Screening of Ester-Forming and Hydrolyzing Enzymes Linked to Pheromone Production in Ips typographus (Linnaeus, 1758)

Screening of Ester-Forming and Hydrolyzing Enzymes Linked to Pheromone Production in Ips typographus (Linnaeus, 1758) | 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 Screening of Ester-Forming and Hydrolyzing Enzymes Linked to Pheromone Production in Ips typographus (Linnaeus, 1758) Jaroslav Strádal, Rajarajan Ramakrishnan, Ondřej Lukšan, Michal Tupec, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7583299/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Mar, 2026 Read the published version in BMC Genomics → Version 1 posted 9 You are reading this latest preprint version Abstract Background: The bark beetle Ips typographus (Coleoptera: Curculionidae: Scolytinae) is a major pest of spruce trees in Central Europe. Its ecological success is mediated by a male-produced aggregation pheromone, which includes the monoterpene cis -verbenol. Cis -verbenol is biosynthesized from host-derived α-pinene, but can also be released through enzymatic cleavage of verbenyl-fatty acyl esters, which are initially produced by young beetles during maturation feeding and stored in their fat bodies. The main objective of this study was to identify the rarely studied ester-forming and hydrolyzing enzymes in I. typographus , and to suggest their possible roles in beetle metabolism. Results: By blasting reference gene set against a newly assembled I. typographus transcriptome and performing phylogenetic analyses, we identified 27 novel ester-modifying genes: 23 carboxylesterases, two (phospho)lipases, one notum-like gene, and one neurolactin-like gene. Full gene structures were described. Based on GC-MS measured production profiles of verbenyl oleate and cis -verbenol across beetle life stages and phenotypes, transcriptome pairs were selected for differential expression analysis. Eight genes were chosen for detailed RT-qPCR expression profiling across sexes, developmental stages, and tissues. Based on these findings, we propose possible roles of genes encoding enzymes in verbenyl-fatty acyl ester metabolism or broader lipid metabolic processes in bark beetles. However, functional validation through enzyme assays and gene silencing will be necessary to confirm their specific roles. Conclusion: Although the functions of these candidate genes remain hypothetical, the identification and structural description of 27 new ester-modifying enzymes provide important insight into this poorly characterized enzyme group in insects. Furthermore, understanding the genetic basis of cis -verbenol biosynthesis in I. typographus may support the development of novel, pheromone-based pest management strategies. European spruce bark beetle carboxylesterase lipase pheromone biosynthesis transcriptomics coleoptera lipid metabolism fatty acyl esters detoxification Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Background The European spruce bark beetle, Ips typographus (Coleoptera: Curculionidae: Scolytinae; Linnaeus 1758), is a major forest pest driving the decline of Norway spruce Picea abies (Pinaceae, H. Karst 1881) forest monocultures in Central Europe. Over the past decade, its outbreaks have intensified due to climate change and human activities, exacerbated by droughts and other forest disturbances. I. typographus uses a coordinated mass-attack strategy to colonize its host, the spruce tree. Male beetles produce an aggregation pheromone that attracts conspecific beetles, enabling the population to collectively overcome the defenses of trees and establish a new generation [ 1 , 2 ]. This pheromone is composed of three key hydroxylated terpenoids. The primary component, 2-methyl-3-buten-2-ol, and the minor component, ipsdienol, are synthesized de novo in the male midgut through the mevalonate pathway, triggered by feeding and mating, respectively [ 3 ]. In contrast, the third behaviorally active compound, ( S )-(−)- cis -verbenol, a cyclic monoterpenoid, is synthesized by feeding males via cytochrome P450 (CyP450)-mediated hydroxylation of (−)- α -pinene, the dominant monoterpene in host spruce resin, which the beetles ingest during feeding [ 4 , 5 ] (Fig. 1 A). In I. typographus , the utilization of cis -verbenol as a pheromone may have co-evolved with detoxification pathways for host-derived terpenes, enhancing the beetles' ability to exploit spruce defenses [ 6 ]. Besides being induced by direct feeding on the host tree, the production of ( S )-(−)- cis -verbenol can also be stimulated in the absence of (−)- α -pinene. Laboratory experiments showed that topical treatment of non-feeding males with juvenile hormone III (JH III) stimulates ( S )-(−)- cis -verbenol synthesis as well [ 7 ]. JH III is widely used to artificially induce de novo biosynthesis of bark beetle aggregation pheromones for experimental purposes [ 8 , 9 ]. These observations led to the hypothesis that, during colonization, males produce cis -verbenol not only through the direct hydroxylation of (−)- α -pinene, but also from alternative internal sources, specifically, cis -verbenyl fatty acyl (FA) esters stored in significant quantities in the fat body [ 3 ] (Fig. 1 B). A similar mechanism has been proposed for the Mountain pine beetle Dendroctonus ponderosae Hopkins, 1902, in which females were found to store the pheromone trans -verbenol in their fat bodies as verbenyl esters [ 6 , 10 ]. The biosynthesis of verbenyl esters in young I. typographus beetles and adult males is presumably catalyzed by either lipase/esterase enzymes or FA transferases. In pheromone-producing adult males, these esters could be hydrolyzed back to yield free cis -verbenol by a male-specific lipase or esterase, potentially under the regulatory control of JH III (Fig. 1 B). In a previous transcriptomic study, three candidate contigs, Ityp_7084, Ityp_9460, and Ityp_11977, were preliminarily selected as possible genes encoding such enzymes [ 3 , 7 ]. These candidates were identified based on their sequence similarity to esterase BT127766.1 from Dendroctonus ponderosae [ 9 , 11 ] as well as their expression patterns across different developmental stages. However, their functional roles remain unconfirmed, as previous analyses did not provide sufficient evidence to validate these contigs. Although ester-forming and -hydrolyzing enzymes likely play central roles in both pheromone biosynthesis and general metabolic processes across many insect taxa, they remain largely understudied. This knowledge gap may stem from their broad substrate specificity and functional redundancy, which complicate thorough experimental characterization. Among the ester-hydrolyzing enzymes documented in insects, the majority belong to the carboxylesterase (CE) family, with relatively limited diversity in enzymatic classes and functions [ 12 – 15 ]. In bark beetles, this pattern is consistent, most identified esterases are also CEs, which are frequently upregulated in response to host plant toxins. This suggests that they may play a role in detoxification by converting hydroxylated compounds into more water-soluble metabolites for excretion or into long-chain esters for storage [ 16 ]. Notably, nine CE have been reported in bark beetles closely related to I. typographus , particularly in the genus Dendroctonus [ 15 ]. In D. armandi , CE gene expression increases upon exposure to host plant compounds, reinforcing their role in plant defense neutralization [ 17 ]. Additional esterases and lipases have been identified in Hymenoptera [ 18 – 20 ], as well as in other insect species, including juvenile hormone esterase in Tribolium [ 21 ] and the antennal esterases in Spodoptera [ 22 ]. These enzymes may be involved in olfactory processing. Some ester-hydrolyzing enzymes have also been studied in the contexts of insecticide resistance [ 23 , 24 ], chemical communication, and lipid metabolism. A comprehensive list of ester-forming and hydrolyzing genes in insects with both experimentally validated and predicted sequences, is summarized in Table 1 , and selected genes served as the reference framework for the current study. In this study, we initially focused on identifying genes in I. typographus that may be involved in both the biosynthesis of verbenyl FA esters and their subsequent hydrolysis to cis -verbenol. Such identification could lend critical support to the currently unproven hypothesis that cis -verbenol, a major aggregation pheromone component in I. typographus , originates from detoxification-related lipid precursors. Additionally, we conducted a more extensive screening of ester-forming and -hydrolyzing enzymes in I. typographus and explored their potential biological functions. 2. Methodology 2. 1. Rearing beetles, treatment, and preparation of samples Norway spruce ( Picea abies L.) logs (20–30 cm DBH, 50 cm length), naturally infested by I. typographus , were collected from a forest near Kostelec nad Černými lesy (Czech University of Life Sciences Prague; 50°00′07.2″ N, 14°50′56.3″ E, Forest CZU) and stored at 4°C until use. The logs were then placed in ventilated plastic containers (55.5 × 39 × 28.5 cm; IKEA, Sweden) under controlled conditions (27 ± 1°C, 70% humidity, 16:8 h light: dark photoperiod). Upon emergence, 150 fully sclerotized individuals, unsexed F0 adults, were transferred to fresh spruce logs (of the same dimensions and origin) to initiate the F1 generation. Developmental stages of the F1 generation were sampled at defined time points: larvae L1, L2, and L3 at 7, 14, and 20 days post-colonization, respectively; pupae at approximately 4 weeks; immature adults (< 24 h post-eclosion); newly emerged adults after exiting the breeding logs; and adult males and females after 24 h of feeding in nuptial chambers excavated in uninfested spruce logs (hereafter referred to as fed males and fed females) Except for larvae and pupae, the collected beetles were sorted by sex based on external morphology and confirmed by reproductive organ dissection, according to [ 25 ]. Subsequently, larvae, pupae, immature, emerged, and fed adult beetles were further processed: For juvenile hormone III (JH III) induction, only newly emerged beetles were used. After sorting by sex, groups of beetles were topically treated with 0.5 µL JH III solution (20 µg/µL in acetone) on the abdomen, while the control group was treated with 0.5 µL of pure acetone. All beetles were then maintained under the previously described laboratory conditions for 8 hours [ 7 ]. Beetles from various life stages and treatment groups were flash-frozen in liquid nitrogen and stored at − 80°C for subsequent analysis. Prior to processing, guts were dissected from all beetles except larvae and pupae, for which dissection was not experimentally feasible. In beetles with dissected guts, the elytra, wings, and legs were further removed. The remaining tissue, referred to in this study as fat body, was used for metabolomic and differential gene expression (DGE) analyses. For RNA isolation, tissues were placed in a droplet of RNAlater (Invitrogen, Carlsbad, CA, USA) and stored at − 80°C for downstream applications. For metabolite production studies, dissected guts were immediately submerged in cold pentane (10 guts per 100 µL), while fat bodies were extracted with chloroform (10 bodies per 1000 µL). 2.2. GC–MS-based determination of verbenyl oleate and cis -verbenol in beetle tissues The separation of compounds in beetle tissue extracts, and target identification, and quantification of verbenyl oleate and cis -verbenol were performed using gas chromatography–mass spectrometry (GC-MS), following the protocols described by [ 3 , 7 ]. Analyses were conducted on an Agilent 7890B GC system (Agilent Technologies, Palo Alto, CA, USA) coupled with a Pegasus 4D Time-of-Flight Mass Spectrometer (LECO, St. Joseph, MI, USA). A programmed temperature vaporization (PTV) injector was used, operated in split mode (10:1) with the following temperature program: 20°C ramped at 8°C/s to 275°C. The separation was achieved on an HP-5MS UI capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness; Agilent). The oven temperature program was: 40°C (1 min hold), ramped at 10°C/min to 210°C, then at 20°C/min to 320°C (6 min hold). Electron ionization was performed at 70 eV, with a scanned mass range of 35–500 Da at an acquisition rate of 10 Hz. Identification of target compounds was confirmed using analytical standards of cis -verbenol and verbenyl oleate, in combination with the NIST 2017 mass spectral library. For quantification, linear calibration curves were constructed based on the respective external standards. 2.3. mRNA isolation and transcript Illumina sequencing RNA was isolated from 16 distinct sample groups representing seven developmental stages. Unsexed whole-body samples included the first-, second-, and third-instar larvae, as well as pupae. In addition, gut and fat body tissues were isolated separately from the following groups: immature females, immature males, emerged females, emerged males, fed females, and fed males. Each of these samples consists of four biological replicates, with each replicate comprising tissues from ten individual beetles. The only exception was fat body tissue from fed beetles, for which nine technical replicates per sex were prepared. Total RNA extraction was performed using the PureLink™ RNA Mini Kit (Invitrogen, Carlsbad, CA, USA), strictly following the manufacturer's protocol. Extracted RNA underwent DNase treatment using the TURBO DNase Kit (Invitrogen, Carlsbad, CA, USA). RNA integrity was assessed by electrophoresis on a 1% agarose gel, and samples were stored at − 80°C until sequencing. Five RNA samples from fat body tissues of fed adult beetles (both sexes) were selected and sent to Novogene Co., Ltd. (UK) for transcriptome sequencing, performed on an Illumina NovaSeq X Plus platform (paired-end sequencing, 150 bp reads). 