Chemosensory System Decoding: Transcriptome-Wide identification and Expression Profiling of Olfactory Genes in Lytta sifanica

Chemosensory System Decoding: Transcriptome-Wide identification and Expression Profiling of Olfactory Genes in Lytta sifanica | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Ecology and Evolution This is a preprint and has not been peer reviewed. Data may be preliminary. 15 March 2025 V1 Latest version Share on Chemosensory System Decoding: Transcriptome-Wide identification and Expression Profiling of Olfactory Genes in Lytta sifanica Authors : Feng Zhou [email protected] , Zhuanxia Li 0009-0004-3550-373X , Jiani Chen 0009-0009-1293-0110 , Xinge Song , Shuning Sun , Yuying Zhang , Liyuan Yao , Yuqin Wang , Xinyu Sun , and Lixia wan Authors Info & Affiliations https://doi.org/10.22541/au.174204366.66023286/v1 Published Ecology and Evolution Version of record Peer review timeline 385 views 170 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The insect olfactory system employs a diverse array of olfactory-related proteins to facilitate odor detection and signal transduction. Their expression and regulation are crucial for mediating communication between themselves and environments. Lytta sifanica (Coleoptera: Meloidae) is one of the most important economic pests and is famous for producing a toxic substance cantharidin. However, the genes underlying olfactory sensation are lacking in this Blister Beetle. In this study, the transcriptomes of adult L. sifanica antennae were sequenced and analyzed. A total of 70 chemosensory genes, including 17 odorant binding proteins (OBPs), 5 chemosensory proteins (CSPs), 13 gustatory receptors (GRs), 17 odorant receptors (ORs), 13 ionotropic receptors (IRs) and 5 sensory neuron membrane proteins (SNMPs) were identified based on sequence homology analysis and phylogenetic reconstruction. The expression patterns of all candidate genes in the antennae of adults, head, mouthparts, pronotum, foreleg tarsus, abdomen skin, wings were confirmed by RT-PCR. The analyses demonstrated that all protein families related to olfaction are widely expressed in examined tissues. However, the expression patterns varied for different families. Eleven members are predominantly expressed in the antennae (Lsif_OBP1/2/19d, Lsif_OR2/20/49b, Lsif_IR2a, Lsif_GR7/127, Lsif_CSP1, Lsif_SNMP2/4). Twenty-two members were exclusively expressed in the mouthparts (Lsif_OBP70/56d2, Lsif_GR12a/21/28a/68a), foreleg tarsus (Lsif_OR6/67c, Lsif_GR12, and Lsif_OBP2) and were abundant in the non-olfactory tissues head (Lsif_OBP99a, Lsif_OR9a/49b, Lsif_IR25a/56e, and Lsif_CSP6), pronotum (Lsif_OBP5/C20, Lsif_Orco3, Lsif_OR13, and Lsif_IR7), abdomen skin (Lsif_SNMP5), suggesting their various functions in the olfactory system of L. sifanica. This research offers an extensive resource for investigating the chemoreception mechanism in beetle L. sifanica. Chemosensory System Decoding: Transcriptome-Wide identification and Expression Profiling of Olfactory Genes in Lytta sifanica Feng Zhou #,* , Zhuan-xia Li # , Jia-ni Chen, Xin-ge Song, Shu-ning Sun, Yu-ying Zhang, Li-yuan Yao, Yu-qin Wang, Xin-yu Sun, Li-xia Wan College of Life Science, Northwest Normal University, Lanzhou, China # These authors contribute equally to this paper . * Corresponding author : Dr. Feng Zhou, College of Life Science Northwest Normal University 967 Anning East Road, Anning District, Lanzhou 730070, China Tel: 86-0931-7970522 Fax: 86- 0931-7971770 E-mail: [email protected] Abstract The insect olfactory system employs a diverse array of olfactory-related proteins to facilitate odor detection and signal transduction. Their expression and regulation are crucial for mediating communication between themselves and environments. Lytta sifanica (Coleoptera: Meloidae) is one of the most important economic pests and is famous for producing a toxic substance cantharidin. However, the genes underlying olfactory sensation are lacking in this Blister Beetle. In this study, the transcriptomes of adult L. sifanica antennae were sequenced and analyzed. A total of 70 chemosensory genes, including 17 odorant binding proteins (OBPs), 5 chemosensory proteins (CSPs), 13 gustatory receptors (GRs), 17 odorant receptors (ORs), 13 ionotropic receptors (IRs) and 5 sensory neuron membrane proteins (SNMPs) were identified based on sequence homology analysis and phylogenetic reconstruction. The expression patterns of all candidate genes in the antennae of adults, head, mouthparts, pronotum, foreleg tarsus, abdomen skin, wings were confirmed by RT-PCR. The analyses demonstrated that all protein families related to olfaction are widely expressed in examined tissues. However, the expression patterns varied for different families. Eleven members are predominantly expressed in the antennae ( Lsif_OBP1/2/19d, Lsif_OR2/20/49b, Lsif_IR2a, Lsif_GR7/127, Lsif_CSP1, Lsif_SNMP2/4 ). Twenty-two members were exclusively expressed in the mouthparts ( Lsif_OBP70/56d2, Lsif_GR12a/21/28a/68a ), foreleg tarsus ( Lsif_OR6/67c, Lsif_GR12, and Lsif_OBP2 ) and were abundant in the non-olfactory tissues head ( Lsif_OBP99a, Lsif_OR9a/49b, Lsif_IR25a/56e, and Lsif_CSP6 ), pronotum ( Lsif_OBP5/C20, Lsif_Orco3, Lsif_OR13, and Lsif_IR7 ), abdomen skin ( Lsif_SNMP5 ), suggesting their various functions in the olfactory system of L. sifanica . This research offers an extensive resource for investigating the chemoreception mechanism in beetle L. sifanica . Keywords Chemosensory gene, Blister beetle, Antennal transcriptome, Gene identification, Gene expression analysis Introduction Insects are the most diverse and widely distributed fauna on earth. Various ecological environments results in they live in a changed chemical surroundings, in which insects should properly understand those chemical signals (Jiang et al., 2020). Food, sex, predators, pathogens, oviposit, and sites to inhabit are sources of characteristic chemical signatures that insects must perceive and respond to. Consequently, their olfactory systems have become complex and diverse, with their related functions being well-developed, tightly regulated, and capable of detecting a wide array of chemical signals in the surrounding environment (Ma et al., 2022). For example, the beetle Brontispa longissima located its food by smelling the volatile compounds from related plants or animals (Bin et al., 2017). Colaphellus bowringi find their mates or predators by identifying the odor through the pheromones released by others (Li et al., 2015). To the contrary, the OBP genes knock-out flies of Drosophila melanogaster showed altered behavioral responses to the odor of the ripe fruit of Morinda citrifolia . Because the gene knock-out induced flies lose the appeal by hexanoic acid and octanoic acid, which were odors from Morinda citrifolia (Zhan et al., 2021). There is no doubt that the olfactory system is vital to insects. Olfactory system of insects is sophisticated, involving a variety of related functional proteins, such as odorant-binding proteins (OBPs), chemosensory proteins (CSPs), olfactory receptors (ORs), ionotropic receptors (IRs), gustatory receptors (GRs), and sensory neuron membrane proteins (SNMPs) (Gu et al., 2015). Every family contains many members and plays different roles in receiving odors for insects. When chemical signals enter the receptive lymph of insects, they will bind to the associated signal molecules. The latter were further presented to OR proteins on the olfactory sensory neuron (OSN), which then converts the chemical signal into electrical signal. After completing their functional role, these signaling molecules will be degraded by corresponding enzymes (Guo et al., 2023). OBPs and CSPs have been considered to play very important roles in the odorant reception in insects. These functional molecules are thought to bind, solubilize, and transport hydrophobic