003e,\u0026nbsp;a multi-instar species with 4-11 molts,\u0026nbsp;frequent molting means a significant demand for chitin synthesis. Chitin can provide physical support, prevent desiccation, protect from physical and chemical damages, and defend against pathogens. On the other hand, chitin is also an important signaling molecule in insects, and is involved in regulating the growth and development of larvae. In addition, the top three\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eenriched pathways involved in the red module were \"fatty acid degradation\", \"oxidative phosphorylation\" and \"phototransduction\". These three pathways were included in the top five pathways enriched for the down-regulated DEGs in the ML vs. YL comparison group, suggesting that oxidative phosphorylation and fatty acid degradation serve energy production in early larval metabolism. Compared to the egg stage, the young larvae require a large amount of energy to support morphogenesis and organ development.\u0026nbsp;At the same time, transition to the larval stage involves autonomous activities, such as feeding and developing vision through\u0026nbsp;phototransduction\u0026nbsp;to continuously perceive a large amount of external environmental information or stimuli to survive.\u0026nbsp;Some pathways enriched in the red module, such as \"drug metabolism-cytochrome \u003cem\u003eP450\u003c/em\u003e\", and \"metabolism of xenobiotics by cytochrome \u003cem\u003eP450\u003c/em\u003e\", suggest that cytochrome\u003cem\u003e\u0026nbsp;P450\u003c/em\u003e may help protect the larvae from damage by other organisms.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe blue module was closely associated with the mature larval stage. The biological functions of the mature larvae focused on metabolism-related processes, especially the biosynthesis and metabolism of amino acids, which are necessary for growth and development. During the development from young larvae to mature adults, the body size of \u003cem\u003eD. frischii\u003c/em\u003e increases after each molt and the focus of the biological functions shifts from chitin synthesis and energy metabolism to amino acid synthesis, to prepare for the increased energy consumption in the post-feeding stage, metamorphosis and growth\u0026nbsp;during\u0026nbsp;the\u0026nbsp;pupal stage. Results also showed that immune-related GO terms were dominant in the up-regulated DEGs of the ML vs. YL comparison group and the down-regulated DEGs of the P vs. ML comparison group. These include \"leukocyte mediated immunity\", \"neutrophil activation involved in immune response\", and \"neutrophil activation\".\u0026nbsp;The GO terms related to cellular stress, such as \"cellular response to oxygen-containing compound\", and \"cellular response to chemical stimulus\" were also enriched for this phase. Since mature larvae usually live in the carcass where many microorganisms breed, a heightened immune response and increased expression of stress-related genes\u0026nbsp;will ensure their survival in this harsh environment.\u003c/p\u003e\n\u003cp\u003eThe brown module was highly associated with the pupal stage, and the top five GO terms enriched for the BP category and the down-regulated DEGs of the A vs. P comparison group were identical, focusing on cell adhesion and morphogenesis. Cell adhesion is the process in which cells attach to neighboring cells through the interaction of specialized molecules on the cell surface and it is speculated that during metamorphosis, \u003cem\u003eD. frischii\u003c/em\u003e undergoes extensive cell rearrangement and tissue remodeling through cell adhesion. However, in the up-regulated DEGs of the P vs. ML comparison group, the top GO terms were cilium and microtubule related.\u0026nbsp;Researchers have found that\u0026nbsp;cilium\u0026nbsp;is an important microtubule-based organelle that influences the cell cycle and regulates various cell life activities, such as stem cell maintenance, differentiation, and asymmetric division\u0026nbsp;(Zheng et al. 2016; Patel and Tsiokas 2021).