2.4. Transcriptome assembly and ester-modifying protein identification De novo transcriptome assembly was conducted using SPAdes v3.15.5, pooling transcriptome data previously published under NCBI BioProject accession number PRJNA679450 (Male fed midgut – MFMg, Male immature midgut – MIMg, Female immature midgut – FIMg, Male immature fat body – MIFb, Female immature fat body – FIFb) [ 3 , 16 , 26 ], along with the transcriptomes from midguts of JH III- and acetone-treated males and females from NCBI Bioproject PRJNA934749 (Male JHIII midgut – MJMg, Male acetone midgut – MAMg, Female JHIII midgut – FJMg, Female acetone midgut – FAMg) [ 7 ], and newly generated RNA-seq data from the fat bodies fed adult beetles (Male fed fat body – MFFb, Female fed fat body – FFFb). Prior to assembly, raw sequencing reads were quality-filtered using Trimmomatic v0.39, which removed adapter sequences and trimmed low-quality bases. The quality of the resulting assembly was evaluated using RNAquast v2.3.1 [ 27 ] and BUSCO v5.5.0 [ 28 ], in eukaryotic transcriptome mode, employing the insecta_odb10 database. A manual inspection was performed to further confirm assembly completeness and reliability. Next, raw reads were aligned to the reference genome NCBI accession number GCA_016097725.1 [ 29 ] using STAR v2.7.6a [ 30 ]. Transcript abundance was estimated using the RSEM method (implemented via Trinity v2.15.1 scripts) and featureCounts v2.0.3 [ 31 ], following manual annotation of transcripts. Prediction of open reading frames (ORFs) from the assembled transcripts was carried out using TransDecoder.LongOrfs v5.7.1, and the resulting ORF set was again assessed with BUSCO using library insecta_odb10. Putative verbenyl ester-forming and -hydrolyzing enzyme genes were identified through local protein-protein alignment using BLAST v2.9.0+. Candidate sequences were filtered to retain those with a minimum of 50% sequence similarity and alignment lengths of ≥ 200 amino acids. Redundant hits and apparent artefacts were manually removed. To identify functionally relevant esterase genes, multiple sequence alignment was performed using Clustal Omega, MUSCLE, and MAFFT, all within SeaView v4.7 and AliView v1.28 [ 32 , 33 ]. Reference protein sequences were retrieved from GenBank and included both experimentally validated and computationally predicted sequences. To broaden our search and account for in insect uncharacterized functional enzyme classes with acyl esterification functions, we extended our references homology-based queries using sequences derived from phylogenetically distant taxa, including plants and vertebrates (Table 1 ). Table 1 Reference carboxylesterase (CE) and related ester-modifying protein sequences retrieved from GenBank. Accession numbers, source organisms, sequence types, and gene descriptions are provided, along with citation information. Both experimentally validated and predicted sequences from diverse taxa were included. Hydrolase and transferase sequences are marked with H and T, respectively. Enzyme class Accession No. of gene in GenBank [literature reference] Common name of the organism Scientific name of organism Description in database Used for BLAST H QWW26267.1 [ 34 ] Chinese White Pine Beetle Dendroctonus armandi Tsai & C-L. Li, 1959 (Z)-6-nonen-2-ol dehydrogenase H AYN64423.1 [ 15 ] Chinese White Pine Beetle Dendroctonus armandi Tsai & C-L. Li, 1959 carboxylesterase H AYN64424.1 [ 15 ] Chinese White Pine Beetle Dendroctonus armandi Tsai & C-L. Li, 1959 carboxylesterase H AYN64425.1 [ 15 ] Chinese White Pine Beetle Dendroctonus armandi Tsai & C-L. Li, 1959 carboxylesterase H AYN64426.1 [ 15 ] Chinese White Pine Beetle Dendroctonus armandi Tsai & C-L. Li, 1959 carboxylesterase H AYN64427.1 [ 15 ] Chinese White Pine Beetle Dendroctonus armandi Tsai & C-L. Li, 1959 carboxylesterase H AYN64428.1 [ 15 ] Chinese White Pine Beetle Dendroctonus armandi Tsai & C-L. Li, 1959 carboxylesterase H AYN64429.1 [ 15 ] Chinese White Pine Beetle Dendroctonus armandi Tsai & C-L. Li, 1959 carboxylesterase H AEE62728.1 [ 11 ] Mountain Pine Beetle Dendroctonus ponderosae Hopkins, 1902 unknown H UUB32789.1 [ 35 ] Red Turpentine Beetle Dendroctonus valens LeConte, 1857 carboxylesterase COEA1 H UUB32825.1 [ 35 ] Red Turpentine Beetle Dendroctonus valen s LeConte, 1857 carboxylesterase COEM1 H NM_001193294.1 [ 21 ] Red Flour Beetle Tribolium castaneum (Herbst, 1797) juvenile hormone esterase (Tcjhe) H NM_168643.3 [ 36 ] Common Fruit Fly Drosophila melanogaster Meigen, 1830 notum (Notum) H MW699017.1 [ 20 ] Fiery-tailed Bumble Bee Bombus ignitus Smith, 1869 venom carboxylesterase (vCaE) H NM_001287565.1 [ 18 ] Buff-tailed Bumble Bee Bombus terrestris (Linnaeus, 1758) lipase member H-A-like (LOC100646090) H XM_012694947.3 [ 37 ] Domesticated Silkmoth Bombyx mori (Linnaeus, 1758) lipase member H-A (LOC101742752) PLLG H XM_026443853.1 [ 19 ] European Honeybee Apis mellifera Linnaeus, 1758 lipase member H-A (LOC727193) H XM_006566867.3 [ 19 ] European Honeybee Apis mellifera Linnaeus, 1758 venom carboxylesterase-6-like (LOC408395) H KU360126.1 [ 38 ] Chinese Tussar Moth Antheraea perny i (Guérin-Méneville, 1855) lipase-related protein LRP H JF728804.1 [ 39 ] Beet Armyworm Spodoptera exigua (Hübner, 1808) antennal esterase CXE11 H UFA27653.1 [ 40 ] Tobacco budworm Heliothis virescens (Fabricius,1777) LipX protein, partial NO H UFA27658.1 [ 40 ] Tobacco budworm Heliothis virescens (Fabricius,1777) LipZ protein, partial NO H AAW28928.1 [ 41 ] Lesser Grain weevil Sitophilus oryzae (Linnaeus, 1763) pectin methylesterase NO H A0A0M3KKW3.1 [ 42 ] Black-bellied hornet Vespa basalis Smith, 1852 Phospholipase A1 NO H AAG42021.2 [ 43 ] Tobacco hornworm Manduca sexta (Linnaeus, 1763) juvenile hormone esterase precursor NO H ACV60237.1 [ 12 ] African cotton leafworm Spodoptera littoralis (Boisduval, 1833) antennal esterase CXE10 NO H UFA27654.1 [ 40 ] Tobacco budworm Heliothis virescens (Fabricius,1777) Est1 protein, partial NO H AAB67728.1 [ 44 ] Australian sheep blowfly Lucilia cuprina (Wiedemann, 1830) E3, carboxylesterase NO H CAA83643.1 [ 45 ] Southern house mosquito Culex quinquefasciatus Say, 1823 serine esterase NO H ACV60234.2 [ 46 ] African cotton leafworm Spodoptera littoralis (Boisduval, 1833) antennal esterase CXE7 NO H JAI18199.1 [ 47 , 48 ] Light brown apple moth Epiphyas postvittana (Walker, 1863) Carboxylesterase, partial NO H CAA83122.1 [ 49 ] Fungus Moesziomyces antarcticus (Sporobolomyces antarcticus Goto Sugiy. & Iizuka, 1969) lipase B NO H ALV82133.1 [ 50 ] Fruit fly Drosophila melanogaster Meigen, 1830 esterase 6 H AAF54915.1 [ 51 ] Fruit fly Drosophila melanogaster Meigen, 1830 acetylcholine esterase, isoform A T NM_001077781.1 [ 52 ] Zebrafish Danio rerio (Hamilton, 1822) zDHHC palmitoyltransferase 15b (zdhhc15b) T AY512893.1 [ 53 ] Common Apple Malus domestica (Suckow) Borkh. alcohol acyl transferase AAT3 T AF149919.1 [ 54 ] Jojoba Tree Simmondsia chinensis (Link) C.K. Schneid. wax synthase T KJ626344.1 [ 55 ] Chinese gooseberry Actinidia eriantha Benth. alcohol acyltransferase (AT9) T AY056316.1 [ 56 ] Thale Cress Arabidopsis thaliana (L.) Heynh. wax ester synthase/diacylglycerol acyltransferase WSD1 (At5g37300) T AY947638.1 [ 57 ] Modern Human Homo sapiens Linnaeus, 1758 acyl-CoA wax alcohol acyltransferase (AWAT1) T BC034944.1 [ 58 ] Modern Human Homo sapiens Linnaeus, 1758 zinc finger, DHHC-type containing 20 Phylogenetic analysis of the identified esterase sequences was conducted using IQtree v2.2.2.6 [ 59 ]. The best-fit substitution model was selected with ModelFinder, based on AIC/BIC criteria. Phylogenetic trees were inferred using Maximum Likelihood (ML) with 10,000 bootstrap replicates to assess branch support. The resulting trees were visualized and annotated using FigTree v1.4.4 [ 60 ]. Protein structure prediction was carried out using ColabFold [ 61 ]. The best models were aligned in PyMOL (Schrödinger, USA) and inspected for protein fold and active site conservation. The figures were prepared in UCSF ChimeraX [ 62 ]. 2.5. Differential gene expression analysis Differential gene expression (DGE) pairwise analysis was performed using the DESeq2 package [ 63 ]. To identify genes whose expression patterns align with hypothesized functional roles and to address the core research questions, transcriptome pairs for comparison were selected (Table 2 ) based on found production profiles of cis -verbenol and verbenyl oleate across life stages, tissues, and sexes of I. typographus ([ 3 , 7 ]; see Fig. 2 ). RNA-seq for various tissues were accessed again from BioProject accession number PRJNA679450 [ 3 , 16 , 26 ], Bioproject PRJNA934749 [ 7 ], and newly generated RNA-seq data from the fat bodies of fed adult beetles from this study PRJNA1321731. To elucidate the presence of a male-specific gene coding for an enzyme that hydrolyses FA esters into cis -verbenol, gene expression was expected to be specifically upregulated in the guts of fed or JH III-treated males. To support the hypothesis, we analyzed differential gene expression in the following pairwise comparisons: MFMg vs. FAMg; MFMg vs. MAMg; MJMg vs. FAMg; MJMg vs. MAMg. To identify the ester-forming gene responsible for synthesizing verbenyl fatty acid esters, candidate genes were expected to be overexpressed in the fat bodies of both sexes of immature beetles and adult males, but downregulated in the fat bodies of adult females. To investigate this, the following comparisons were made: FIFb vs. L1Wb; MIFb vs. L1Wb, and for downregulation: FFFb vs. FIFb. To identify ester-hydrolyzing enzymes active in newly emerged beetles, their genes were hypothesized to be upregulated in the fat bodies of both sexes. However, since RNAseq data from fat bodies of newly emerged beetles were not available, expression was inferred from comparisons involving immature and larval whole bodies: FIFb vs. L1Wb; MIFb vs. L1Wb. Alternatively, if the hydrolytic process occurred in the gut, comparisons were made between guts of immature and adult beetles: FIMg vs. FAMg; MIMg vs. FAMg. Finally, to investigate the tissue-specific expression of the searched metabolic genes, we compared expression in midguts and fat bodies from individuals of the same developmental stage and sex, or between sexes. The full list of experimental groups used for DGE analysis is provided in Table 2 , with additional details in Supplementary Table S3 . Table 2 Pairwise comparisons of experimental groups used for differential gene expression (DGE) of 26 selected enzymes across two tissues, various developmental stages, and sexes in Ips typographus. Leading vs. Control Leading vs. Control Leading vs. Control Male Fed Midgut (MFMg) vs. Male Acetone Midgut (MAMg) Male Fed Fatbody (MFFb) vs. Female Fed Fatbody (FFFb) Male Fed Fatbody (MFFb) vs. Male Fed Midgut (MFMg) Male Fed Midgut (MFMg) vs. Female Acetone Midgut (FAMg) Male Fed Fatbody (MFFb) vs. Male Immature Fatbody (MIFb) Male Immature Fatbody (MIFb) vs. Male Immature Midgut (MIMg) Male JHIII Midgut (MJMg) vs. Male Acetone Midgut (MAMg) Female Fed Fatbody (FFFb) vs. Female Immature Fatbody (FIFb) Female Immature Fatbody (FIFb) vs. Female Immature Midgut (FIMg) Male JHIII Midgut (MJMg) vs. Female Acetone Midgut (FAMg) Male Fed Fatbody (MFFb) vs. Larvae L1 Whole body (L1Wb) Male Immature Midgut (MIMg) vs. Female Acetone Midgut (FAMg) Female Fed Fatbody (FFFb) vs. Larvae L1 Whole body (L1Wb) Female JHIII Midgut (FJMg) vs. Female Acetone Midgut (FAMg) Male Immature Fatbody (MIFb) vs. Male Acetone Midgut (MAMg) Female Immature Midgut (FIMg) vs. Female Acetone Midgut (FAMg) Female Immature Fatbody (FIFb) vs. Female Acetone Midgut (FAMg) Male Immature Fatbody (MIFb) vs. Larvae L1 Whole body (L1Wb) Female Immature Fatbody (FIFb) vs. Larvae L1 Whole body (L1Wb) Statistical significance was determined using an adjusted p-value (padj). For each gene, the log2 fold change (log2FC) was calculated. Genes with log2FC > 3.5 and a padj < 0.05 were considered differentially expressed. A heatmap was constructed using Z-score normalized expression values to visualize expression profiles. Hierarchical clustering was based on Euclidean distance and average linkage. The heatmap was generated using the pheatmap package in R [ 64 ]. 2.6. cDNA synthesis and RT-qPCR of candidate genes Based on differential gene expression (DGE) analysis of the transcriptomic data from relevant comparison pairs (Table 2 , Chap. 2.5), candidate ester-forming/hydrolysing genes that were significantly upregulated in at least two comparisons (adjusted p-value 3.5) were selected. Furthermore, these genes were either upregulated or downregulated in specific comparison groups designed to address the core questions of this study (Chap. 2.5). From this analysis, seven carboxylesterase genes, Ityp-CE9, Ityp-CE10, Ityp-CE13, Ityp-CE15, Ityp-CE16, Ityp-CE20, Ityp-CE21, and one lipase gene, Ityp-Lip1, were selected for validation using reverse transcription quantitative PCR (RT-qPCR). Transcript levels of the selected candidate genes were quantified across developmental stages, between sexes, and tissues. Unsexed whole-body samples from larval instars (L1–L3) and pupae were analyzed, along with dissected gut and fat body tissues from immature, newly emerged, and fed adults of both sexes. Total RNA from all relevant samples was isolated, quantified, and purified as previously described. One microgram of RNA was used as a template for first-strand cDNA synthesis using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, ThermoFisher Scientific, USA), following the manufacturer’s protocol. The reverse transcription reaction was carried out in a total volume of 20 µL, with three technical replicates per sample. These replicates were pooled following the reaction, and the resulting cDNA was stored at − 20°C until use. Gene-specific primers were designed using the PrimerQuest™ Tool (Integrated DNA Technologies, www.idtdna.com ), based on the target gene sequences (Supplementary Table S1 ). Primer parameters were optimized for a melting temperature of ~ 60°C, GC content ~ 55%, and primer length of ~ 22 base pairs, and RT-qPCR reactions were performed using SYBR™ Green Universal Master Mix (Applied Biosystems™, Thermo Fisher Scientific, USA). The cycling conditions on the real-time PCR system were set to initial denaturation at 95°C for 3 minutes, followed by 40 cycles of 95°C for 3 seconds and 60°C for 34 seconds, as described by [ 65 ] and [ 3 ]. The ribosomal protein L6 (RPL6) gene was selected as the internal reference due to its stable expression across different life stages and between sexes [ 66 ]. For each target gene and the reference gene, four biological replicates were analyzed, each with two technical replicates. The Ct cycle threshold (Ct) value for each biological replicate was calculated as the average of its technical replicates. Relative gene expression (transcript abundance) levels were calculated using the 2^(-ΔCt) method [ 67 ], where ΔCt corresponds to the difference between the Ct values of the target gene and the reference gene RPL6 (referred to as normalized expression). For reactions in which no amplification was detected across all four biological replicates, the Ct value was set to 40 (the maximum cycle number) to represent undetected expression [ 68 , 69 ]. 2.7. Statistical analysis of verbenyl oleate and cis -verbenol production and RT-qPCR expression data Production of verbenyl oleate and cis -verbenol across different life stages of I. typographus , and following JHIII treatment, was analyzed based on data adapted from [ 3 , 26 ] and [ 7 ], respectively. Depending on the dataset, statistical evaluation was performed using a one-way analysis of variance (ANOVA) followed by Fisher’s least significant difference (LSD) test. Independent Student's t-tests were performed after qPCR to evaluate differences in gene expression between sexes. In addition, to assess intra-sexual variation across developmental stages, a one-way ANOVA followed by Tukey’s Honest Significant Difference (HSD) post-hoc test was used for multiple comparisons. All statistical analyses were performed using Microsoft Excel (Microsoft Corporation, 2018), with significance set at p < 0.05. Statistical assumptions, including normality and homogeneity of variances, were checked prior to analysis. The p -values derived from the t -tests and ANOVA were used to determine whether the observed differences were statistically significant. The null hypothesis assumed no significant differences in CE and LIP expression levels between the compared groups. 