odorants on the sensilla of antennae. IRs are involved in perception of humidity and temperature. GRs are thought to be related to the perception of sugars, bitter tasting compounds, non-volatile compounds, and carbon dioxide. SNMPs may play important roles in sensing pheromones (Andersson et al., 2013). These molecules in pathways are the universal evolutionary patterns of olfactory systems in insects. However, their composition is not invariable among different populations. They are variations in numbers or sequences in family members to meet their specific needs or habits, which gives an important significance to the in-depth investigation of olfactory genetic variations. L. sifanica is a typical agricultural insect and belongs to the Coleoptera: Meloidae family. It has harmful effects on plants such as Sophora japonica , willows, poplars with destroying the leaves, sucking the sap, and ultimately affecting their growth(PanGuo-dong, 2018). They are also important predators of locusts and other Orthoptera insects, due to their larval habit of eating locust eggs. Interestingly, its adults will secrete an irritating defense substance called cantharidin (C 10 H 12 O 4 ), which is biosynthesized in their secretory gland with pungent smell (Aoun et al., 2018). W hen beetles are threatened , this chemical can erode from the skin to form blisters, so they are commonly called “Blister Beetle”. Nowadays, cantharidin is used for treating human diseases, such as tumors, cancer, and shows broad medical application values (Lin et al., 2016). However, it remains unclear how olfactory genes contribute to predator defense mechanisms involving cantharidin and influence foraging interactions with plants and other animals (Fratini et al., 2022). Research on the genetic basis of the olfactory system in beetles began with the red flour beetle, Tribolium castaneum. Other beetles, including scolytid beetles and Rhynchophorus ferrugineus , have also been studied, focusing on olfactory genes at various functional levels. Like other populations, beetles have evolved typical olfactory genes and formed their families (Guan et al., 2020). However, their olfactory genes show a partial conservation and remarkable divergence in functional evolution, which could explain some differentiation of olfactory senses in ecological adaptation. In the current study, we focused on the hereditary basis of the olfactory system in L. sifanica. Among related blister beetles, only a few members have had their olfactory genes explored . For example, Hycleus Cichorii and Hycleus phaleratus have been fully sequenced and analyzed the olfactory-related genes in antenna by using genome and transcriptome methods. However, the gene identification in other blister beetles remains limited. Currently, there is still a lack of in-depth investigation on olfactory genes in L. sifanica , with only a small fraction of olfactory gene members have been sequenced (Wu et al., 2018). In this study, we investigate the olfactory genetic system of L. sifanica using transcriptome sequencing technology. Overall, seventy olfactory-related genes in L. sifanica were identified. The sequence characteristics and evolutionary patterns in beetle populations were explored within and between species through the analysis of sequence data and phylogenetic trees of each olfactory gene family. Their expression patterns were further analyzed across multiple tissues using RT-PCR experiments combined with FPKM value analysis. This work is the first to identify the chemosensory gene families in the beetle species L. sifanica and to analyze their related expression profiles, laying the foundation for future research into their olfactory mechanisms. Materials and Methods Insects The experimental materials used in this study were antenna of L. sifanica . The adult L. sifanica were collected in Shifogou (103°37′04″N, 35°9′32″E) of Lanzhou city, GanSu province of China in early May 2023 ( Figure 1A ). The local temperature at the time of insect capture was 22℃, humidity was 30% and the average altitude was 2225 meters. The sampled beetles feeding the leaves of Lonicera chrysantha were shown in picture ( Figure 1B ). A total of 105 adult L. sifanica were captured followed by morphological identification. The identified insects were then collected to clean plastic containers with feeding leaves of L. chrysantha. Experimental samples collection and RNA isolation In the laboratory, the insects were immediately placed in the freezer (−80℃) for about 5 minutes for immobilization. Then, sixty adult beetles including male and female with similar size and healthy activity were selected and their antennae were dissected by using surgical scissors. The remaining body part of beetles was used for the tissue-specific expression analysis. All antennae tissues were immediately frozen in liquid nitrogen and subsequently stored at −80℃ for further use. All samples of beetle antennae were further divided into 3 groups (20 beetle antennae each group) to isolate the total RNA. TRIzol reagent (Life Technologies) was used to extract total RNAs following the manufacturer’s protocol. RNA samples were treated with RNase-free DNase I (Life Technologies) to remove any genomic DNA. Next, the RNA samples for each group were evaluated by 1% agarose gel electrophoresis and Agilent 2100 Bioanalyzer (Agilent Technologies). High-quality RNA samples were used for library preparation. cDNA Library Construction and Transcriptome sequencing Ten µg of total RNA each sample was used to isolate mRNA with oligo (dT) magnetic beads. The cDNA library was constructed using NEBNext mRNA Library Prep Reagent Set (NEB, Ipswich, MA, USA) following the manufacturer’s protocols. The library was sequenced on Illumina HiSeq 2000 (Illumina, San Diego, CA, USA) at BioMarker company (Beijing, China). Libraries were constructed with 1.5 µg purified RNA using a TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, United States) following the manufacturer’s instructions. The libraries were paired-end sequenced using the Illumina HiSeq 2000 platform. Clean reads were obtained after primers and adapters were removed from raw data. Transcriptome Data Assembly The clean data with high quality obtained from the Illumina platform were then assembled by Trinity (Version: r2013-11-10. Parameters: –min contig_length (the minimum length of assembled contigs): 200; –group_pairs_distance (insert size): 500 bp). The other parameters were set to the default values after combining all clean reads. The largest assembled sequences were deemed to be “unigenes”. Reads are fragmented into smaller pieces, known as K-mers. These K-mers are then used as seeds to be extended into contigs, and components are constructed based on contig overlaps. The open reading frames (ORFs) of sequences were predicted using an online program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Functional Annotation The sequences of Unigenes were annotated using databases including NR, Swiss-Prot, GO, COG, KOG, eggNOG, and KEGG. The annotation of unigenes was performed using NCBI BLASTx searches against the Nr protein database, with an E-value threshold of 1e-5. The BLAST results were then imported into the Blast2GO pipeline for GO annotation. The longest ORF for each unigene was determined using the NCBI ORF Finder tool (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The amino acid sequences of unigenes were predicted and the related sequences were annotated by blasting against the Pfam database by HMMER. TransDecoder was used for amino acid prediction. The thresholds were set with a BLAST E-value no larger than 1e-5 and an HMMER E-value no larger than 1e-10. KEGG Orthology of unigenes was obtained by KOBAS2.0. Genes Identification To identify the genes related chemosensory proteins in L. sifanica , a total of 1186 nucleotide sequences of chemosensory