\u0026nbsp;Therefore,\u0026nbsp;cilium\u0026nbsp;dynamics are closely related to cellular processes, such as the cell cycle and differentiation, and it is speculated that the process of complete metamorphosis is mainly mediated by\u0026nbsp;cilium\u0026nbsp;and microtubule activity.\u003c/p\u003e\n\u003cp\u003eThe black module was closely associated with the adult stage. The enriched GO terms and KEGG pathways in both the black module and the up-regulated DEGs of the A vs. P comparison group were significantly related to energy metabolism, including \"ATP synthesis coupled proton transport\", and \"TCA cycle\". This suggests an increased energy consumption in adult \u003cem\u003eD. frischii\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eExpression characteristics of olfactory-related genes\u003c/p\u003e\n\u003cp\u003eInsect ecology, to a strong degree, depends on the chemosensory modalities of smell and taste\u0026nbsp;(Walker et al. 2022)\u0026nbsp;and the olfactory system plays an important role in guiding behaviors, including feeding, mating, and oviposition\u0026nbsp;(Al-Jalely et al. 2021).\u0026nbsp;For example, with \u003cem\u003eD. frischii\u003c/em\u003e, male adults are first attracted by the odor emitted by decaying carcasses or stored materials. After confirming through olfaction that the environment is suitable for offspring development, male adults will release sex hormones to attract females to mate. Eventually, females, attracted to the pheromones, will mate and lay their eggs on carcasses or storage rich in organic matter. However, little is known about the molecular mechanism of olfactory reception in insects. In \u003cem\u003ePachyrhinus yasumatsui\u0026nbsp;\u003c/em\u003e(Kono and Morimoto 1960)(Coleoptera: Curculionidae), 113 genes were identified to be involved in chemosensory functions\u0026nbsp;(Hong et al. 2023). In\u003cem\u003e\u0026nbsp;Chilo sacchariphagus\u0026nbsp;\u003c/em\u003e(Bojer, 1856)(Lepidoptera: Crambidae), a key pest of sugarcane, 72 candidate chemosensory genes were identified from different tissues and genders\u0026nbsp;(Liu et al. 2021). Here, a total of 78 candidate genes were associated with the sense of smell, including 32 olfactory receptors, four odorant receptors, 19 \u003cem\u003eOBPs\u003c/em\u003e, and 23 chemosensory proteins. Among them, 49 olfactory genes were differentially expressed during the developmental process of \u003cem\u003eD. frischii\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eMost olfactory-related genes maintained low expression levels during the egg stage. Subsequently, these genes showed divergent expression patterns as development progressed. Notably, almost all the \u003cem\u003eOBPs\u003c/em\u003e were up-regulated at the adult stage. Insect \u003cem\u003eOBPs\u003c/em\u003e are a class of small soluble proteins that\u0026nbsp;facilitate\u0026nbsp;the binding and transport of small molecules and can be found in various tissues. In the Japanese pine sawyer \u003cem\u003eMonochamus alternatus\u003c/em\u003e Hope, 1842 (Coleoptera: Cerambycidae), an apparent gradient expression pattern of \u003cem\u003eOBP19\u003c/em\u003e was detected. The binding protein was highly and specifically expressed in the antennae and played an essential role in the detection of camphene\u0026nbsp;(Li et al. 2022). Throughout the development of \u003cem\u003eSpodoptera frugiperda\u0026nbsp;\u003c/em\u003e(Smith, 1797) (Lepidoptera: Noctuidae), the transcription of \u003cem\u003eOBP27\u003c/em\u003e steadily increased and the transcripts of this gene were abundant in the fat body and male reproductive organs\u0026nbsp;(Han et al. 2023). Thus, data suggests that the up-regulated expression levels of \u003cem\u003eOBPs\u003c/em\u003e in the adult stage ensure a faster detection of the corpse or breeding site. Investigating these olfactory-related genes will help reveal the molecular mechanism of odorant recognition in \u003cem\u003eD. frischii\u0026nbsp;\u003c/em\u003eand may aid in the development of novel control strategies for this species.