3. Results 3.1 Production of verbenyl oleate and cis -verbenol in different life stages, sexes, and tissues, and their induction by JHIII To elucidate the phenotypic context of final metabolite production and identify the life stages, sexes, and tissues in which the studied ester-forming/hydrolyzing genes are predicted to be upregulated, we examined the production patterns of verbenyl oleate and cis -verbenol across different phenotypes and treatments in I. typographus using GC-MS analysis ([ 3 ], Fig. 2 A, B). The highest concentrations of verbenyl oleate, representing the pool of stored verbenyl esters, were detected in the fat bodies of immature beetles, irrespective of sex. After beetles emerged from their host tree, verbenyl oleate levels declined sharply, becoming undetectable in the fat bodies of females and decreasing to approximately 25% of the pre-emergence concentration in males. Verbenyl oleate was only detectable in adult males. In feeding males, the life stage associated with peak production of the aggregation pheromone cis -verbenol, fat body levels of verbenyl oleate were further reduced, compared to newly emerged males. The highest concentrations of cis -verbenol were observed in immature beetles of both sexes. However, after emergence, it was detected exclusively in males, with the highest levels found in feeding males, which are known to actively produce aggregation pheromones. To investigate the regulatory role of JH III in the biosynthesis of these compounds, hormone treatments were applied. JH III increased verbenyl oleate levels in the male fat body but caused a slight suppression in the male gut. Unexpectedly, JH III also induced verbenyl oleate production in females, in both the fat body and gut, although at lower concentrations than in males. For cis -verbenol, significant induction was observed only in the guts of JH III-treated males, with no response in females. Additionally, a notable increase in cis -verbenol content was observed in the fat bodies of feeding males, reinforcing their role as the primary pheromone-producing stage. 3.2. Transcriptome assembly To establish a reliable database for identifying candidate ester-forming/hydrolyzing genes, we compiled and pooled relevant publicly available and newly generated transcriptomic datasets to assemble a comprehensive reference transcriptome. BUSCO analysis using the insecta_odb10 lineage dataset (1,367 orthologs) indicated a highly complete assembly. In transcriptome mode, 99.1% of Benchmarking Universal Single-Copy Orthologs (BUSCOs) were classified as complete (1,355/1,367), comprising 5.8% single-copy and 93.3% duplicated orthologs. Only 0.7% were fragmented, and 0.2% were missing. To further validate the assembly’s coding content, BUSCO analysis was performed in protein mode using TransDecoder-predicted ORFs. This revealed 96.9% complete BUSCOs (1,324 out of 1,367), including 14.3% single-copy and 82.6% duplicated genes. Only 1.8% were fragmented, and 1.3% were missing. These findings confirm that the transcriptome assembly is both structurally complete and rich in intact protein-coding sequences, affirming its reliability for downstream analyses. 3.3. Identification and Phylogenetic Analysis of Esterase Orthologs. To identify putative transcripts involved in ester formation and hydrolysis, we conducted a series of protein-protein local alignments using the predicted ORFs from our transcriptome assembly. These were compared against known ester-forming and hydrolyzing enzymes previously identified in bark beetles, other insect species, and functionally characterized enzymes from various taxa (see Table 1 ). based on following manual curation and gene annotation, we identified a total of 23 carboxylesterases, 2 lipases, one neurolactin-like, and one notum-like ortholog, all containing conserved catalytic triads comprising serine-histidine-glutamate/aspartate residues. The candidates are further reported as Ityp-CE1 to Ityp-CE24 and Ityp-LIP1, Ityp-LIP2, and Ityp-altCEA, Ityp-altCEB. The transcript sequence of Ityp-CE1-2 mapped with 100% homology into two distinct genomic loci represented by contigs JADDUH010000016.1 and JADDUH010000023.1 while keeping similar gene structure, thus pointing to local chimeric misassembly in draft genome GCA_016097725.1. Full sequence identifiers are provided in Supplementary Table S2 . Sequences of candidate genes were filtered based on alignment length and homology, with redundant or partial entries excluded. Phylogenetic reconstruction using IQtree v2.2.2.6 placed the candidates into three main clades A-C, containing 33 previously characterized esterases from various taxa (Fig. 3 B). The putative I. typographus esterases are represented by small genes, mostly ranging up to 5kb in size and sharing conserved structure with 9–11 protein-coding exons interspersed with short introns (Fig. 3 A). While the genes in clades A and B are structurally conserved with only a few exceptions, such as Ityp-CE10 and Ityp-CE11 containing longer intronic regions or Ityp-CE19 with only 8 protein-coding exons, the gene structures represented in clade C are more relaxed. (Fig. 3 A) All Ityp-CE genes encode proteins of more than 500 amino acids, which distinguishes them from putative lipases with size of roughly 330 residues. Smaller product sizes, Ityp-LIP1 and Ityp-LIP2, are given by a lower number of protein coding exons (7 and 6, respectively), and the genes also differ significantly in sequence homology (Fig. 3 A and B). The phylogenetic tree (Fig. 3 B) demonstrates that many of the newly identified esterase candidates cluster into well-supported clades alongside known insect esterases, particularly those from closely related bark beetle species. Clade A contains eleven newly identified gene sequences from I. typographus , including Ityp-CE9 and Ityp-CE10, which clustered closely with a group of previously characterized esterases from Dendroctonus armandi (AYN64424.1, AYN64425.1, AYN64427.1, AYN64429.1; Table 1 , Fig. 3 B) and Dendroctonus valens (UUB32789.1, Table 1 , Fig. 3 B). Clade B includes eight sequences and is subdivided into two well-supported branches. The first branch consists of five genes (Ityp-CE12 to Ityp-CE16) that cluster near Dendroctonus ponderosae esterase (AEE62728.1, Table 1 , Fig. 3 B). The second branch comprises Ityp-CE17 to Ityp-CE19, which cluster around D. armandi esterase AYN64428.1 (Table 1 , Fig. 3 B). Clade C contains the remaining five carboxylesterase sequences (Ityp-CE20 to Ityp-CE24). These sequences mostly fall outside of the main bark beetle carboxylesterase clusters, with the exception of two D. armandi sequences and one from Tribolium castaneum (Table 1 , Fig. 3 B), which cluster close to Ityp-CE20 . This clade predominantly includes carboxylesterase-like sequences reported in more distantly related insect orders, including Hymenoptera, Lepidoptera, and Diptera. Notably, Ityp-CE22, Ityp-CE23, and Ityp-CE24 cluster near carboxylesterases from Spodoptera spp., Lucilia spp. , and Culex spp. (Table 1 , Fig. 3 B). In addition to carboxylesterases, we also identified two novel lipases, Ityp-LIP1 and Ityp-LIP2, based on sequence similarity to functionally characterized insect lipases. Ityp-LIP1 clustered most closely with lepidopteran lipases from Bombyx mori and Antheraea pernyi , while Ityp-LIP2 grouped with hymenopteran lipases from Apis mellifera and Vespa basalis (Table 1 , Fig. 3 B). Lastly, we examined two additional genes via BLAST that resemble non-typical esterases. A neurolactin-like gene previously reported in D. valens clustered closely with Ityp-altCEB, while a notum-like gene from Drosophila melanogaster clustered with Ityp-altCEa (Table 1 , Fig. 3 B). Thus, based on sequence analyses and predicted function, Ityp-altCEA and Ityp-altCEB were excluded from further analyses as these two enzymes are most likely to act on protein or as membrane-associated proteins. Ityp-altCEA is a closely related sequence with features of NOTUM-like and palmitoleoyl-protein carboxylesterases, likely targeting lipid modifications on proteins. Ityp-altCEB shows similarity to neurotactin-like proteins and may adopt an α/β-hydrolase fold, but is predicted as a membrane-associated protein. A) Structure of 24 esterase-like (red arrow) and 2 lipase-like (orange arrow) candidate genes identified in I. typographus genome assembly GCA_016097725.1. Protein-coding exons are represented by black boxes, and genomic coordinates in contigs marked in italics are given by numbers surrounding the arrows. Candidate genes used for qPCR analyses are highlighted in dark color. B) Phylogenetic analysis of esterase orthologs illustrating relationships of I. typographus esterases to reference genes. A maximum-likelihood tree was reconstructed in IQ‐TREE v2.2.2.6 using the VT + G4 amino‐acid substitution model. Branch support was assessed with 10,000 nonparametric bootstrap replicates. Terminal labels indicate species abbreviations (e.g., Athal, Darm, Amel) and GenBank accession numbers. Colors represent taxonomic order; previously characterized enzymes are marked with red stars and described based on reported functions. Capital letters A-C represent three main clades of I. typographus carboxylesterase paralogs. The tree is presented in the form of a cladogram with transformed branches, and the scale bar represents the mean number of substitutions per site. Lipase B from the fungus Moesziomyces antarcticus was used as an outgroup protein. On the level of predicted protein structure, both CE and LIP proteins show distinct architectures (Fig. 4 A). They all share an 11-β-sheet core surrounded by α-helices, but the CE proteins are generally larger. All proteins contain a putative catalytic triad consisting of serine-histidine-glutamate/aspartate residues (Fig. 4 B). 3.4. Gene-Specific Expression Patterns 3.4.1. NGS-based Gene Expression Analysis To investigate gene expression dynamics across life stages, treatments, and sexes in I. typographus , we performed gene expression analysis using both publicly available and newly generated RNA-seq datasets. To visualize expression patterns across all inspected tissues, development stages and sexes, we generated a heatmap representing TPM-normalized expression of identified candidate genes transformed to Z-scores (Fig. 5 ). Guided by these patterns and results from pairwise differential gene expression analyses according to Table 2 (for complete results see Supplementary Table S3 ), we identified several candidate esterases and lipases genes that may be involved in verbenyl ester hydrolysis and/or biosynthesis. Carboxylesterase-coding genes Ityp-CE9, Ityp-CE10, Ityp-CE13, Ityp-CE15, Ityp-CE20, and Ityp-CE21 were selected for further analyses based on their differential expression profiles (Fig. 6 ). All six showed significant upregulation in at least two comparisons (padj 3.5). In addition, two further genes were included: Ityp-LIP1, representing a different enzyme class, and Ityp-CE16, chosen for its distinct expression profile relative to the other candidates. Their expression patterns are as follows: Ityp-CE9 exhibited a broad expression profile, with the highest expression detected in the fat bodies of immature beetles of both sexes. Expression was especially prominent in fat bodies of both sexes of immature beetles (Fig. 5). This tissue specificity was further supported by pairwise differential expression analysis, with significant upregulation in MIFb vs. MAMg (log₂FC = 5.29, padj = 1.36×10 −15 ) and in FIFb vs. FAMg (log₂FC = 3.71, padj = 3.56×10 −14 ). Notably, Ityp-CE9 expression was also induced in male midguts under feeding and JHIII treatment conditions. Its expression was significantly elevated in the MFMg vs. MAMg (log₂FC = 2.57, padj = 0.00008) and in the MJMg vs. MAMg (log₂FC = 1.75, padj = 0.00001). No comparable induction was observed in female midgut tissues (Fig. 6A, Tab. S3). Ityp-CE10 exhibited a similar expression trend to Ityp-CE9, although its overall transcript levels were lower. This gene showed the highest expression in fat bodies, particularly in immature beetles (Fig. 5). Significant upregulation was observed in FIFb vs. FAMg (log₂FC =5.44, padj = 5.16×10 −23 ) and MIFb vs. MAMg (log₂FC = 7.62, padj =2.09×10 −22 ). Furthermore, despite its lower expression levels there, Ityp-CE10 was also upregulated in the midguts of JHIII-treated males and immature females. Specifically, significant upregulation was detected in the MFMg (log₂FC = 2.68, padj = 0.03) and MJMg (log₂FC = 1.69, padj = 0.00003), compared to the MAMg control. Upregulation was also found in the midguts of immature beetles when compared with midguts of acetone-treated adults, FIMg vs. FAMg (log₂FC = 2.79, padj = 00003) and MIMg vs. FAMg (log₂FC = 3.38, padj = 6.99×10 −8 ) (Fig. 6B, Tab. S3). Ityp-CE13 was expressed broadly across tissues, with the highest levels observed in the immature fat bodies of both sexes (Fig. 5). After DGE, expression is demonstrably higher in the immature female fat bodies than midguts FIFb vs. FIMg (log₂FC = 2.68, padj = 0.0019), than in female acetone-treated midguts FIFb vs. FAMg (log₂FC = 2.67, padj = 1.15×10 −9 ), and as well the larvae FIFb vs. L1WB (log₂FC = 1.76, padj =1.26×10 −7 ). The lowest expression occurred in the male fed midguts, where it was strongly downregulated compared to the male acetone-treated midguts MFMg vs. MAMg (log₂FC = –6.60, padj = 9.37×10 −52 ). Nonetheless, slight upregulation can be observed in the immature and JHIII-treated male midguts when compared to acetone-treated female midguts MIMg vs FAMg log₂FC = 1.98, padj =0.004) and MJMg vs FAMg log₂FC = 1.56, padj =0.00009), respectively (Fig.6C, Tab. S3). Ityp-CE15 showed the highest expression in the fat bodies of immature beetles (Fig. 5). This was demonstrated by significant upregulation in comparisons such as MIFb vs. L1Wb (log₂FC = 4.50, padj = 0.001) and FIFb vs. L1Wb (log₂FC = 3.14, padj = 0.004), as well as in other DGE comparisons where fat bodies of fed beetles were compared to control groups. No significant upregulation was observed in any midgut comparisons (Fig. 6D, Tab. S3). Unlike most of the analysed genes in this study, Ityp-CE16 was predominantly expressed in the midguts of beetles rather than in the fat bodies, with particularly high expression in the midguts of acetone-treated males and immature females (Fig. 5). This was evidenced by its significant downregulation in comparisons between fat bodies and midguts, for example, in MIFb vs. MIMg (log₂FC = -8.89, padj = 4.74×10 −37 ) and FIFb vs. FIMg (log₂FC = -10.5, padj =5.04×10 −56 ). A similar pattern of downregulation was observed across all comparison pairs where fat bodies (from both sexes and treatments) were compared with midguts (from both acetone-treated