proteins of other beetles were downloaded from NCBI and severed as bait sequences. The related species included Agrilus planipennis (14), Dastarcus helpophoroides (37), Dendroctonus adjunctus (32), Dendroctonus ponderosae (68), Dendroctonus valens (46), H. cichorii (374), H. phaleratus (286), Ips typographus (41), Monochamus alternatus (55), and T. castaneum (233) . All above beetle sequences were carried out sequence similarity searching with nucleotide sequence database of L. sifanica by local BLASTx. The candidate sequences with high similarity to the bait chemosensory proteins were further aligned with the Nr database of NCBI to determine their homology. Next, all identified sequences were performed protein family analysis according to the pattern characterization of OBPs, CSPs, ORs, GRs, IRs, and SNMPs, respectively. Finally, all identified sequences were combined using BLASTx and BLASTn software and produced a non-redundant gene set for manual examination. Besides, the prediction of signaling peptide for odor-binding and chemosensory proteins were carried out using SignalP 6.0 - DTU Health Tech. Expression levels of different olfactory genes in the antennae of L. sifanica were assessed using FPKM values. All identified chemosensory proteins were used to identify motif patterns. The MEME (Version 5.1.1) on the line server (https://meme-suite.org/meme/) was used for discovery and analysis of the motifs with the following criteria: minimum width = 6, maximum width = 10, and the maximum number of motifs to discover = 8. Phylogenetic Analysis To further explore the evolutionary patterns of chemosensory proteins in L. sifanica , we rebuilt the phylogenetic tree using olfactory-related genes identified in L. sifanica and other beetle species. Amino acid sequences of identified OBPs, CSPs, ORs, IRs, GRs, and SNMPs from L. sifanica were aligned together with proteins from other Diptera, such as H. cichorii, H. phaleratus, D. ponderosae, and T. castaneum . Firstly, the multiple sequence alignments were performed for all members of each family of chemosensory proteins by using ClustalX 2.0. The matrixes of sequence alignments were further to estimate the best-fitting substitution model using IQtree software. Consensus rooted trees were reconstructed based on a maximum-likelihood (ML) analysis with Statistical methods and 1000 bootstrap trials by using the iTOL online servers. Phylogenetic trees were colored and arranged by using Fig Tree (Version: 1.4.2). RT-PCR To investigate the expression profiles of identified chemosensory proteins in different tissues of L. sifanica, we dissected the head, pronotum, foreleg tarsus, abdomen skin, wings, mouthparts, and antennae of L. sifanica and extracted total RNA independently using Trizol TM Reagent (invitrogen). Next, the cDNA samples of different tissues were synthesized using Oligo(dT) 15 Primer (Promega). The expression profiles of OBPs, ORs, CSPs, SNMPs, GRs, and IRs among various tissues were initially evaluated using RT-PCR. ATP8 was evaluated and used as the most stable reference gene for gene expression profiling analysis ( Table S5 ). RT-PCR reactions were conducted using 2 × F8 FastLong PCR MasterMix (Aidlab). RT-PCR products were analyzed by electrophoresis in 1% agarose gels. Statistical Analysis Data collected from these assays were subjected to analysis of variance and perform the figures using InStat software (GraphPad Inc, San Diego). The significant difference analysis of each gene among various tissues was determined using a one-way nested analysis of variance (ANOVA), following by Duncan’s new multiple range test (α = 0.05).Values are presented as mean±SE. Transcriptome sequencing and assembly process In total, approximately 18.12 Gb clean data were generated in antennae of L. sifanica. After filtering adapters and low-quality raw sequences, the data of each sample had reached 5.73Gb. The Q30 base percentage of all clean data exceeded 90.36% with GC content of 37.91%. The clean reads from all three samples were assembled into a single transcriptome, yielding 66,945 transcripts with an average length of 1,572 bp. A total of 35,136 unigenes were screened, with an N50 of 1,629 bp. Length distribution analysis indicated 8,214 unigenes, which accounted for 23% of all unigenes, were longer than 1 kb (Figure S1) . The clean reads were deposited at the National Center for Biotechnology Information (NCBI) – Sequence Read Archive (SRA) database with the submission number: PRJNA1221373. Functional Annotation and Species homology analysis A total of 17,094 unigenes (48.7% of total unigenes) were annotated using multiple databases including COG, GO, KEGG, KOG, Pfam, Swissprot, TrEMBL, eggNOG, NR, and the number (%) were 3,984 (23%), 14,079 (82%), 12,053 (70%), 9,942 (58%), 11,277 (65%), 7544 (44%), 15914 (93%), 12526 (73%), and 15801 (92%) respectively (Figure S2A). Among the annotated unigenes matched to the Nr database using the Blastx algorithm (≤ E-value of 1e-5), the highest match percentage (35.39%) was identified with sequences of T. castaneum followed by sequences of Asbolus verrucosus (21.15%) and Bombus terrestris (5.38%). The number (proportion) of genes annotated to other species in all annotated genes as shown in. Gene ontology (GO) analysis was used to classify unigenes into different functional categories. In the “biological process” category, the subcategory “cellular” process was the most abundant assignment followed by “metabolic” and “biological regulation” processes. Within the “molecular function” category, the subcategory “binding” and “catalytic activity” process were the most represented. In the “cellular component” category, the subcategory “cellular anatomical entity”, “protein-containing complex”, “intracellular” was the most represented (Figure S2B) . Candidate Odorant binding proteins (OBPs) analysis Seventeen candidate OBPs were identified in the antennal transcriptome of L. sifanica with an average sequence length of the 138 amino acids. Sequence identities of predicted OBPs with those from other beetles in the NCBI-nr database ranged from 0.9% to 35.39%, with an average of 9.10%. Among identified unigenes of OBPs, eleven members had a full-length ORFs encoding proteins and encoded a putative signal peptide at the N terminal region. Remaining members had partial sequences of ORF without successfully predicted signal peptides ( TableS1: sheet1 ). In general, OBPs can be classified into several different groups. Classic OBPs are characterized by six cysteine residues at conserved positions. The Plus-C class has 4 to 6 additional cysteines, whereas the Minus-C class has lost cysteine residues, generally C2 and C5 (Li et al., 2015). According to these criteria, five OBPs ( Lsif_OBP8 , Lsif_OBP17 , Lsif_OBP56d1 , Lsif_OBP5, and Lsif_OBP70 ) were classified as classic OBPs. Seven OBPs ( Lsif_OBP6 , Lsif_OBP99a , Lsif_OBP56d2 , Lsif_OBP2 , Lsif_OBPC20 , Lsif_OBP7, and Lsif_OBPC20 ) and five OBPs ( Lsif_OBP83a1 , Lsif_OBP69a , Lsif_OBP83a2 , Lsif_OBP1, and Lsif_OBP19d ) were classified as the Minus-C and Plus-C OBPs respectively ( Figure 2A ). All identified OBPs were further analyzed for motifs and found eight their members in their family ( Figure S3 ) . Motif 1 was presented in all OBP proteins, except for Lsif_OBP7 . Motif 2 and motif 3 were employed by eight and ten OBP proteins, respectively. Other motifs were found in fewer than six sequences . The motif 1, 2, 3 and 5 have all highly conserved cysteine residues (C), and the other motifs also have some conserved amino acids such as Serine (S), Tyrosine (Y), and Aspartic acid (D), which makes the structural domain of OBPs relatively conserved. All motifs demonstrated the largely consistent and partially variable evolutional patterns of conservated regions in sequences of OBPs family in L. sifanica ( Figure S3 ). To illustrate the evolutionary relationships