\u003c/p\u003e\n\u003cp\u003eExpression characteristics of insect hormone biosynthesis related DEGs\u003c/p\u003e\n\u003cp\u003eTwo major hormones, \u003cem\u003eJH\u003c/em\u003e and \u003cem\u003e20E\u003c/em\u003e, regulate insect growth and development according to their precisely coordinated titers, which are controlled by both biosynthesis and degradation pathways\u0026nbsp;(Zhang et al. 2017). The \u003cem\u003eJHs\u003c/em\u003e are a family of sesquiterpenoid molecules that are secreted into the hemolymph, and play a key role in preventing larval precocious metamorphosis, maintaining the larval state, controlling adult sexual development, and promoting insect egg maturation\u0026nbsp;(Cai et al. 2022). Enzymes involved in the biosynthesis of \u003cem\u003eJH\u003c/em\u003e include \u003cem\u003eFPPS\u003c/em\u003e, \u003cem\u003eFOHSDR\u003c/em\u003e, \u003cem\u003eALDH\u003c/em\u003e, juvenile hormone acid methyltransferase (\u003cem\u003eJHAMT\u003c/em\u003e), and \u003cem\u003eCYP15A1\u003c/em\u003e, where \u003cem\u003eJHAMT\u003c/em\u003e is a rate-limiting enzyme of \u003cem\u003eJH\u003c/em\u003e biosynthesis in insects. It transfers the methyl group of S-adenosyl methionine to either the carboxyl group of \u003cem\u003eJH\u003c/em\u003e acids or the farnesoic acid to produce \u003cem\u003eJH\u003c/em\u003e (Zhang et al. 2022). In this study, among the 42756 annotated transcripts, we did not find transcripts encoding for \u003cem\u003eJHAMT\u003c/em\u003e. Another key enzyme-encoding gene, \u003cem\u003eCYP15A1\u003c/em\u003e, was reported to encode for the cytochrome \u003cem\u003eP450\u003c/em\u003e enzyme responsible for the epoxidation of methyl farnesoate to \u003cem\u003eJH\u003c/em\u003e (Marchal et al. 2011). The strong up-regulation of two transcripts encoding for \u003cem\u003eCYP15A1\u003c/em\u003e found here might contribute to \u003cem\u003eJH\u003c/em\u003e accumulation in promoting egg maturation.\u003c/p\u003e\n\u003cp\u003eIn multicellular organisms, most of the developmental transitions are driven by steroid hormones. Steroid hormone \u003cem\u003e20E\u003c/em\u003e is secreted by prothoracic glands and transported to the target organs via the hemolymph\u0026nbsp;(Lee et al. 2022). Previous studies showed that \u003cem\u003e20E\u003c/em\u003e is actually regulated by six \u003cem\u003eP450\u003c/em\u003e genes (five \u003cem\u003eP450\u003c/em\u003e genes belonging to the Halloween family and a \u003cem\u003eCYP18A1\u003c/em\u003e gene) in model insects\u0026nbsp;(Liu et al. 2020). In \u003cem\u003eManduca sexta\u003c/em\u003e (Linnaeus, 1763) (Lepidoptera: Sphingidae), \u003cem\u003eCYP306A1\u003c/em\u003e, \u003cem\u003eCYP302A1\u003c/em\u003e, and \u003cem\u003eCYP315A1\u003c/em\u003e, which mediate the final hydroxylation in the biosynthesis of ecdysone, were selectively expressed in the prothoracic glands, and changes in their expression correlate with the haemolymph ecdysteroid titer during the fifth (final) larval instar. Transcript levels of \u003cem\u003eCYP314A1\u003c/em\u003e, the 20-hydroxylase, which converts ecdysone into the more active \u003cem\u003e20E\u003c/em\u003e, closely parallels the enzyme activity measured in vitro\u0026nbsp;(Rewitz et al. 2006). \u003cem\u003eCYP18A1\u003c/em\u003e has been shown to play a key role in insect steroid hormone inactivation through 26-hydroxylation\u0026nbsp;(Li et al. 2014). In \u003cem\u003eBombyx mori\u003c/em\u003e (Linnaeus, 1758) (Lepidoptera: Bombycidae), overexpression of \u003cem\u003eCYP18A1\u003c/em\u003e resulted in developmental arrest during the final instar larval stage. Also, the \u003cem\u003e20E\u003c/em\u003e titers in the transgenic \u003cem\u003eB. mori\u003c/em\u003e expressing \u003cem\u003eCYP18A1\u003c/em\u003e were lower compared to the levels in the control group\u0026nbsp;(Li et al. 2014). In this study, the expression of \u003cem\u003eCYP315A1\u003c/em\u003e and \u003cem\u003eCYP314A1\u003c/em\u003e were up-regulated and the expression of \u003cem\u003eCYP18A1\u003c/em\u003e was down-regulated in the pupal stage, suggesting that these