and fed beetles of both sexes). Moreover, Ityp-CE16 also showed elevated expression levels in the whole bodies of larvae, as shown in comparisons such as MIFb vs. L1Wb (log₂FC = -6.76, padj = 2.71×10 − 25 ) and FIFb vs. L1Wb (log₂FC = -10.54, padj = 9.31×10 − 44 ) (Fig. 6E, Tab. S3) Ityp-CE20 exhibited strong expression primarily in the midguts of acetone- and juvenile hormone-treated females, as well as in the fat bodies of feeding beetles of both sexes (Fig. 5). This was supported by its significant downregulation in the pairwise comparison FIFb vs. FAMg (log₂FC = -3.01, padj = 8.18×10 − 25 ), and in all transcriptome comparisons where acetone-treated female midguts were used as the reference. Additionally, Ityp-CE20 was clearly expressed in the fat bodies of feeding beetles, as indicated by strong upregulation in comparisons such as FFFb vs. L1Wb (log₂FC = 5.86, padj = 4.40×10 − 17 ) and MFFb vs. L1Wb (log₂FC = 5.78, padj = 8.45×10 − 12 ). In contrast, its expression in larval tissues and in the midguts of juvenile or immature beetles remained consistently low (Fig. 6F, Tab. S3). Ityp-CE21 exhibited high expression levels in the midguts of immature beetles of both sexes, as well as in the fat bodies of feeding males (Fig. 5). This was demonstrated by significant upregulation in DGE comparisons such as FIMg vs. FAMg (log₂FC = 4.29, padj = 3.54×10 −14 ) and MIMg vs. FAMg (log₂FC = 3.94, padj = 2.74×10 −22 ). Conversely, when midguts of immature beetles were used as reference in other comparisons, Ityp-CE21 log₂FC was constantly negative, confirming elevated expression in this tissue. High expression in the fat bodies of fed males was further supported by the comparison MFFb vs. MFMg (log₂FC = 2.55, padj = 5.51×10 −19 ) (Fig. 6G, Tab. S3) Ityp-LIP1 exhibited low overall expression across all tissues and treatments. The highest transcript levels were detected in the fat bodies of immature beetles and in the midguts of immature individuals (Fig. 5). This was supported by significant upregulation in pairwise comparisons FIFb vs. FAMg (log₂FC = 2.63, padj = 0.0001) for fat bodies, and FIMg vs. FAMg (log₂FC = 3.61, padj = 0.009), MIMg vs. FAMg (log₂FC = 3.91, padj = 0.00001) for midguts (Fig. 6H, Tab. S3). Overall, the majority of analyzed genes showed their highest expression in fat bodies, particularly in immature beetles (Ityp-CE9, -CE10, -CE13, -CE15, -CE20). Ityp-CE16 differed from this pattern by being predominantly expressed in midguts across sexes and treatments. Ityp-CE21 displayed stage-dependent expression, with strong activity in immature midguts of both sexes. This pattern is similar to the expression of Ityp-LIP1 with minor differences. 3.4.2. RT-qPCR of the selected candidates in different life stages, tissues, and sexes To further verify transcriptional dynamics of the eight selected candidate genes, we performed RT-qPCR analyses across multiple developmental stages and tissues of I. typographus . Expression was examined in whole bodies of larval and pupal stages, as well as in the gut and fat body tissues of adult beetles from both sexes (Fig. 7). Supporting data, including Ct values and results of statistical analyses, are provided in Table S4. RT-qPCR expression profile of Ityp-CE9 in whole-body samples of juvenile beetle life stages was similar across all larval stages, followed by a marked decline in pupae (Fig. 7A1). In fat body tissue, expression patterns paralleled those in the gut, with generally similar levels between sexes at all stages. Although the emerged and fed males showed higher values than females, these differences were not statistically significant. Across life stages, expression was highest in immature beetles, dropped in emerged beetles, and rose again in fed beetles, with a trend toward higher levels in fed males (Fig. 7A2). In gut tissue, immature males exhibited slightly lower expression than females, but the difference was not significant. In emerged and fed adults, expression levels were not sex-specific (Fig. 6A3). Ityp-CE10 was expressed in samples from whole bodies of juvenile beetles relatively constantly across larval stages L1 to L3 before increasing significantly in L3 and then sharply decreasing in pupae (Fig. 7B1). In fat body tissue, immature beetles showed the highest expression levels in both sexes, with values significantly greater than those of emerged and fed individuals. Emerged adults displayed reduced expression, while fed beetles showed a modest increase. Expression was generally similar between males and females within each stage, although immature males exhibited slightly higher levels than females ( p = 0.04) (Fig. 7B2). In gut tissue, expression was again highest in immature adults of both sexes, significantly exceeding that of emerged and fed stages. Emerged and fed individuals exhibited reduced expression with no major sex-specific differences (Fig. 7B3). RT-qPCR expression level of Ityp-CE13 in whole bodies of young beetles increased steadily from L1 to L3 larvae, then dropped sharply to near-zero in pupae (Fig.7C1), resembling patterns seen in Ityp-CE9 (Fig. 6A1), Ityp-CE10 (Fig. 7B1). In fat body tissue, expression rose again in immature beetles of both sexes, although significantly more in males ( p = 0.018). In newly emerged adults, levels dropped sharply, but females still expressed significantly more than males ( p = 0.0005). In fed adults, expression increased again, with higher levels in males than females (Fig. 7B2), again resembling expression of Ityp-CE9 (Fig. 7A2), Ityp-CE10 (Fig. 7B2). In gut tissue, Ityp-CE13 was highly expressed in immature beetles of both sexes, with no significant sex difference. Expression declined in newly emerged adults and remained low in fed beetles, with a non-significant decrease in fed males (Fig. 7C3). In whole-body samples, Ityp-CE15 expression was notably high only in L3 larvae, with very low levels in L1, L2, and pupae (Fig. 7D1). In fat body tissue, expression was generally higher than in gut tissue, with a trend toward higher levels in males. The only significant sex-based difference occurred in the immature stage with higher expression in male tissue ( p = 0.016). Across life stages, expression increased from immature to emerged adults, then declined after feeding, significantly so in females (Fig. 7D2). In gut tissue, immature females expressed more than immature males ( p = 0.035). Female expression declined progressively from immature to emerged to fed adults, whereas males peaked at the emerged stage (Fig. 7D3). Expression of Ityp-CE16 in juvenile beetles increased from L1 to L3, followed by a sharp decline to near-zero levels in pupae (Fig. 6E1), resembling patterns seen in Ityp-CE9 (Fig. 7A1), Ityp-CE10 (Fig. 7B1), and Ityp-CE13 (Fig. 7C1). In fat body tissue, expression remained low at all three adult stages, with a significant male bias in newly emerged adults ( p = 0.023), consistent with the profile of the above genes (Fig. 7E2). In gut tissues, Ityp-CE16 was the most highly expressed among all tissues and showed exceptionally high transcript levels compared to other genes in the beetle midgut (Fig. 7E3). In females, expression peaked in immature beetles, dropped sharply in newly emerged adults, and rose again in fed adults, a trend also seen in Ityp-CE9 (Fig. 7A3), Ityp-CE10 (Fig. 7B3), and Ityp-CE13 (Fig. 7C3). In males, expression was similarly high in immature beetles, decreased in newly emerged adults (less sharply than in females), and, unlike females, declined further after feeding. This profile, though at much higher absolute levels, resembled that of Ityp-CE13 (Fig. 7E3). Ityp-CE20 was expressed in the whole bodies of young beetles, mostly in L1 larvae and lower in L2, L3, and pupae (Fig. 7F1). In fat body tissue, expression was equally low in immature beetles of both sexes, but increased several-fold in newly emerged females, reaching significantly higher levels than in males ( p =0.016). In fed adults, expression dropped again, equalizing between sexes (Fig. 7F2). In gut tissue, females showed a similar profile: expression increased sharply in newly emerged adults, reaching levels several-fold higher than in males ( p = 0.050), but was sex-equal and lower in immature and fed beetles (Fig. 7F3). The last from carboxylesterases Ityp-CE21 expression in whole bodies of young beetles was uniformly low across larval stages (L1-L3) but rose sharply in pupae (Fig. 7G1), although high variability among pupae rendered the difference non-significant. In fat body tissue, clear sex-specific differences were present at all stages. Immature females expressed more than males ( p = 0.029), while in emerged ( p = 0.032) and fed adults ( p = 0.026), expression was higher in males. In females, expression peaked in the immature stage; in males, it peaked in the emerged stage (Fig. 7G2). In gut tissue, overall expression was very low. The only significant sex-specific difference occurred in the immature stage ( p = 0.0003), with higher expression in males. In males, expression remained consistent across stages; in females, it was low in immature and emerged beetles, with a slight increase after feeding (Fig. 7G3). Similarly to the transcriptome data, the expression profile of Ityp-Lip1 was consistently lower than that of the other carboxylesterases analyzed in this study. In whole bodies of larvae and pupae, Ityp-Lip1 expression was below the detection limit of the method used (Fig. 7H1). In the fat body tissue, expression was higher than in other samples, and peaked in both sexes during the emerged stage. Furthermore, in fed adults, expression in the fat body was significantly higher in males (p= 0.035) (Fig. 7H2). In the gut tissues, overall expression levels were lower than in the fat bodies, and expression in the guts of feeding females and immature males was below the detection limit. The highest expression could again be observed in the guts of the emerged stage of both sexes. A significant sex-specific difference was observed in the guts of the immature stage, where females exhibited higher expression levels than males ( p = 0.011) (Fig. 7H3). 4. Discussion 4.1. Production profiles of verbenyl oleate and cis -verbenol as indicators of the genetic basis of their biosynthesis The highest verbenyl oleate concentrations were found in the fat bodies of immature beetles of both sexes that had not yet left the bark and did not require pheromonal communication [3]. This may show that, at this stage, verbenyl oleate may be produced as a detoxification product of α-pinene ingested during maturation feeding of juvenile beetles (larvae, pupae, immature). Based on this, we initially investigated immature beetles for enzymes catalyzing verbenyl ester formation. A sharp decline in verbenyl oleate content after emergence in both sexes, though males retained or produced small amounts [3], led us to examine newly emerged beetles for ester-hydrolyzing enzymes that could release stored energy. In feeding males, the key stage for cis -verbenol pheromone production, fat body concentrations of verbenyl oleate were lower than in newly emerged males. This suggests partial hydrolysis of stored esters to release free cis -verbenol for pheromone production [3]. This interpretation was supported by the effect of JH III, which stimulated cis -verbenol production in the gut of emerged males while reducing verbenyl oleate levels in the same tissue [7]. These findings indicate ester cleavage as a source of pheromonal cis -verbenol and guided our search for an adult male-specific hydrolyzing enzyme. Changes in cis -verbenol levels and residual verbenyl oleates in the gut suggest that both esterification and hydrolysis may occur there, alongside α-pinene detoxification by hydroxylation. However, the exact tissue specificity of these processes remains unresolved. 4.2 Selection of candidate genes To disentangle the enzyme classes involved in the metabolism of monoterpenyl fatty acid esters in I. typographus , we focused our gene-level analysis on known insect ester-forming and -hydrolysing enzymes, primarily carboxylesterases, along with lipases and acyltransferases to a lesser extent. To broaden the scope and capture potentially uncharacterized or divergent enzyme families, we expanded our homology-based searches to include reference sequences from phylogenetically distant organisms, such as fish, plants, and humans. This approach allowed us to identify 27 putative ester-modifying genes in I. typographus , all encoding enzymes with a catalytic triad. Most were carboxylesterases (23), with two lipases and two other distinct enzymes, but no acyltransferases met the similarity criteria. All identified genes were closely related in sequence to those from bark beetles or other insects. The 27 candidate genes were ubiquitously expressed across sexes, developmental stages, tissues, and in response to juvenile hormone (JH) treatment. The criteria established for pairwise comparison of gene expression across different transcriptomes led to the selection of eight candidate genes for further investigation. These included seven carboxylesterases, Ityp-CE9,10,13,15,16,20,21, and one lipase, Ityp-Lip1. 