of identified OBPs in L. sifanica, t he phylogenetic tree was constructed using 278 protein sequences of OBPs from 11 beetle species. The results showed that all OBPs were built a tree with eight distinct branches (I-Ⅷ). All 17 OBPs of L. sifanica were distributed along various branches, and each was clustered with at least one ortholog from other beetles according to their types or classifications ( Figure 2B ) . For example, seven OBPs ( Lsif_OBP5 , Lsif_OBP1 , Lsif_OBP70 , Lsif_OBP19d , Lsif_OBPC20 , Lsif_OBP69a , and Lsif_OBP5 ) were clustered with their orthologs of other beetles, respectively. Lsif_OBP7 and Lsif_OBP17 , Lsif_OBP2 and Lsif_OBP56d2 , Lsif_OBP6, and Lsif_OBP8 clustered together and were separately clustered with their orthologs of other beetles, respectively. Lsif_OBP99a , Lsif_OBP83a1 , Lsif_OBP56d1 , Lsif_OBP83a, and Lsif_OBP99a were located on the root of tree and not clustered with other homologs of beetles. It is possible that these sequences are new to evolution, or that the sequences are partial loss of sequences during assembly and annotation. Candidate Chemoseneory Proteins (CSP) analysis A total of five CSPs ( Lsif_CSP1 , Lsif_CSP2 , Lsif_CSP3 , Lsif_CSP4 , and Lsif_CSP6 ) were identified in the antennal tissue of L. sifanica, with sizes ranging from 117 to 163 amino acids . Among them, four members ( Lsif_CSP1 , Lsif_CSP2 , Lsif_CSP3 , and Lsif_CSP6 ) had a complete open reading frame (ORFs) and a predicted signal peptide at the N terminal region, except for Lsif_CSP4 , which was incompletely assembled due to a lack of a 5’ terminus (Table S1: sheet2) . All these CSPs have the highly conserved pattern of four cysteine residues (Cys-X6-Cys-X18-Cys-X 2-Cys, where X represents any amino acid), which is characteristic of CSP gene family (Figure 3A) . All candidate CSP genes had four conserved cysteines in the corresponding positions and a conserved OSD domain (InterPro: IPR005055). For CSP family of L. sifanica, a total of eight motifs were identified. Motif 1 was present in all CSP members. Their structure shows three cysteine residues, which is consistent with the structure of the insect CSP gene family. Motif 2 were appeared in four of five CSP members. Both of two members were highly conserved. Other motifs were partially appeared in CSP family members ( Figure S4 ). Motif analysis results clearly provide us the structural characteristics of OBP sequences, and different conserved segments. The phylogenetic tree was constructed using the amino acid sequences of 103 CSPs from nine species ( L. sifanica, I. typographus, A. planipennis, T. castaneum, D. valens, M. alternatus, D. ponderosae, D. adjunctus, and D. helpophoroides ). All CSPs formed four clades (A-D) and five CSP members in L. sifanica were clustered with at least one beetle ortholog and were shown in Clade B and D. Among the five candidate CSPs, two members (Lsif_CSP6 and Lsif_CSP3) clustered together with T. castaneum CSPs (Tcas_CSP1 and Tcas_CSP20) separately. The other two members (Lsif_CSP1 and Lsif_CSP2) of L. sifanica were clustered together with D. ponderosae (Dpon_CSP1 and Dpon_CSP20), only Lsif_CSP4 was clustered together with Dval_CSP5 ( Figure 3B ). Thus, the CSPs of L. sifanica we found are homologous to CSPs in beetles. However, there are no CSP members of L. sifanica located on branch A and C, which may suggest that some members are lacked in evolutionary branches, in which other beetles’ CSP are expanded. Candidate Gustatory Receptors (GRs) analysis Thirteen candidate GRs were identified in the antennal transcriptome of L. sifanica , with an average sequence length of 175 amino acids. However, only five of them contained a complete open reading frame (ORFs) and the remaining sequences were incomplete due to a lack of a 5’or 3’ terminus. Some GRs encoded longer ORFs than those of other members (Table S1: sheet3) . We identified a total of three motifs in the GR family of L. sifanica . Only motif 1 was predicted for all GRs of L. sifanica , except Lsif_GR7. Motif 2 and 3 were rarely appeared in only two sequences . This result suggested low conserved regions in sequences between members of GRs family in L. sifanica ( Figure S5 ). A GR phylogenetic tree based on 224 protein sequences from eight beetles ( L. sifanica, H. cichorii, H. phaleratus, M. alternatus, D. ponderosae, D. valens, I. typographus, and D. helpophoroides ) was then constructed. All the GRs of L. sifanica were clustered with their orthologs from other beetles into five distinct clades. Four GR proteins ( Lsif_GR12 , Lsif_GR12a, Lsif_GR64f, and Lsif_GR7 ) were grouped with their homologs in the sugar GRs clade. Three members ( Lsif_GR2, Lsif_GR127, and Lsif_GR28a ) were clustered with known fructose receptors of other beetles and formed a separate branch. Lsif_GR24 and Lsif_GR2a were clustered together with other known Carbon dioxide GRs and formed a clade. The remaining three GRs are distributed in different branches and show homology with GR orthologs of other beetle species, which are significantly expanded in GR family ( Figure 4 ). Therefore, the GR members we identified contain a richer functional classification, although there is a clear GR expansion with other species of Meloidae. Candidate Ionotropic Receptors (IR) analysis A total of thirteen putative IRs were identified from the antennal transcriptome of L. sifanica, with the average sequence length of 256 amino acids . Among these putative IRs, only two members contained a complete open reading frame (ORFs), and the remaining IRs were incomplete due to a lack of a 5’ or 3’ terminus. Except for the Lsif_IR2a , there were no signal peptides were predicted in other IR sequences (Table S1: sheet5) . The prediction of the transmembrane structure showed that only Lsif_IR_56e demonstrated four transmembrane structures. The predicted motif showed that a total of 6 motif in the IR family of L. sifanica . Nevertheless, all motifs were partially present in members of IRs family and showed inconsistent orders except for Lsif_IR19 , other IR members possessed less than four of six motifs, which suggested that low conservation in evolution of IR family in L. sifanica ( Figure S6 ). Based on IRs of L. sifanica combined with the four beetle species ( H. Cichorii, H. Phaleratus, D. helophoroides, and M. alternatus ), the phylogenetic analysis was constructed with six clades. Two IRs ( Lsif_IR_25a and Lsif_IR6 ) of L. sifanica clustered separately with orthologs of coreceptor IRs from other beetles. Lsif_IR13 and Lsif_IR76b were separately clustered with their orthologs of other beetles in two clades of antennal IRs. Lsif_IR56e clustered with orthologs of other beetles in the clades 41a expression IRs. The other nine IR members of L. sifanica were clustered with their orthologs of other beetles and formed a separate clade A, which is located at root of phylogenetic tree ( Figure 5 ) . These results illustrate that the IR members we identified are homologous genes of the IR family of other blister beetles, in which there is a significant expansion of IRs in these species. Candidate Odorant Receptors (ORs) analysis A total of seventeen ORs were analyzed from the antennal transcriptome of L. sifanica , with an average sequence length of 306 amino acids. Nine putative ORs had complete open reading frame (ORFs) that encoded 107-478 amino acids. The remaining ORs were incomplete due to a lack of a 5’ or 3’ terminus. Three ORco ( Lsif_ORco1, Lsif_ORco2, and Lsif_ORco3 ) were identified with full-length ORFs that encoded 228-478 amino acids (Table S1: sheet5) . The predicted transmembrane structure showed that six members have 6 to 7 transmembrane domains (TMDs), which is characteristic of typical insect ORs (Cheema et al., 2021). However, the others showed less than five TMDs in sequence of amino acids, which may be caused by incomplete ORFs ( Figure S9 ). We found 8 motifs