genes were transcriptionally regulated to support the high \u003cem\u003e20E\u003c/em\u003e biosynthesis activity that produces the ecdysteroid pulses triggering the pupation of \u003cem\u003eD. frischii\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA heterodimeric complex of two nuclear receptors, \u003cem\u003eEcR\u003c/em\u003e and \u003cem\u003eUSP\u003c/em\u003e, transduces \u003cem\u003e20E\u003c/em\u003e signaling to modulate insect growth and development\u0026nbsp;(Yu et al. 2023). In \u003cem\u003eApis mellifera\u003c/em\u003e Linnaeus (1758) (Hymenoptera: Apidae), \u003cem\u003e20E\u003c/em\u003e induced \u003cem\u003eEcR\u003c/em\u003e expression and RNAi knockdown of the \u003cem\u003eEcR\u003c/em\u003e gene lead to a delay in larval transition to the pupal stage. In the presence of \u003cem\u003e20E\u003c/em\u003e, \u003cem\u003eEcR\u003c/em\u003e can bind to\u003cem\u003e\u0026nbsp;USP\u003c/em\u003e and increase the expression of \u003cem\u003e20E\u003c/em\u003e-inducible genes for pupal-adult development\u0026nbsp;(Song et al. 2023). These early ecdysone-inducible genes include\u003cem\u003e\u0026nbsp;E74\u003c/em\u003e, \u003cem\u003eE75\u003c/em\u003e, \u003cem\u003eE93\u003c/em\u003e, and \u003cem\u003eHR3\u003c/em\u003e. For example,\u003cem\u003e\u0026nbsp;E74\u003c/em\u003e is a key transcription factor induced by \u003cem\u003e20E\u003c/em\u003e, which plays a role in many physiological events during insect growth and development, including vitellogenesis, organ remodeling, and new tissue formation, as well as programmed cell death and metamorphosis\u0026nbsp;(Zhang et al. 2022). More specifically, \u003cem\u003eE74\u003c/em\u003e is encoded within the \u003cem\u003e74EF\u003c/em\u003e early puff and consists of two overlapping transcription units, \u003cem\u003eE74A\u003c/em\u003e and \u003cem\u003eE74B\u003c/em\u003e. In \u003cem\u003eDrosophila\u003c/em\u003e, mutations in \u003cem\u003eE74A\u003c/em\u003e and \u003cem\u003eE74B\u003c/em\u003e are lethal during prepupal and pupal development, which is consistent with the critical role their gene products play in metamorphosis\u0026nbsp;(Fletcher et al. 1995). In \u003cem\u003eSitobion avenae\u0026nbsp;\u003c/em\u003e(Fabricius, 1775) (Hemiptera: Aphididae), as a major component of the insect ecdysone signaling pathway, the expression of ecdysone-inducible \u003cem\u003eE75\u003c/em\u003e was low in the adult stage, but high in the pseudo embryo and nymphal stages\u0026nbsp;(Zheng et al. 2023). In this study, we speculate that the strong up-regulation of these ecdysone-inducible genes (\u003cem\u003eE74\u003c/em\u003e, \u003cem\u003eE75\u003c/em\u003e, \u003cem\u003eE78\u003c/em\u003e, and \u003cem\u003eE93\u003c/em\u003e) is because of a rapidly increasing \u003cem\u003e20E\u003c/em\u003e pulse, leading to undergoing extensive metamorphic remodeling in the pupae.\u003c/p\u003e\n\u003cp\u003eIn summary, \u003cem\u003eD. frischii\u003c/em\u003e exhibits unique and important biological functions at all developmental stages. In the egg stage, the biological functions of \u003cem\u003eD. frischii\u003c/em\u003e mainly focus on cell division, DNA replication, and microtubule-related processes. In the young larvae, the biological functions of \u003cem\u003eD. frischii\u003c/em\u003e mainly include cuticle development, energy metabolism, and phototransduction. In the mature larval stage, the main biological functions are transformed into amino acid synthesis and metabolism, immunity, and stress. In the pupal stage, the biological functions of \u003cem\u003eD. frischii\u003c/em\u003e were mainly focused on cilium assembly and movement, cell adhesion, and morphogenesis. In the adult stage, its main biological function is mainly related to energy metabolism. Transcription of most odorant binding proteins was up-regulated in the adult stage, and many genes encoding for the ecdysone-inducible protein were up-regulated in the pupal stage.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China [grant number 31872258, 32070508, and 82002007] and Priority Academic Program Development of Jiangsu Higher Education.