4.3 Description and putative functions of selected esters-forming/hydrolyzing genes Ityp-CE9, Ityp-CE10: Ityp-CE9 and Ityp-CE10, both located in clade A of the phylogenetic tree, differ in gene structure: Ityp-CE9 contains 9 exons, while Ityp-CE10 has 11 exons with longer intronic regions, suggesting an evolutionarily older origin [70, 71]. Both genes showed their highest expression in the fat bodies of immature beetles of both sexes and lower expression in the midguts. Pairwise comparisons revealed notable upregulation of expression in the guts of fed and JH III-treated males, compared to both control and newly emerged males. This expression pattern aligns with observed metabolic changes: elevated levels of cis -verbenol and reduced levels of verbenyl oleate in the guts of fed and JH III-treated males (Fig. 2A–B; [3, 7]). Ityp-CE9 and Ityp-CE10 clustered phylogenetically with carboxylesterases from Dendroctonus armandi (AYN64424.1, AYN64425.1, AYN64427.1, AYN64429.1; Tab. 1, Fig. 3B) and Dendroctonus valens (UUB32789.1; Tab. 1, Fig. 3B), genes suggested to be involved in detoxification. Notably, Ityp-CE9 is closely related to Darm-AYN64429.1, whose function in monoterpene detoxification was particularly highlighted when D. armandi was exposed to α-pinene [15]. RT-qPCR profiling of Ityp-CE9 and Ityp-CE10 across life stages, sexes, and tissues (midgut and fat body) in I. typographus revealed highly similar expression patterns, indicating a correlation between their expression and feeding activity in beetles. Although both genes showed higher expression in the midguts and fat bodies of adult males than females, this difference was not always statistically significant. According to this, the trend suggests that these enzymes may be involved in male-specific hydrolysis of verbenyl esters to release cis -verbenol in the gut. However, no strong overexpression in the gut of feeding males was observed. While it remains possible that male-specific hydrolysis could occur in the fat bodies, it is more plausible that these enzymes are involved in the biosynthesis of monoterpenyl FA esters, likely as part of a detoxification mechanism active during juvenile beetle stages in response to monoterpene exposure. In adults, the male-biased expression could reflect a sex-specific metabolic function, as these monoterpenyl FA esters are absent in females' fat bodies. Since Ityp-CE9 and Ityp-CE10 , initially promising for alternative cis -verbenol production [3], appear instead to function in general detoxification, we propose a new hypothesis: rather than searching for a hydrolytic enzyme in fed male midguts, the key may lie in identifying an ester-forming gene that is downregulated during feeding . This gene would normally convert cis -verbenol into ester-bound forms, and its downregulation would reduce ester formation, allowing free cis -verbenol to accumulate in guts of feeding males for pheromone use. Gene expression data point to Ityp-CE13 and later discussed Ityp-CE15, especially Ityp-CE16, as promising candidates. Ityp-CE13: Carboxylesterase Ityp-CE13 clustered in clade B, closely with Dpon-AEE62728.1 from Dendroctonus ponderosae , a gene previously implicated in the biosynthesis of trans -verbenol, likely through the formation or hydrolysis of verbenyl esters [6, 11]. The gene contains 11 exons and has a relatively short intronic region. In transcriptome comparisons, Ityp-CE13 showed an expression profile similar to Ityp-CE9 and Ityp-CE10, with the highest levels in the fat bodies of immature beetles of both sexes and detectable expression in the midguts of acetone-treated and JHIII-treated males. A notable exception was the strong downregulation observed in the midguts of feeding males. qPCR confirmed a similar expression pattern to that of the previously described genes Ityp-CE9 and Ityp-CE10: expression peaked in the fat bodies of immature beetles, declined after emergence, and rose again in fed beetles. Midgut expression followed the same general pattern, again with the exception of markedly low levels in fed male midguts. This sex-specific suppression of Ityp-CE13 after feeding in adult male gut is unexpected and may be linked to pheromone production, for example, by switching off further detoxification of cis -verbenol to its esters to increase the availability of cis -verbenol for pheromone communication in males. However, the underlying mechanism remains unclear. A possible compelling role for Ityp-CE9 , Ityp-CE10 , and Ityp-CE13 may lie in the biosynthesis of verbenyl esters during the immature stages , when these compounds are the most abundant. Supporting this, all three carboxylesterases showed elevated expression in the fat body of both sexes at this developmental phase. Ityp-CE15: Even though carboxylesterase Ityp-CE15 has a similar length and the same number of exons (11) as Ityp-CE13, and clusters closely with it in clade B, also near Dpon-AEE62728, it exhibits distinct expression profiles compared to Ityp-CE13, both in DGE analysis and qPCR. Among the selected carboxylesterases analyzed here so far, Ityp-CE15 consistently exhibited lower overall expression in the compared transcriptomes. Pairwise DGE comparisons indicated that Ityp-CE15 is sex-neutral, strongly fat body–specific, and developmentally enriched in immature beetles, with no detectable feeding or hormonal responsiveness. qPCR showed the highest expression in fat bodies, similar to that observed for Ityp-CE13, but notably, Ityp-CE15 displayed higher expression in the fat body of newly emerged beetles compared to immature individuals. This temporal pattern supports its candidacy as a hydrolytic esterase potentially involved in cleaving monoterpenyl FA esters during the developmental transition from the energy-acquiring, feeding immature stage to the non-feeding, emerged adult stage, when beetles must locate a new host tree and initiate mating. The male-biased expression further suggests a sex-specific metabolic role, potentially associated with the reduced FA ester content observed in adult females. These findings make the Ityp-CE15 a candidate for carboxylesterase catalysing the hydrolysis of verbenyl esters in newly emerged beetles, potentially to mobilise stored energy reserves, but also its downregulation in the feeding male gut makes it possible to be involved in cis -verbenol enrichment. Ityp-CE16: Another carboxyl esterase gene, Ityp-CE16, also clusters within clade B, positioned near Ityp-CE13, Ityp-CE15, and Dpond AEE62728. Its gene structure is relatively compact, with short intronic regions and only nine exons. Among all Ityp-CE/LIP genes analyzed in this study, Ityp-CE16 was the only one to exhibit significantly higher expression in midgut tissues of both sexes, in contrast to the fat-body or mixed-tissue enrichment observed for most other genes. The generally robust expression peaked in the midguts of immature and acetone-treated beetles of both sexes, as shown by pairwise comparisons, and was unaffected by JHIII treatment. RT-qPCR results confirmed that midgut expression in both immature and adult beetles was approximately an order of magnitude higher than in the corresponding fat bodies, with slightly higher levels in emerged males than in females. Although Ityp-CE16 is midgut-specific, its expression pattern does not correspond with the production curves of cis -verbenol or verbenyl oleate in fed males or after JHIII treatment, making it an unlikely candidate for the male gut-specific gene with verbenyl esters hydrolytic function, but still may be involved in cis -verbenol enrichment as discussed above. Ityp-CE20: Ityp-CE20 clusters within the distant clade C, alongside carboxyl-like esterases from various insect orders. Its closest relative sequence is a putative carboxylesterase from D. armandi Darm-AYN64426.1 [15], with nearby branches including a juvenile hormone esterase from Tribolium castaneum Tcas-NM_001193294.1 [21]. On the sister branch was clustering venom carboxylesterases from Bombus ignitus Bign-MW699017.1 [20], esterase 6 from D. melanogaster Dmel-ALV82133.1, and carboxylesterase from Epiphyas postvittana Epos-JAI18199.1 [47, 48]. The gene contains moderately long intronic regions and consists of 10 exons. Transcriptome data revealed that Ityp-CE20 is most abundantly expressed in the midguts of both immature and adult beetles, with particularly high levels in the midguts of acetone-treated females, considered equivalent to emerged females. Elevated expression was also observed in the fat bodies of feeding beetles. RT-qPCR analysis confirmed that expression peaks in the fat bodies and midguts of emerging females, exceeding levels in males and other life stages. This sex- and stage-specific increase in expression among females may be linked to preparation for future reproduction, possibly by enhancing energy reserves, initiating female-specific metabolic pathways, or processes associated with juvenile hormone biosynthesis. From the perspective of verbenyl ester metabolism, its involvement appears unlikely, as neither verbenyl esters nor cis -verbenol were detected in emerged females [3]. Ityp-CE21: Ityp-CE21 also clusters with carboxyl-like esterases in clade C and groups near Darm-AYN64423.1, a putative carboxylesterase from D. armandi [15], and is closely related to a venom carboxylesterase from Apis mellifera [19]. The gene comprises 10 exons and features exceptionally long intronic regions, second only to Ityp-CE10 among the carboxylesterases analysed, which may indicate an older evolutionary origin [70, 71]. Although expression varied between sexes and life stages in immature and adult beetles, with a general bias toward males, notable upregulation was observed only during the pupal stage. This expression pattern suggests a possible role in developmental processes, potentially related to hormonal regulation or lipid metabolism during the metamorphosis from pupa to pre-adult, within endocrine-associated metabolic pathways, rather than in direct detoxification or pheromone biosynthesis. Ityp-Lip1: The only selected lipase, Ityp-LIP1, was phylogenetically grouped within a monophyletic branch of reference lipases that is distinct from carboxylesterases. Lipase has a different gene structure and a different shape of binding site than studied carboxylesterases. It has 7 exons and long intronic regions, similar to Ityp-CE10 and Ityp-CE21. It clustered most closely with lepidopteran lipases from Bombyx mori and Antheraea pernyi [37, 38]. Expression in gut tissues and whole-body samples was generally low. Transcriptome data showed significant upregulation of Ityp-LIP1 in the immature beetles, but RT-qPCR demonstrated that Ityp-LIP1 expression is highest in the fat bodies of emerged beetles of both sexes. However, the stage- and sex-specific expression of Ityp-Lip1, though relatively modest, suggests it may play a specialized role, possibly related to the reduced fatty acid ester content observed in adult females, similar to the function proposed for Ityp-CE15. Tissue location of verbenyl FA esters forming/hydrolysis: The precise localization of verbenyl fatty acid ester formation or hydrolysis remains an open question. While previous studies, e.g., [15], [16], [35], [72], focused on carboxylesterase roles in gut detoxification, our findings support a broader hypothesis: detoxification of monoterpenols and hydrolysis of verbenyl esters may also occur in the fat body, as all genes were expressed in both tissues. However, caution is warranted, since the anatomical proximity of gut and fat body may lead to tissue cross-contamination during dissection, possibly explaining low-level expression in gut samples [3]. Future perspectives: Despite extensive analyses, none of the eight candidate genes could be conclusively linked to a specific enzymatically catalyzed step in the metabolism of verbenyl esters, presumed precursors in an alternative biosynthetic pathway for pheromonal cis -verbenol, leaving key mechanistic questions unresolved. To better understand this understudied and technically challenging enzyme group, central to bark beetle metabolism and pheromone biosynthesis, complementary investigative approaches are essential. Future work should prioritize functional characterization of top candidate genes, starting with cloning into bacterial expression vectors and conducting enzymatic activity assays using verbenyl esters as substrates, with acylglycerols and wax esters as controls [73–75]. These findings should be validated through RNAi-mediated gene knockdowns to assess in vivo function [76]. Together, these approaches will clarify the functional diversity and biological roles of ester-forming and -hydrolyzing enzymes, not only in I. typographus but also across other insect species. 5. Conclusion Although ester-forming and ester-breaking enzymes are believed to play key roles in many metabolic processes, they are still poorly understood in insects. Comprehensive gene sequencing and functional studies for these enzymes are rare. In this study, we used both phylogenetic analysis and differential gene expression data to discover 27 previously uncharacterized genes in the bark beetle I. typographus that likely code for ester-forming or -hydrolyzing enzymes. These include 23 carboxylesterases, two (phospho)lipases, one notum-like gene, and one neurolactin-like gene. From this group, eight genes were selected for more detailed expression analysis. These were seven carboxylesterases (Ityp-CE9, CE10, CE13, CE15, CE16, CE20, CE21) and one lipase (Ityp-Lip1). Their potential roles were investigated with a focus on identifying genes possibly involved in the metabolism of verbenyl FA esters—suspected intermediate compounds in the biosynthesis of the pheromone cis -verbenol. To support this, we compared gene expression across different beetle life stages and forms (phenotypes) that vary in their verbenyl oleate and cis -verbenol production. While the functional involvement of these eight candidates in pheromone metabolism remains hypothetical, their expression patterns provide a valuable foundation for further study. Future functional assays and gene silencing experiments will be essential to validate their roles. Furthermore, given the central importance of aggregation pheromones in I. typographus behaviour, elucidating the genetic underpinnings of pheromonal cis -verbenol biosynthesis may support the development of targeted pest management strategies that modulate beetle populations rather than eradicate them, thereby helping to maintain ecological balance. Abbreviations MFMg Male Fed Midgut MIMg Male Immature Midgut FIMg Female Immature Midgut L1WB Larvae1 Whole Body MJMg Male JHIII Midgut MAMg Male Acetone Midgut FJMg Female JHIII Midgut FAMg Female Acetone Midgut MIFb Male Immature Fat Body FIFb Female Immature Fat Body MFFb Male Fed Fat Body FFFb Female Fed Fat Body. Declarations Ethics approval No specific permits were required. All experiments were conducted on insects ( Ips typographus ) that are neither endangered nor protected species within the European Union. According to EU Directive 2010/63/EU on the protection of animals used for scientific purposes, research involving invertebrates such as insects does not require formal ethical approval. Consent to participate All authors consented to their participation in this research. Consent for publication All authors consent to the publication of this manuscript. Competing interests The authors declare no competing interests. Funding Jaroslav Strádal, Rajarajan Ramakrishnan and Anna Jirošová were funded by the funding agency Czech Science Foundation GACR 23–07916 S, Czech Republic. Jaroslav Strádal was funded by the student Internal Grant Commission [IGA_A_32_24, JAROSLAV STRADAL] at the Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, Prague, Czech Republic. Ondřej Lukšan, computational resources were provided by the e-INFRA CZ project (ID:90254), supported by the Ministry of Education, Youth and Sports of the Czech Republic and High Performance Computing Service Group at IOCB, CAS. Author Contribution JSt: Conceptualization, beetle rearing, collection, treatments, dissections, genomic analyses, method development, statistical analyses, manuscript drafting, and writing. RR: Treatments, metabolomic data analysis, method development, genomic analyses, manuscript drafting. OL: Genomic analyses, method development, bioinformatic data processing, manuscript drafting, and writing. MT: Conceptualization, genomic, transcriptomic and phylogenetic analyses, method development, manuscript drafting, and writing. JSy: Beetle rearing, collection, treatments, manuscript drafting. SM: Genomic analyses, method development, manuscript editing. IP: Conceptualization, manuscript editing and review, supervision. AJ: Conceptualization, study design, metabolomic data analysis, formal analysis, manuscript writing, editing, review, supervision. Acknowledgement We thank Forest CZU enterprise for providing biological material. Data Availability All data generated or analysed during this study are included in this published article and its supplementary information files. Publicly available datasets used were sourced from NCBI Bioprojects under accession numbers PRJNA679450, PRJNA934749 and PRJNA1321731. Newly identified putative esterase sequences are available under accession numbers PX172087, PX172088, PX172089, PX172090, PX172091, PX172092, PX172093, PX172094, PX172095, PX172096, PX172097, PX172098, PX172099, PX172100, PX172101, PX172102, PX172103, PX172104, PX172105, PX172106, PX172107, PX172108, PX172109, PX172110, PX172111, PX172112, PX172113, PX172114 and the new *Ips typographus* transcriptome assembly as well as predicted protein structures are available at [https://doi.org/10.5281/zenodo.17063688](https:/doi.org/10.5281/zenodo.17063688) [77]. References Hlásny T, Zimová S, Merganičová K, Štěpánek P, Modlinger R, Turčáni M. Devastating outbreak of bark beetles in the Czech Republic: Drivers, impacts, and management implications. Ecol Manag. 