in the OR family of L. sifanica. Motif 1-3 are the most conserved and are all located in nine OR members. However, five OR members ( Lsif_OR 1, Lsif_OR4 , Lsif_OR10 , Lsif_OR14, and Lsif_OR17 ) showed 1-2 motifs in their sequences, which may be due to a lack of a 5’ or 3’ terminus ( Figure S7 ). The motif locations suggested that the C terminalis of OR family in L. sifanica is more conservative. A phylogenetic tree was constructed using identified ORs in L. sifanica along with known members from the other eight beetles, I. typographus , H. Cichorii , H. phaleratus , T. castaneum , D. valens , D. ponderosae , D. helophoroides , and M. alternatus . The result demonstrated that 444 proteins clustered in the phylogenetic tree and formed six evolutionary clades (I-VI). Each of ORs in L. sifanica was clustered with their orthologs from other beetles and formed distinct branches. Two ORco ( Lsif_ORco1 and Lsif_ORco2 ) of L. sifanica were clustered with orthologs from other beetles and formed separate a clade VI in root of phylogenetic tree, which showed high conserved and diverged early from other OR members in evolution, which were consistent with findings in other Coleoptera species (Gu et al., 2015). Several species-specific clades were also observed, which clustered from extended members of ORs from same beetle species, such as T. castaneum and H. cichorii ( Figure 6 ). Totally, the OR members of L. sifanica we identified were homologous from other beetles, but the expansion in the number of family members was not found in this beetle species. Candidate Sensory Neuron Membrane Proteins (SNMP) analysis Five candidate SNMPs were identified in the antennal transcriptome of L. sifanica . Among them, four members ( Lsif_SNMP1 , Lsif_SNMP2 , Lsif_SNMP3, and Lsif_SNMP4 ) showed a complete open reading frame (ORFs) that encoding 154 to 568 amino acids (Table S1: sheet6) . The motif analysis showed that five SNMP proteins of L. sifanica have 8 motif structures. Each motif was partially present in family members of SNMPs. Lsif_SNMP1 , Lsif_SNMP2, and Lsif_SNMP4 have the same number of motifs, which are 1-8. Lsif_SNMP2 and Lsif_SNMP5 showed less than three motifs in each sequence , which may be caused by incomplete ORF ( Figure S8 ). To reveal the homologous relationships of all putative SNMPs in L. sifanica with other beetle gene sets, the phylogenetic tree was constructed based on 36 protein sequences from eight beetles ( H. cichorii , T. castaneum , A. planipennis, and I. typographus ). Five distinct clades (Clade A-E) were clustered in the phylogenetic tree. The five SNMPs of L. sifanica were clustered with their orthologs of other beetles and distributed among three different branches (Clade A, B, D). Among them, Lsif_SNMP3, Lsif_SNMP1, and Lsif_SNMP4 were orthologs of Hcic_SNMP3, Apla_SNMP1 , and Tcas_SNMP1a, respectively ( Figure 7 ). In summary, the SNMP members we found in L. sifanica cover the corresponding gene members of the other beetles with high evolutional conservation and employed more family members than the other blister beetles. Tissue-Specific Expression patterns of olfactory-related protein in L. sifanica To further illustrate the expression patterns of identified olfactory-related proteins, the relative expression levels (FPKM values) in the transcriptome of the antennae were analyzed for all members of related families. RT-PCR was used to characterize the expressional patterns of identified olfactory-related proteins in multiple tissues of L. sifanica including antennae, mouthparts, head (without antennae and mouthparts), pronotum, foreleg tarsus, abdomen skin and wings. For OBP proteins, RT-PCR validated that five OBPs ( Lsif_OBP83a2 , Lsif_OBP2 , Lsif_OBP19d , Lsif_OBP83a1, and Lsif_OBP1 ) were almost exclusively expressed in antennae. Among them, Lsif_OBP83a2 , Lsif_OBP83a 1, and Lsif_OBP19d were highly expressed with FPKM values ≥ 1,000 ( Figure S10e ). Lsif_OBP2 and Lsif_OBP1 demonstrated high expression in foreleg tarsus and pronotum, respectively. Four members ( Lsif_OBP99a , Lsif_OBP7 , Lsif_OBPC20, and Lsif_OBP5 ) were exclusively expressed in the head or pronotum, respectively. Three members ( Lsif_OBPC70 , Lsif_OBP56d2, and Lsif_OBP56d1 ) were exclusively expressed in the mouthparts. Two members ( Lsif_OBP69a and Lsif_OBP17 ) were specifically and exclusively expressed in four tissues (head, foreleg tarsus, antennae, and mouthparts or wings). The remaining OBPs, including Lsif_OBP3 were abundant in all analyzed tissues ( Figure 8A ). These results showed that the OBP family members of L. sifanica can be expressed in multiple tissues, but antennae followed by the mouthparts are dominant expressed tissues, in which some members are specifically expressed. For OR family, RT-PCR proved that four members ( Lsif_OR20 , Lsif_OR2 , Lsif_OR49b, and Lsif_OR14 ) were almost exclusively expressed in antennae, despite some members showing low FPKM values less than 2, except for Lsif_OR14 with 42.15 (Figure S10f) . Lsif_OR49b and Lsif_OR14 also showed higher expression in head and foreleg tarsus, respectively. Other four members ( Lsif_OR6 , Lsif_OR67c , Lsif_OR19, and Lsif_ORco2 ) were almost exclusively expressed in foreleg tarsus. Lsif_OR19 also showed higher expression in head. Among them, Lsif_OR19 and Lsif_ORco2 also showed relatively high FPKM values in antennae with 37.40 and 281.57, respectively. Lsif_OR9a showed almost exclusively expressed in the head. Five OR members ( Lsif_OR4 , Lsif_OR4a , Lsif_OR15 , Lsif_OR13, and Lsif_ORco3 ) showed higher expression in the pronotum. Other OR members showed constitutive expression in multiple tissues ( Figure 8B ). These results show that the ORs of L. sifanica are also expressed in multiple tissues, but the antennae and foreleg tarsus tend to be the tissues where Ors are specifically expressed. For the IR family, three IRs ( Lsif_IR2a , Lsif_IR7, and Lsif_IR16 ) showed remarkable tissue-specific expression in antennae, pronotum and foreleg tarsus, respectively. Four other IRs ( Lsif_IR56e , Lsif_IR22, and Lsif_IR76b ) showed higher expression in the head, but Lsif_IR 25a, Lsif_IR 22 and Lsif_IR76b were also highly expressed in mouthparts and antennae or pronotum and foreleg tarsus. Other members showed higher expression in the mouthparts, but also constitutive expression in multiple tissues ( Figure 8C ). However, only three members ( Lsif_IR6 , Lsif_IR76b, and Lsif_IR25a ) were highly expressed in the antennae of L. sifanica . Other members were lowly expressed with FPKM values less than 10 (Figure S10b) . Overall, we found that the IRs of L. sifanica are widely expressed in several tissues, but IRs showed the highest expression in the mouthparts, and some members were specific expressed in head tissue. For the GR family, Lsif_GR127 and Lsif_GR7 were almost exclusively expressed in antennae, despite have low FPKM values less than 2. Lsif_GR12 and Lsif_GR65f were mainly expressed in foreleg tarsus and pronotum, respectively. Five GR members ( Lsif_GR12a , Lsif_GR21 , Lsif_GR28a , Lsif_GR68a, and Lsif_GR24 ) were mainly expressed in the mouthparts, but Lsif_GR24 was also higher expressed in head and pronotum. The remaining GRs were abundant in multiple tissues. Almost all members of L. sifanica were lowly expressed with FPKM values less than 10 (Figure S10f). Totally, the GRs in L. sifanica were expressed in multiple tissues, but dominantly expressed in the mouthparts ( Figure 8D ). For CSPs expression analysis, we find that all members showed highly expression in antennae of L. sifanica (Figure S10d) . For example, Lsif_CSP2 , Lsif_CSP3, and Lsif_CSP4 showed FPKM values more than 300. However, RT-PCR further validated that Lsif_CSP1 was almost exclusively expressed in the antennae. Lsif_CSP2 was more highly expressed in other tissues including mouthparts and foreleg tarsus. Other members (Lsif_CSP4 , Lsif_CSP6, and