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China [grant number 31872258, 32070508, and 82002007] and Priority Academic Program Development of Jiangsu Higher Education.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGH and ZG conceived experiments and wrote manuscript. YW (Yu Wang) designed experiments. LL, YL, SS, RZ, YG (Yundi Gao), YG (Yi Guo) and YW (Yinghui Wang) conducted experiments. GH and ZG analyzed data. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during the current study are available in the NCBI repository, PRJNA1067750.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAl-Jalely BH, Wang PH, Liao YL, Xu W (2021) Identification and characterization of olfactory genes in the parasitoid wasp \u003cem\u003eDiadegma semiclausum\u003c/em\u003e (Hell\u0026eacute;n) (Hymenoptera: Ichneumonidae). B Entomol Res 112:187-196. https://doi.org/10.1017/S0007485321000675\u003c/li\u003e\n \u003cli\u003eAltner H, Prillinger L (1980) Ultrastructure of invertebrate chemo-, thermo-, and hygroreceptors and its functional significance. 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Nat Commun 7:11874. https://doi.org/10.1038/ncomms11874\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Dermestes frischii, developmental stage, RNA-seq, SMRT sequencing, Transcriptome, WGCNA","lastPublishedDoi":"10.21203/rs.3.rs-4206363/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4206363/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eDermestes frischii\u003c/em\u003e Kugelann, 1792 is a storage pest worldwide, and is important for estimating the postmortem interval in forensic entomology. However, because of the lack of transcriptome and genome resources, population genetics and biological control studies on \u003cem\u003eD. frischii\u003c/em\u003e have been hindered. Here, single-molecule real-time sequencing and next-generation sequencing were combined to generate the full-length transcriptome of the five developmental stages of \u003cem\u003eD. frischii\u003c/em\u003e, namely egg, young larva, mature larva, pupa, and adult. A total of 41665 full-length non-chimeric sequences and 59385 non-redundant transcripts were generated, of which 42756 were annotated in public databases. By comparing the transcripts from adjacent developmental stages, 24376, 11802, 20726, and 13262 differentially expressed genes were identified, respectively. Using the weighted gene co-expression network analysis, gene co-expression modules related to the five developmental stages were constructed and screened, and the genes in these modules subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The expression patterns of the DEGs related to olfaction and insect hormone biosynthesis were also explored. Transcription of most odorant binding proteins was up-regulated in the adult stage, suggesting they are important for foraging in adults. Many genes encoding for the ecdysone-inducible protein were up-regulated in the pupal stage. The results of the qRT-PCR were consistent with the RNA-seq results. This is the first full-length transcriptome sequencing of dermestids, and the data obtained here is vital for understanding the stage-specific development and olfactory system of \u003cem\u003eD. frischii\u003c/em\u003e, providing valuable resources for storage pest and forensic research.\u003c/p\u003e","manuscriptTitle":"Full-length transcriptome-referenced analysis reveals developmental and olfactory regulatory genes in Dermestes frischii","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-09 09:59:30","doi":"10.21203/rs.3.rs-4206363/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e70dbce6-a4d9-4cb0-bbc5-8ce4645e6ee5","owner":[],"postedDate":"April 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-03T13:53:09+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-09 09:59:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4206363","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4206363","identity":"rs-4206363","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}