2021;490:119075. https://doi.org/10.1016/j.foreco.2021.119075 . Huang J, Kautz M, Trowbridge AM, Hammerbacher A, Raffa KF, Adams HD, et al. Tree defence and bark beetles in a drying world: carbon partitioning, functioning and modelling. 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Overview on the expression of toxic gene products in Escherichia coli. Curr Protoc Protein Sci. 2007. https://doi.org/10.1002/0471140864.ps0519s50 . Chap. 5:Unit 5.19. Sandstrom P, Welch WH, Blomquist GJ, Tittiger C. Functional expression of a bark beetle cytochrome P450 that hydroxylates myrcene to ipsdienol. Insect Biochem Mol Biol. 2006;36:835–45. https://doi.org/10.1016/j.ibmb.2006.08.004 . Fang S, Chang X, Chen H, Wu Z, Shi J. Cloning and RNAi-mediated functional characterization of two Monochamus alternatus chitinase genes. Pest Manag Sci Epub 2025 Jul 29. https://doi.org/10.1002/ps.70013 Lukšan O, Strádal J, Tupec M, Jirošová A. Ips typographus transcriptome assembly and predicted protein structures [dataset]. Zenodo; 2025. https://doi.org/10.5281/zenodo.17063688 . Additional Declarations No competing interests reported. Supplementary Files TableS2EsteraseAbbreviations.xlsx TableS2EsteraseAbbreviationsFINAL.xlsx TableS3DGE.xlsx SupplementaryArchiveS1.zip TableS4qPCRCtValuesandStatisticalAnalysis.xlsx.xlsx TableS1PrimerSequencesqPCR.xlsx Cite Share Download PDF Status: Published Journal Publication published 24 Mar, 2026 Read the published version in BMC Genomics → Version 1 posted Editorial decision: Revision requested 09 Dec, 2025 Reviews received at journal 04 Dec, 2025 Reviewers agreed at journal 07 Nov, 2025 Reviews received at journal 30 Oct, 2025 Reviewers agreed at journal 13 Oct, 2025 Reviewers invited by journal 21 Sep, 2025 Editor assigned by journal 11 Sep, 2025 Submission checks completed at journal 11 Sep, 2025 First submitted to journal 10 Sep, 2025 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. 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1","display":"","copyAsset":false,"role":"figure","size":273246,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eA. Production of pheromonal cis-verbenol in adult males feeding on spruce trees by the direct hydroxylation of spruce-source (−)-α-pinene catalyzed by CyP450. B. Suggested pathway for cis-verbenol production via hydrolysis of alternative source verbenyl esters. The formation of verbenyl esters in pre-emerged beetles of both sexes and adult males is putatively catalyzed by FA transferases (FATs) or esterases, and the hydrolytic release of cis-verbenol in adult calling males is putatively catalyzed by lipase/esterase. C. Structure of cis-verbenyl oleate.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7583299/v1/b06466099c3569dc43039a0f.jpeg"},{"id":92748875,"identity":"acdf9488-b869-4b62-b974-5c455cc55ccd","added_by":"auto","created_at":"2025-10-03 20:33:06","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":741656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eA) Content of verbenyl oleate (in fat body) and cis-verbenol (in gut) in different life stages of I. typographus males (right) and females (left). Adapted from \u003c/em\u003e[3, 26]\u003cem\u003e. B) Content of verbenyl oleate (in both fat body and gut) and cis-verbenol (only in gut) of treated I. typographus males (right) and females (left) by juvenile hormone III or by acetone as a control (N = 3), statistical significance is denoted as *** for p \u0026lt; 0.001, ** for p \u0026lt; 0.01, and * for p \u0026lt; 0.05.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7583299/v1/76f59eb3a15f94ae35046eff.jpeg"},{"id":92748882,"identity":"7f60ac70-261f-4882-9747-bd5965926bdd","added_by":"auto","created_at":"2025-10-03 20:33:06","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":601258,"visible":true,"origin":"","legend":"\u003cp\u003eGene structure and phylogenetic clustering of Ips typographus carboxylesterases.\u003c/p\u003e\n\u003cp\u003eA) Structure of 24 esterase-like (red arrow) and 2 lipase-like (orange arrow) candidate genes identified in I. typographus genome assembly GCA_016097725.1. Protein-coding exons are represented by black boxes, and genomic coordinates in contigs marked in italics are given by numbers surrounding the arrows. Candidate genes used for qPCR analyses are highlighted in dark color. B) Phylogenetic analysis of esterase orthologs illustrating relationships of I. typographus esterases to reference genes. A maximum‐likelihood tree was reconstructed in IQ‐TREE v2.2.2.6 using the VT+G4 amino‐acid substitution model. Branch support was assessed with 10,000 nonparametric bootstrap replicates. Terminal labels indicate species abbreviations (e.g., Athal, Darm, Amel) and GenBank accession numbers. Colors represent taxonomic order; previously characterized enzymes are marked with red stars and described based on reported functions. Capital letters A-C represent three main clades of I. typographus carboxylesterase paralogs. The tree is presented in the form of a cladogram with transformed branches, and the scale bar represents the mean number of substitutions per site. Lipase B from the fungus Moesziomyces antarcticus was used as an outgroup protein.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7583299/v1/75ae3da7517e47fdaaad28eb.jpeg"},{"id":92748883,"identity":"07b699c1-5cb1-49fd-9f75-180a4348ddba","added_by":"auto","created_at":"2025-10-03 20:33:07","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2555536,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eOverall architecture of CE and LIP proteins from I. typographus. A) Representative AlphaFold 2-predicted models of Ityp-CE9 (left) and Ityp-LIP1 (right), with predicted catalytic residues shown as black sticks. B) Close-up at the external surface of Ityp-CE9 (left) and Ityp-LIP1 (right) with a substrate pocket and a catalytic triad. See Supplementary Archive S1 for the remaining predicted models.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7583299/v1/46428577faae2ee56f91c3e5.jpeg"},{"id":92749314,"identity":"fcc0db68-cd79-43eb-9817-c6c69df2303d","added_by":"auto","created_at":"2025-10-03 20:41:07","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":651474,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHeat map illustrating the relative expression profiles of Ityp-esterase genes across distinct tissue types and developmental stages. Each cell represents Z-score transformed value of TPM-normalized expression of a gene in a given sample, where red hues indicate upregulation and blue hues indicate downregulation relative to the mean expression across all samples.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7583299/v1/119d9f953bd58aa4bd0b3e67.jpeg"},{"id":92748899,"identity":"33d31112-0665-488f-8a39-639ba500563d","added_by":"auto","created_at":"2025-10-03 20:33:07","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":892587,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDifferential expressions of eight selected genes (A) Ityp-CE9, (B) Ityp-CE10, (C) Ityp-CE13, (D) Ityp-CE15, (E) Ityp-CE16, (F) Ityp-CE20, (G) Ityp-CE21, (H) Ityp-Lip1 across developmental and treatment groups. Bars represent log₂ fold change (log₂FC) relative to the respective control group. \u003c/em\u003eDGE pairwise analysis was performed using the DESeq2 package [63]. \u003cem\u003eAsterisks indicate statistically significant differences (* = p \u0026lt; 0.05, *\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003e*\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e = \u003c/strong\u003e\u003c/em\u003e\u003cem\u003ep \u0026lt; 0.01, * * * = p \u0026lt; 0.001).\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAbbreviations: MFMg – Male Fed Midgut; MIMg – Male Immature Midgut; FIMg – Female Immature Midgut; L1WB – Larvae1 Whole Body; MJMg – Male JHIII Midgut; MAMg – Male Acetone Midgut; FJMg – Female JHIII Midgut; FAMg – Female Acetone Midgut; MIFb – Male Immature Fat Body; FIFb – Female Immature Fat Body; MFFb – Male Fed Fat Body; FFFb – Female Fed Fat Body.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7583299/v1/3b3d955b66a78bdfd8a89fa0.jpeg"},{"id":92748893,"identity":"e55f022f-7ee4-42ad-8431-c9be7875f34a","added_by":"auto","created_at":"2025-10-03 20:33:07","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":831415,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRelative expression of four candidate genes across developmental stages and tissues, measured using the 2^(-ΔCt) method. A) Whole-body samples from larval instars (L1–L3) and pupae. B) Fat body tissue from immature, emerged, and fed adults, separated by sex. C) Gut tissue from immature, emerged, and fed adults, separated by sex. In panels B and C, female samples are shown on the left and male samples on the right for each developmental stage. Boxes represent the distribution of normalized expression values (2^(-ΔCt). Different lowercase letters indicate statistically significant differences among developmental stages within each sex (Tukey’s HSD, p \u0026lt; 0.05). Asterisks denote significant differences between sexes within the same stage (Student’s t-test, * = p \u0026lt; 0.05, * * = p \u0026lt; 0.01, * * * = p \u0026lt; 0.001).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7583299/v1/d92dbb7c4fb62acd7279bc08.jpg"},{"id":105755968,"identity":"9b26bc41-5517-408c-9f3d-6d2229b51ddb","added_by":"auto","created_at":"2026-03-30 16:33:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8605012,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7583299/v1/c80c5e73-3fa7-47ab-834b-c96a371218f4.pdf"},{"id":92748872,"identity":"b483a6e6-f1ee-49ad-abf5-3291698f89b4","added_by":"auto","created_at":"2025-10-03 20:33:06","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":14353,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2EsteraseAbbreviations.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7583299/v1/ff2e09ec9e21ecfdb9c9bd82.xlsx"},{"id":92748871,"identity":"be0901ab-6f35-4989-bbe1-3886834f5519","added_by":"auto","created_at":"2025-10-03 20:33:06","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11428,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2EsteraseAbbreviationsFINAL.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7583299/v1/798995370b14ee82f14b8f5a.xlsx"},{"id":92748876,"identity":"45ce9fe9-86f5-4656-a5cf-823eb97b8eb3","added_by":"auto","created_at":"2025-10-03 20:33:06","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":26346,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3DGE.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7583299/v1/0cec27f5a47ca516537a18f9.xlsx"},{"id":92748879,"identity":"5aa24441-d02c-42f9-95c5-c76777902afa","added_by":"auto","created_at":"2025-10-03 20:33:06","extension":"zip","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1891481,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryArchiveS1.zip","url":"https://assets-eu.researchsquare.com/files/rs-7583299/v1/0939d0ea3965aafd3a254c4a.zip"},{"id":92748892,"identity":"7da2c2c8-362c-4998-9b99-58889c3ad1e1","added_by":"auto","created_at":"2025-10-03 20:33:07","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1501464,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4qPCRCtValuesandStatisticalAnalysis.xlsx.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7583299/v1/789b3330336c5082b8712663.xlsx"},{"id":92749312,"identity":"0040426c-9de0-46b6-81f3-84657e22553c","added_by":"auto","created_at":"2025-10-03 20:41:06","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":15847,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1PrimerSequencesqPCR.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7583299/v1/d4d0be308d40d6f4d670544f.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Screening of Ester-Forming and Hydrolyzing Enzymes Linked to Pheromone Production in Ips typographus (Linnaeus, 1758)","fulltext":[{"header":"1. Background","content":"\u003cp\u003eThe European spruce bark beetle, \u003cem\u003eIps typographus\u003c/em\u003e (Coleoptera: Curculionidae: Scolytinae; Linnaeus 1758), is a major forest pest driving the decline of Norway spruce \u003cem\u003ePicea abies\u003c/em\u003e (Pinaceae, H. Karst 1881) forest monocultures in Central Europe. Over the past decade, its outbreaks have intensified due to climate change and human activities, exacerbated by droughts and other forest disturbances.