Lsif_CSP3 ) showed constitutive expression in multiple tissues ( Figure 8E ). These results suggest that although the CSPs of L. sifanica show high expression in the antennae, it does not exhibit the specific expressed in this part of the body . For the expression analysis of SNMPs, RT-PCR further validated that both members ( Lsif_SNMP2 and Lsif_SNMP4 ) were almost exclusively expressed in the antennae with high FPKM values ≥ 300 (Figure S10a) . Lsif_SNMP5 and Lsif_SNMP3 are two members that were mainly expressed in abdomen skin and wings respectively. Lsif_SNMP1 expressed in multiple tissues ( Figure 8F ). These results indicate that the SNMPs of L. sifanica shows high expression in the antennae . Discussion Olfaction related genes usually play important roles in the insects’ life (Zhou, et al，2004). They are capable of detecting chemical signals in their surrounding environment and relaying these signals through olfactory-related genes to their nervous system, thereby regulating vital activities such as orientation, mating behavior, foraging strategies, and predator avoidance (Zhang et al., 2021) . For example, in above study of olfactory genes of flies, it is a chain of multiple olfactory genes that transmits such olfactory information. Conversely, the high expression of certain olfactory receptor genes may be closely related to the species’ efficient odor molecule recognition capabilities. In Curculio Dieckmanni, it was discovered that Cdie_OR13 and Cdie_OR15 exhibit a pronounced female-biased expression (Ma et al., 2022). Therefore, this category of olfactory genes is essential for them when seeking mates or searching for oviposition sites. Therefore, understanding the genetic basis of these olfactory genes is crucial for comprehending their behavioral traits. In this study, we focused on the genetic bases of chemosensory system in Lytta sifanica , and 70 related proteins, including OBPs, CSPs, SNMPs, ORs, IRs, and GRs family members, were identified based on the antennal transcriptome. Evolutionary relationships of these genes with those from other blister beetles and Coleoptera were analyzed along with comparison of family characteristics. Surprisingly, these olfactory genes are not all highly expressed in the antennae, instead, many specifically highly expressed olfactory gene members have been found in multiple tissues such as the mouthparts, head, and foreleg tarsus. These genetic characteristics and unique expression patterns may reflect the intriguing life activities of L. sifanica . OBPs of insects are indispensable in odor processing, facilitating the transport of odorant molecules through the sensillar lymph and serving as the liaison between the external environment and olfactory receptors (Zafar, et al, 2022). Their functions usually rely on three stable disulfide bonds formed by six highly conserved Cys residues, which are remarkable features in their family identification (Zhou, et al, 2004). Seventeen OBPs were identified in the L. sifanica antennal transcriptome through homology searching, and further distributed to subclades of six Minus-C, six Plus-C and five Classic-C members. The motif patterns exhibited the main characteristics of the OBPs, in addition to highly conserved residues in all OBPs. This is generally consistent with the characteristics of the entire Coleoptera (Vieira and Rozas, 2011). However, compared to other beetles, the antennal transcriptome of the L. sifanica lacks the dimer-OBP subtype of OBP genes. This may be because the dimer-OBP subtype genes are not present in the antennae of blister beetles but are instead found in other tissues. Compared to existing studies on I. typographus and D. ponderosae , the L. sifanica beetle has an additional Classic-C subtype, which may be related to certain biological functions (Andersson et al., 2013). Previous studies have suggested that Minus-C OBPs are more abundant in insects, whereas Plus-C OBPs are more abundant in higher species (Spinelli et al., 2012; VieiraRozas, 2011). The number of these subtypes in the blister beetle transcriptome analysis is generally consistent, whereas in studies of blister beetles and other species, the Minus-C type of OBP is the most abundant (Capinera et al., 1985). This may be because this subtype of OBP genes is primarily present in the antennae, where they play a role, possibly related to odor recognition functions. The phylogenetic analysis of OBPs from several beetles showed that the Minus-OBP type in this blister beetle forms an independent branch with homologs from other beetles, while the classic-OBP and Plus-OBP appear to have lower intraspecific homology and are located at the base of the phylogenetic tree, which reflects the sequence conservation within subtypes of Minus-OBP genes during evolution. Tissue-specific expression does not seem to be directly related to subtypes. However, we found that OBPs of each subtype in L. sifanica are specifically expressed in the antennae. The antennae appear to be a key tissue for OBP expression. For example, in Tenebrio molitor, approximately half of the OBPs are predominantly expressed in the antennae (Liu et al., 2015) . Additionally, many members are specifically expressed in the labial palps, while others exhibit widespread constitutive expression. Recent studies have shown that OBPs also play a role in non-chemoreceptive tissues, such as pheromone glands, where they are involved in pheromone release (Dani et al., 2011; Jacquin-Joly et al., 2001) . These OBPs with tissue-specific expression in non-antennal tissues warrant further functional investigation. CSPs are conserved family of small binding proteins which can bind pheromone compounds (Agnihotri, et al，2022). We totally identified five CSP members in the antennae of this blister beetle. The high-level similarities found in Blastp best-hit results demonstrated that CSPs were highly conserved proteins insects. Comparing Lsif_CSPs (five CSPs) gene numbers with those Coleopteran species, there were fewer than in Leptinotarsa decemlineata (15 CSPs) and Glnea cantor (14 CSPs) (Liu et al., 2012), and similar number of CSP gene in Callosobruchus maculatus (Wang et al., 2017). Their amino acid sequence revealed a typical four-cysteine motif at conserved positions, conforming to the CSP model of C1-X6–8-C2-X16–21-C3-X2-C4 (X represents any amino acid) (Dippel et al., 2014). The Motif analysis shows that the second, third, and fourth cysteines are all included in the Motif1 structure, while the first cysteine is included in the Motif2. This is consistent with the multiple sequence alignment results of L. sifanica. This indicates that the CSP gene of L. sifanica is highly conserved in structure, suggesting its critical role in the life activities reported on other beetles (Wanner et al., 2004) . In the phylogenetic tree, five Lsif_CSPs were distributed in different branches, suggesting that these may have undergone rapid evolution in adaptation to ecological changes. Lsif_CSP1, Lsif_CSP2, Lsif_CSP3, and Lsif_CSP4 cluster near the root of the phylogenetic tree, indicating that these CSP genes are relatively conserved during evolution (Capinera et al., 1985) . Such genes typically have less specialized functions, retaining more of their original roles and are likely widely present across organisms, having undergone minimal divergence or adaptation (Wang et al., 2017) . In contrast, Lsif_CSP6 clusters near the terminal branches of the phylogenetic tree ( Figure 3B ) , suggesting that this gene is a specialized version derived from ancestral genes. It may have experienced more mutations or adaptations, allowing it to play a specific role in certain organisms or functions, reflecting higher specificity or specialization, and possibly significant changes in structure or function (Wanner et al., 2004) .This is further supported by tissue-specific expression analyses. For