\u003c/p\u003e\u003cp\u003e\u003cem\u003eI. typographus\u003c/em\u003e uses a coordinated mass-attack strategy to colonize its host, the spruce tree. Male beetles produce an aggregation pheromone that attracts conspecific beetles, enabling the population to collectively overcome the defenses of trees and establish a new generation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This pheromone is composed of three key hydroxylated terpenoids. The primary component, 2-methyl-3-buten-2-ol, and the minor component, ipsdienol, are synthesized \u003cem\u003ede novo\u003c/em\u003e in the male midgut through the mevalonate pathway, triggered by feeding and mating, respectively [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In contrast, the third behaviorally active compound, (\u003cem\u003eS\u003c/em\u003e)-(\u0026minus;)-\u003cem\u003ecis\u003c/em\u003e-verbenol, a cyclic monoterpenoid, is synthesized by feeding males via cytochrome P450 (CyP450)-mediated hydroxylation of (\u0026minus;)-\u003cem\u003eα\u003c/em\u003e-pinene, the dominant monoterpene in host spruce resin, which the beetles ingest during feeding [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In \u003cem\u003eI. typographus\u003c/em\u003e, the utilization of \u003cem\u003ecis\u003c/em\u003e-verbenol as a pheromone may have co-evolved with detoxification pathways for host-derived terpenes, enhancing the beetles' ability to exploit spruce defenses [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Besides being induced by direct feeding on the host tree, the production of (\u003cem\u003eS\u003c/em\u003e)-(\u0026minus;)-\u003cem\u003ecis\u003c/em\u003e-verbenol can also be stimulated in the absence of (\u0026minus;)-\u003cem\u003eα\u003c/em\u003e-pinene. Laboratory experiments showed that topical treatment of non-feeding males with juvenile hormone III (JH III) stimulates (\u003cem\u003eS\u003c/em\u003e)-(\u0026minus;)-\u003cem\u003ecis\u003c/em\u003e-verbenol synthesis as well [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. JH III is widely used to artificially induce \u003cem\u003ede novo\u003c/em\u003e biosynthesis of bark beetle aggregation pheromones for experimental purposes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThese observations led to the hypothesis that, during colonization, males produce \u003cem\u003ecis\u003c/em\u003e-verbenol not only through the direct hydroxylation of (\u0026minus;)-\u003cem\u003eα\u003c/em\u003e-pinene, but also from alternative internal sources, specifically, \u003cem\u003ecis\u003c/em\u003e-verbenyl fatty acyl (FA) esters stored in significant quantities in the fat body [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). A similar mechanism has been proposed for the Mountain pine beetle \u003cem\u003eDendroctonus ponderosae\u003c/em\u003e Hopkins, 1902, in which females were found to store the pheromone \u003cem\u003etrans\u003c/em\u003e-verbenol in their fat bodies as verbenyl esters [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe biosynthesis of verbenyl esters in young \u003cem\u003eI. typographus\u003c/em\u003e beetles and adult males is presumably catalyzed by either lipase/esterase enzymes or FA transferases. In pheromone-producing adult males, these esters could be hydrolyzed back to yield free \u003cem\u003ecis\u003c/em\u003e-verbenol by a male-specific lipase or esterase, potentially under the regulatory control of JH III (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eIn a previous transcriptomic study, three candidate contigs, Ityp_7084, Ityp_9460, and Ityp_11977, were preliminarily selected as possible genes encoding such enzymes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These candidates were identified based on their sequence similarity to esterase BT127766.1 from \u003cem\u003eDendroctonus ponderosae\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] as well as their expression patterns across different developmental stages. However, their functional roles remain unconfirmed, as previous analyses did not provide sufficient evidence to validate these contigs.\u003c/p\u003e\u003cp\u003eAlthough ester-forming and -hydrolyzing enzymes likely play central roles in both pheromone biosynthesis and general metabolic processes across many insect taxa, they remain largely understudied. This knowledge gap may stem from their broad substrate specificity and functional redundancy, which complicate thorough experimental characterization. Among the ester-hydrolyzing enzymes documented in insects, the majority belong to the carboxylesterase (CE) family, with relatively limited diversity in enzymatic classes and functions [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn bark beetles, this pattern is consistent, most identified esterases are also CEs, which are frequently upregulated in response to host plant toxins. This suggests that they may play a role in detoxification by converting hydroxylated compounds into more water-soluble metabolites for excretion or into long-chain esters for storage [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Notably, nine CE have been reported in bark beetles closely related to \u003cem\u003eI. typographus\u003c/em\u003e, particularly in the genus \u003cem\u003eDendroctonus\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In \u003cem\u003eD. armandi\u003c/em\u003e, CE gene expression increases upon exposure to host plant compounds, reinforcing their role in plant defense neutralization [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAdditional esterases and lipases have been identified in Hymenoptera [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], as well as in other insect species, including juvenile hormone esterase in \u003cem\u003eTribolium\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and the antennal esterases in \u003cem\u003eSpodoptera\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These enzymes may be involved in olfactory processing. Some ester-hydrolyzing enzymes have also been studied in the contexts of insecticide resistance [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], chemical communication, and lipid metabolism. A comprehensive list of ester-forming and hydrolyzing genes in insects with both experimentally validated and predicted sequences, is summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, and selected genes served as the reference framework for the current study.\u003c/p\u003e\u003cp\u003eIn this study, we initially focused on identifying genes in \u003cem\u003eI. typographus\u003c/em\u003e that may be involved in both the biosynthesis of verbenyl FA esters and their subsequent hydrolysis to \u003cem\u003ecis\u003c/em\u003e-verbenol. Such identification could lend critical support to the currently unproven hypothesis that \u003cem\u003ecis\u003c/em\u003e-verbenol, a major aggregation pheromone component in \u003cem\u003eI. typographus\u003c/em\u003e, originates from detoxification-related lipid precursors. Additionally, we conducted a more extensive screening of ester-forming and -hydrolyzing enzymes in \u003cem\u003eI. typographus\u003c/em\u003e and explored their potential biological functions.\u003c/p\u003e"},{"header":"2. Methodology","content":"\u003cp\u003e2. 1. Rearing beetles, treatment, and preparation of samples\u003c/p\u003e\u003cp\u003eNorway spruce (\u003cem\u003ePicea abies\u003c/em\u003e L.) logs (20\u0026ndash;30 cm DBH, 50 cm length), naturally infested by \u003cem\u003eI. typographus\u003c/em\u003e, were collected from a forest near Kostelec nad Čern\u0026yacute;mi lesy (Czech University of Life Sciences Prague; 50\u0026deg;00\u0026prime;07.2\u0026Prime; N, 14\u0026deg;50\u0026prime;56.3\u0026Prime; E, Forest CZU) and stored at 4\u0026deg;C until use. The logs were then placed in ventilated plastic containers (55.5 \u0026times; 39 \u0026times; 28.5 cm; IKEA, Sweden) under controlled conditions (27\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 70% humidity, 16:8 h light: dark photoperiod).\u003c/p\u003e\u003cp\u003eUpon emergence, 150 fully sclerotized individuals, unsexed F0 adults, were transferred to fresh spruce logs (of the same dimensions and origin) to initiate the F1 generation. Developmental stages of the F1 generation were sampled at defined time points: larvae L1, L2, and L3 at 7, 14, and 20 days post-colonization, respectively; pupae at approximately 4 weeks; immature adults (\u0026lt;\u0026thinsp;24 h post-eclosion); newly emerged adults after exiting the breeding logs; and adult males and females after 24 h of feeding in nuptial chambers excavated in uninfested spruce logs (hereafter referred to as fed males and fed females)\u003c/p\u003e\u003cp\u003eExcept for larvae and pupae, the collected beetles were sorted by sex based on external morphology and confirmed by reproductive organ dissection, according to [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Subsequently, larvae, pupae, immature, emerged, and fed adult beetles were further processed:\u003c/p\u003e\u003cp\u003eFor juvenile hormone III (JH III) induction, only newly emerged beetles were used. After sorting by sex, groups of beetles were topically treated with 0.5 \u0026micro;L JH III solution (20 \u0026micro;g/\u0026micro;L in acetone) on the abdomen, while the control group was treated with 0.5 \u0026micro;L of pure acetone. All beetles were then maintained under the previously described laboratory conditions for 8 hours [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Beetles from various life stages and treatment groups were flash-frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent analysis. Prior to processing, guts were dissected from all beetles except larvae and pupae, for which dissection was not experimentally feasible. In beetles with dissected guts, the elytra, wings, and legs were further removed. The remaining tissue, referred to in this study as fat body, was used for metabolomic and differential gene expression (DGE) analyses.\u003c/p\u003e\u003cp\u003eFor RNA isolation, tissues were placed in a droplet of RNAlater (Invitrogen, Carlsbad, CA, USA) and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for downstream applications. For metabolite production studies, dissected guts were immediately submerged in cold pentane (10 guts per 100 \u0026micro;L), while fat bodies were extracted with chloroform (10 bodies per 1000 \u0026micro;L).\u003c/p\u003e\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\u003ch2\u003e2.2. GC\u0026ndash;MS-based determination of verbenyl oleate and \u003cem\u003ecis\u003c/em\u003e-verbenol in beetle tissues\u003c/h2\u003e\u003cp\u003eThe separation of compounds in beetle tissue extracts, and target identification, and quantification of verbenyl oleate and \u003cem\u003ecis\u003c/em\u003e-verbenol were performed using gas chromatography\u0026ndash;mass spectrometry (GC-MS), following the protocols described by [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Analyses were conducted on an Agilent 7890B GC system (Agilent Technologies, Palo Alto, CA, USA) coupled with a Pegasus 4D Time-of-Flight Mass Spectrometer (LECO, St. Joseph, MI, USA). A programmed temperature vaporization (PTV) injector was used, operated in split mode (10:1) with the following temperature program: 20\u0026deg;C ramped at 8\u0026deg;C/s to 275\u0026deg;C. The separation was achieved on an HP-5MS UI capillary column (30 m \u0026times; 0.25 mm i.d., 0.25 \u0026micro;m film thickness; Agilent). The oven temperature program was: 40\u0026deg;C (1 min hold), ramped at 10\u0026deg;C/min to 210\u0026deg;C, then at 20\u0026deg;C/min to 320\u0026deg;C (6 min hold). Electron ionization was performed at 70 eV, with a scanned mass range of 35\u0026ndash;500 Da at an acquisition rate of 10 Hz.\u003c/p\u003e\u003cp\u003eIdentification of target compounds was confirmed using analytical standards of \u003cem\u003ecis\u003c/em\u003e-verbenol and verbenyl oleate, in combination with the NIST 2017 mass spectral library. For quantification, linear calibration curves were constructed based on the respective external standards.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.3. mRNA isolation and transcript Illumina sequencing\u003c/h2\u003e\u003cp\u003eRNA was isolated from 16 distinct sample groups representing seven developmental stages. Unsexed whole-body samples included the first-, second-, and third-instar larvae, as well as pupae. In addition, gut and fat body tissues were isolated separately from the following groups: immature females, immature males, emerged females, emerged males, fed females, and fed males. Each of these samples consists of four biological replicates, with each replicate comprising tissues from ten individual beetles. The only exception was fat body tissue from fed beetles, for which nine technical replicates per sex were prepared.\u003c/p\u003e\u003cp\u003eTotal RNA extraction was performed using the PureLink\u0026trade; RNA Mini Kit (Invitrogen, Carlsbad, CA, USA), strictly following the manufacturer's protocol. Extracted RNA underwent DNase treatment using the TURBO DNase Kit (Invitrogen, Carlsbad, CA, USA). RNA integrity was assessed by electrophoresis on a 1% agarose gel, and samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until sequencing.\u003c/p\u003e\u003cp\u003eFive RNA samples from fat body tissues of fed adult beetles (both sexes) were selected and sent to Novogene Co., Ltd. (UK) for transcriptome sequencing, performed on an Illumina NovaSeq X Plus platform (paired-end sequencing, 150 bp reads).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Transcriptome assembly and ester-modifying protein identification\u003c/h2\u003e\u003cp\u003e\u003cem\u003eDe novo\u003c/em\u003e transcriptome assembly was conducted using SPAdes v3.15.5, pooling transcriptome data previously published under NCBI BioProject accession number PRJNA679450 (Male fed midgut \u0026ndash; MFMg, Male immature midgut \u0026ndash; MIMg, Female immature midgut \u0026ndash; FIMg, Male immature fat body \u0026ndash; MIFb, Female immature fat body \u0026ndash; FIFb) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], along with the transcriptomes from midguts of JH III- and acetone-treated males and females from NCBI Bioproject PRJNA934749 (Male JHIII midgut \u0026ndash; MJMg, Male acetone midgut \u0026ndash; MAMg, Female JHIII midgut \u0026ndash; FJMg, Female acetone midgut \u0026ndash; FAMg) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and newly generated RNA-seq data from the fat bodies fed adult beetles (Male fed fat body \u0026ndash; MFFb, Female fed fat body \u0026ndash; FFFb). Prior to assembly, raw sequencing reads were quality-filtered using Trimmomatic v0.39, which removed adapter sequences and trimmed low-quality bases.\u003c/p\u003e\u003cp\u003eThe quality of the resulting assembly was evaluated using RNAquast v2.3.1 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and BUSCO v5.5.0 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], in eukaryotic transcriptome mode, employing the insecta_odb10 database. A manual inspection was performed to further confirm assembly completeness and reliability.\u003c/p\u003e\u003cp\u003eNext, raw reads were aligned to the reference genome NCBI accession number GCA_016097725.1 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] using STAR v2.7.6a [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Transcript abundance was estimated using the RSEM method (implemented via Trinity v2.15.1 scripts) and featureCounts v2.0.3 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], following manual annotation of transcripts.\u003c/p\u003e\u003cp\u003ePrediction of open reading frames (ORFs) from the assembled transcripts was carried out using TransDecoder.LongOrfs v5.7.1, and the resulting ORF set was again assessed with BUSCO using library insecta_odb10. Putative verbenyl ester-forming and -hydrolyzing enzyme genes were identified through local protein-protein alignment using BLAST v2.9.0+. Candidate sequences were filtered to retain those with a minimum of 50% sequence similarity and alignment lengths of \u0026ge;\u0026thinsp;200 amino acids. Redundant hits and apparent artefacts were manually removed. To identify functionally relevant esterase genes, multiple sequence alignment was performed using Clustal Omega, MUSCLE, and MAFFT, all within SeaView v4.7 and AliView v1.28 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eReference protein sequences were retrieved from GenBank and included both experimentally validated and computationally predicted sequences. To broaden our search and account for in insect uncharacterized functional enzyme classes with acyl esterification functions, we extended our references homology-based queries using sequences derived from phylogenetically distant taxa, including plants and vertebrates (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eReference carboxylesterase (CE) and related ester-modifying protein sequences retrieved from GenBank. Accession numbers, source organisms, sequence types, and gene descriptions are provided, along with citation information. Both experimentally validated and predicted sequences from diverse taxa were included. Hydrolase and transferase sequences are marked with H and T, respectively.