example, Lsif_CSP3 and Lsif_CSP4 are expressed in multiple tissues, indicating their broad functional roles. Meanwhile, Lsif_CSP6 is highly expressed in the head and mouthparts, suggesting that this gene is likely involved in prey capture and host plant detection . However, Lsif_CSP1 displayed obvious antenna-specific expression, indicating that it may play important roles in antennal pheromone recognition. GRs are have also been found in various insect species and mostly expressed in gustatory receptor neurons in taste organs and are involved in detection of sugars, bitter compounds, CO 2 and some pheromones (Chen, et al，2019) . Therefore, insects often need to evolve different members to fulfill these functions. In this study, a total of thirteen GRs family members were identified in antennae of L. sifanica. These sequences maintained a certain degree of conservation within the species, a characteristic that was also supported by motif analysis. The GR genes of L. sifanica showed a conserved motif1 structure, like the GR gene structures of other Coleoptera beetles. In phylogenetic tree, they can cluster on the same branch as the corresponding GRs of other beetles and is supported by different types based on the detectable pheromones. Lsif_GR2a and Lsif_GR 24 are grouped in the clade of carbon dioxide receptor families , and the position on the phylogenetic tree is consistent with that in previous studies of other insects, they may be involved in carbon dioxide Receiving and transmitting (Wu et al., 2020). Lsif_GR 12a, Lsif_GR 64f, Lsif_GR 7, and Lsif_GR 12 were clustered with the sugar receptors of H.cichorii and H.phaleratusr , they may play an roles in host plant selection. However, the specific functions of these olfactory genes still require further experimental validation. H. Cichorii and H. phaleratus have obvious GR expansion in phylogenetic tree , and these members rarely appear in other beetles. The number of GRs in L. sifanica was lower than that in H. Cichorii (102) and H. phaleratus (86) (Wu et al., 2020), presumably because antennae are not major gustatory organs for GR gene expression, which really were confirmed by the results of specific expressed in mouthparts of L. sifanica in our research. Most GR genes of L. sifanica are abundantly expressed in the mouthparts , which was consistent with their roles in the taste recognition (Wu et al., 2018) . Interestingly, Lsif_GR127 and Lsif_GR7 seem to be mainly expressed in the antennae, while Lsif_GR12 is mainly expressed in the foreleg tarsus. These members maybe valuable in exploring the specificity of taste recognition in L. sifanica . Really, there were no report that found GR genes specifically expressed in foreleg tarsus in blister beetles (Chen et al., 2014). IRs are ligand-gated ion channels that mediate the majority of excitatory neurotransmission (Wu et al., 2020). Like other beetles with rich in IR receptor, a total of thirteen IR members in L. sifanica were found in this study. This is considerably lower than the numbers found in H.cichorii (50 IRs), H. phaleratus (45 IRs) (Wu et al., 2020), but greater than those in beetles Plagiodera versicolora (7 IRs) (Liu, 2021) and D. valens (3 IRs) (Gu et al., 2015). IRs are more highly conserved in insects than ORs and GRs (Croset et al., 2010), can be divided into divergent species-specific IRs and conserved antennal IRs (Benton et al., 2009). All the IR members identified in L. sifanica here have orthologs in other Coleoptera and can divided to different subgroups supported in phylogenetic tree. Like Orco, some IR members (IR8a and IR25a) in beetles really act as the co-receptors since they are co-expressed along with other IRs (Zhao et al., 2020). Lsif_IR6 and Lsif_IR25a may belong to the co-expression IR members in L. sifanica, duo to the clustered with clade of IR8a and IR25a rather than other members in the phylogenetic tree. To find evidence regarding expression levels, we conducted a tissue expression analysis and find most of the IRs in L. sifanica are highly expressed in the Mouthparts, which is consistent with the results of other reports (Wang et al., 2023). Lsif_IR2a displayed antennae-specific expression, which is consistently conserved among insect orders and function in olfaction in insects, Lsif_IR56e and Lsif_IR 25a are also expressed in the head. This is quite different from the reports on IR56e and IR25a in other beetles. It is very likely due to the differences in olfactory receptors among species with distant genetic relationships, or it may be caused by the inconsistent evolutionary environments . Remaining Lsif_IRs are expressed in multi-tissue which are divergent IRs and indicate their involvement in taste and food assessment (Croset et al., 2010). The expression level of IR genes in the antennae was relatively low FPKM values among all the olfactory genes. Some IR genes maybe not detected in the antennae in the electrophoresis map. Concerning IRs, the fact that more than one gene has been found clustering in the cladeA unexpected. A possible explanation is that these fragments are indeed part of one large IR gene that may result from one IR gene duplication in L. sifanica. The odorant receptors in insects are expressed primarily in olfactory sensory neurons and served as key players in insect olfactory reception (Wicher，D，2018). When odorant molecules interact with insects’ olfactory sensory neurons, the neural signal was initiated by interacting of odorant molecules with related receptors (Dobritsa, et al，2003). They can determine the sensitivity and specificity of odorant reception, being the centerpiece of the peripheral olfactory (Xia et al., 2008). Therefore, OR proteins are very diverse and often reflect the ecological niche of the various species (Cheema et al, 2021). Like other insects, there are a total of eighteen ORs members were found in antenna of L. sifanica , the number of which was lower than in H. Cichorii (149) and H. phaleratus (89) (Wu et al., 2020), but greater than in Galeruca daurica (10 ORs) (Li et al., 2017). Lsif_ORs were on the same branch and highly homologous with ORco of other species. This was consistent with fact that ORco genes are highly conserved in insect. Motif analysis showed that motif1and motif2 in coleopteran ORs had high similarity in these genes, indicating that they maybe perform the same function as HcicORco , such as could help other Lsif_ ORs localize to the dendritic membrane or better associate with odor molecules (Ma et al., 2019; Wu et al., 2020). Interestingly, different with other beetle, we found three Orco members in L. sifanica and showed different tissue expression . Among them, Lsif_OR co ( Lsif_ORco1 and Lsif_ORco2 ) were on the same branch and were highly homologous with Orco of H. Cichori and other species, this is like findings reported in other insects (Zhang et al., 2024). These two gene members further functional work in Lsif_OR co would allow a better understanding of ion channel formation. However, the other member Lsif_ Orco3 were clustered with other ORs in different branch and mainly expressed in pronotum tissues. This member was not reported by previous papers of beetles maybe suggested potential special functions. However, it may be misleading if one compares the number of genes identified in the genome with that of transcripts in a specific tissue at different life stages. Because some of the OR genes might be expressed only in the larva (Engsontia et al., 2008) or the non-sensory tissues (Gong et al., 2007). The difference in numbers and expressed locations of ORs may be due to the different chemical ecology for these species. Interestingly, we found that many OR members were only expressed in foreleg tarsus. This is basically consistent with the expression patterns of other beetles’ ORs. Because some of the OR genes might be expressed only in the larva (Engsontia et al., 2008) or the non-sensory tissues (Gong et al., 2007). The difference in numbers and expressed locations of ORs may be due to the different chemical ecology for these species. SNMPs are transmembrane domain proteins, and their main function is the recognition and transport of lipophilic odor molecules (Vogt et al., 2009). SNMP genes are expressed in the dendrite membrane of olfactory receptor neurons, and they play an important role in pheromone recognition (Forstner et al., 2008). Most studies have shown two SNMP genes in insects, namely, SNMP1 and SNMP2. the SNMP genes were missing or rare in members for many beetles (CassauKrieger, 2021). However, multiple SNMPs have been identified in a variety of Coleopteran species (Wang et al., 2023). Five SNMPs were identified in the antennal transcriptomes of L. sifanica , the number is greater than in D. ponderosae and I. typographus (3 SNMPs) (Andersson et al., 2013). In order to explore whether intragenomic duplication has occurred, phylogenetic analysis showed that each member of SNMPs clustered branches with their homologs of various beetle species rather than two close blister beetles, the reason of which maybe orthologs of Lsif_ SNMPs is lacked in other beetles H. Cichorii and H. phaleratus . Gene replications maybe inversely appeared in intraspecies of above beetles. With tissue-specific expression analysis, Lsif_ SNMP4 and Lsif_ SNMP2 exhibited antennae-specific expression, although they are distributed in different branches. Inversely, Lsif_ SNMP5 and Lsif_ SNMP3 were dominantly expressed in abdomen skin or wings in L. sifanica , all of which indicated that different members may be important in different chemoreception functions for L. sifanica . In summary, many olfactory-related genes of L. sifanica were firstly identified based on transcriptomic analysis. Every member showed distinct family characteristics of Meloidae or beetles supported by sequence and evolution analysis. However, the numbers of 70 related protein identified in this species were less than the close related beetle species H.cichorii (149 ORs, 102 GRs, 50 IRs) and H.phaleratusr (89 ORs, 86 GRs, 45 IRs), but is similar to beetle I.typographus (15 OBPs, 6 CSPs, 3 SNMPs, 7 IRs, 43 ORs) (Andersson et al., 2013). One reason for this difference is that our dataset was derived from transcriptomes rather than genome data for their closely-related species H.cichorii and H.phaleratusr (Wu et al., 2018). Another reason may be that the genes specifically expressed in other chemosensory organs were not found in our antennal transcriptome analyses. Additionally, lack of larva’s antennal maybe induced the integrality of mined family genes in adult L. sifanica, due to differential expression may exist between developmental stages in insect olfactory organs (Cao et al., 2014). Many open questions or saps in our research can’t be ignored. For example, OR members were remarkably less than that in other blister beetle species (Capinera et al., 1985). whether the result was occurred from gene family contraction or incomplete assembly, because ORs generally have relatively low expression levels (Li et al., 2022), which is still provide additional evidences. Some genes were incomplete annotated, or some new genes could not match in the database used during the assembly process (Arvind et al., 2020). The existence of Orco types remains mysterious (Zhang et al., 2021). It will be part of future studies to investigate if the related candidates form heterodimers with other receptors like GRs or with each other to build functional receptors or if they fulfill a channel function in other processes than olfaction (Christensen et al., 1988). Studies have shown that the expression levels of certain genes in non-olfactory tissues of insects are higher than those in antennae. These members are highly susceptible to omission in the antennal transcriptome analysis. This research only analyzed the expression patterns of olfactory genes in adult L. sifanica and did not carry out functional studies on these genes, so the role of these genes in the olfactory process cannot be clarified for the time being, and subsequent studies will use techniques such as olfactory receptor localization, heterologous expression of proteins, and RNA interference to elucidate the functions of coriander olfactory genes in odor recognition and other physiological processes (Field et al., 2000). Finally, in this experiment, only RT-PCR analysis of the specific expression of different olfactory genes in different tissues was done, and no expression differences between male and female were made, lacking further functional studies of olfactory genes (Xing et al., 2021). Cantharidin (C10H12O4, CTD) (Capinera et al., 1985) synthesized in vivo is a complex physiological process controlled by a variety of factors. However, relevant olfactory genes were not obtained in this study. It is one of the key points to continue to discover olfactory genes and build relationships with the synthesis and perception mechanism of cantharidin in the follow-up work. Funding This study was supported by the National Natural Science Foundation of China (No. 32160127 and No. 32360670). Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions All authors contributed to the study conception and design. Conceived and designed the experiments: Feng Zhou. Wrote the paper: Zhuan-xia Li, Feng Zhou. Analyzed the data: Zhuan-xia Li, Jia-ni Chen. Meanwhile, we would also like to express our gratitude to Xin-ge Song, Shu-ning Sun and Yu-ying Zhang for their assistance in our experiment. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data Availability The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request. Data is available at NCBI GEO, accession numbers: PRJNA1221373. https://www.ncbi.nlm.nih.gov/bioproject/1221373. Reference Andersson, M. N., Grosse-Wilde, E., Keeling, C. I., Bengtsson, J. M., Yuen, M. M., Li, M., Hillbur, Y., Bohlmann, J., Hansson, B. S., & Schlyter, F. (2013). Antennal transcriptome analysis of the chemosensory gene families in the tree killing bark beetles, Ips typographus and Dendroctonus ponderosae (Coleoptera: Curculionidae: Scolytinae). BMC Genomics , 14 , 198. Aoun, O., François, M., Demoncheaux, J.-P., & Rapp, C. (2018). Morning blisters: cantharidin-related Meloidae burns. Journal of Travel Medicine , 25 (1). Arvind, K., Rajesh, M. K., Josephrajkumar, A., & Grace, T. (2020). 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Collection Ecology and Evolution Keywords comparative ecosystem invertebrate laboratory molecular evolution sequencing statistical theory Authors Affiliations Feng Zhou [email protected] Northwest Normal University View all articles by this author Zhuanxia Li 0009-0004-3550-373X Northwest Normal University View all articles by this author Jiani Chen 0009-0009-1293-0110 Northwest Normal University View all articles by this author Xinge Song Northwest Normal University View all articles by this author Shuning Sun Northwest Normal University View all articles by this author Yuying Zhang Northwest Normal University View all articles by this author Liyuan Yao Northwest Normal University View all articles by this author Yuqin Wang Northwest Normal University View all articles by this author Xinyu Sun Northwest Normal University View all articles by this author Lixia wan Northwest Normal University View all articles by this author Metrics & Citations Metrics Article Usage 385 views 170 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Feng Zhou, Zhuanxia Li, Jiani Chen, et al. Chemosensory System Decoding: Transcriptome-Wide identification and Expression Profiling of Olfactory Genes in Lytta sifanica. Authorea . 15 March 2025. DOI: https://doi.org/10.22541/au.174204366.66023286/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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