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnzyme class\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAccession No. of gene in GenBank\u003c/p\u003e\u003cp\u003e[literature reference]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCommon name of the organism\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eScientific name of organism\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eDescription in database\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eUsed for BLAST\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eQWW26267.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChinese White Pine Beetle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eDendroctonus armandi\u003c/em\u003e Tsai \u0026amp; C-L. Li, 1959\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Z)-6-nonen-2-ol dehydrogenase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAYN64423.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChinese White Pine Beetle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eDendroctonus armandi\u003c/em\u003e Tsai \u0026amp; C-L. Li, 1959\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ecarboxylesterase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAYN64424.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChinese White Pine Beetle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eDendroctonus armandi\u003c/em\u003e Tsai \u0026amp; C-L. Li, 1959\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ecarboxylesterase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAYN64425.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChinese White Pine Beetle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eDendroctonus armandi\u003c/em\u003e Tsai \u0026amp; C-L. Li, 1959\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ecarboxylesterase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAYN64426.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChinese White Pine Beetle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eDendroctonus armandi\u003c/em\u003e Tsai \u0026amp; C-L. Li, 1959\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ecarboxylesterase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAYN64427.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChinese White Pine Beetle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eDendroctonus armandi\u003c/em\u003e Tsai \u0026amp; C-L. Li, 1959\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ecarboxylesterase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAYN64428.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChinese White Pine Beetle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eDendroctonus armandi\u003c/em\u003e Tsai \u0026amp; C-L. Li, 1959\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ecarboxylesterase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAYN64429.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChinese White Pine Beetle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eDendroctonus armandi\u003c/em\u003e Tsai \u0026amp; C-L. Li, 1959\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ecarboxylesterase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAEE62728.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMountain Pine Beetle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eDendroctonus ponderosae\u003c/em\u003e Hopkins, 1902\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eunknown\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUUB32789.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRed Turpentine Beetle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eDendroctonus valens\u003c/em\u003e LeConte, 1857\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ecarboxylesterase COEA1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUUB32825.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRed Turpentine Beetle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eDendroctonus valen\u003c/em\u003es LeConte, 1857\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ecarboxylesterase COEM1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNM_001193294.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRed Flour Beetle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eTribolium castaneum\u003c/em\u003e (Herbst, 1797)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ejuvenile hormone esterase (Tcjhe)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNM_168643.3\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCommon Fruit Fly\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eDrosophila melanogaster\u003c/em\u003e Meigen, 1830\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003enotum (Notum)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMW699017.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFiery-tailed Bumble Bee\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eBombus ignitus\u003c/em\u003e Smith, 1869\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003evenom carboxylesterase (vCaE)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNM_001287565.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBuff-tailed Bumble Bee\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eBombus terrestris\u003c/em\u003e (Linnaeus, 1758)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003elipase member H-A-like (LOC100646090)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eXM_012694947.3\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDomesticated Silkmoth\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eBombyx mori\u003c/em\u003e (Linnaeus, 1758)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003elipase member H-A (LOC101742752) PLLG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eXM_026443853.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEuropean Honeybee\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eApis mellifera\u003c/em\u003e Linnaeus, 1758\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003elipase member H-A (LOC727193)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eXM_006566867.3\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEuropean Honeybee\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eApis mellifera\u003c/em\u003e Linnaeus, 1758\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003evenom carboxylesterase-6-like (LOC408395)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eKU360126.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChinese Tussar Moth\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eAntheraea perny\u003c/em\u003ei (Gu\u0026eacute;rin-M\u0026eacute;neville, 1855)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003elipase-related protein LRP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eJF728804.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBeet Armyworm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eSpodoptera exigua\u003c/em\u003e (H\u0026uuml;bner, 1808)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eantennal esterase CXE11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUFA27653.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTobacco budworm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHeliothis virescens\u003c/em\u003e (Fabricius,1777)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLipX protein, partial\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNO\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUFA27658.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTobacco budworm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHeliothis virescens\u003c/em\u003e (Fabricius,1777)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLipZ protein, partial\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNO\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAAW28928.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLesser Grain weevil\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eSitophilus oryzae\u003c/em\u003e (Linnaeus, 1763)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003epectin methylesterase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNO\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA0A0M3KKW3.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBlack-bellied hornet\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eVespa basalis\u003c/em\u003e Smith, 1852\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePhospholipase A1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNO\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAAG42021.2\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTobacco hornworm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eManduca sexta\u003c/em\u003e (Linnaeus, 1763)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ejuvenile hormone esterase precursor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNO\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eACV60237.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAfrican cotton leafworm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eSpodoptera littoralis\u003c/em\u003e (Boisduval, 1833)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eantennal esterase CXE10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNO\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUFA27654.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTobacco budworm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHeliothis virescens\u003c/em\u003e (Fabricius,1777)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eEst1 protein, partial\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNO\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAAB67728.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAustralian sheep blowfly\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eLucilia cuprina\u003c/em\u003e (Wiedemann, 1830)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eE3, carboxylesterase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNO\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCAA83643.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSouthern house mosquito\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eCulex quinquefasciatus\u003c/em\u003e Say, 1823\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eserine esterase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNO\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eACV60234.2\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAfrican cotton leafworm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eSpodoptera littoralis\u003c/em\u003e (Boisduval, 1833)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eantennal esterase CXE7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNO\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eJAI18199.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLight brown apple moth\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eEpiphyas postvittana\u003c/em\u003e (Walker, 1863)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCarboxylesterase, partial\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNO\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCAA83122.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFungus\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eMoesziomyces antarcticus (Sporobolomyces antarcticus Goto Sugiy. \u0026amp; Iizuka, 1969)\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003elipase B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNO\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eALV82133.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFruit fly\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eDrosophila melanogaster\u003c/em\u003e Meigen, 1830\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eesterase 6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAAF54915.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFruit fly\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eDrosophila melanogaster\u003c/em\u003e Meigen, 1830\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eacetylcholine esterase, isoform A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNM_001077781.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eZebrafish\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eDanio rerio\u003c/em\u003e (Hamilton, 1822)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ezDHHC palmitoyltransferase 15b (zdhhc15b)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAY512893.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCommon Apple\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eMalus domestica\u003c/em\u003e (Suckow) Borkh.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ealcohol acyl transferase AAT3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAF149919.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJojoba Tree\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eSimmondsia chinensis\u003c/em\u003e (Link) C.K. Schneid.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ewax synthase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eKJ626344.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChinese gooseberry\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eActinidia eriantha\u003c/em\u003e Benth.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ealcohol acyltransferase (AT9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAY056316.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eThale Cress\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e (L.) Heynh.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ewax ester synthase/diacylglycerol acyltransferase WSD1 (At5g37300)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAY947638.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eModern Human\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHomo sapiens\u003c/em\u003e Linnaeus, 1758\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eacyl-CoA wax alcohol acyltransferase (AWAT1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBC034944.1\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eModern Human\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHomo sapiens\u003c/em\u003e Linnaeus, 1758\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ezinc finger, DHHC-type containing 20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ePhylogenetic analysis of the identified esterase sequences was conducted using IQtree v2.2.2.6 [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The best-fit substitution model was selected with ModelFinder, based on AIC/BIC criteria. Phylogenetic trees were inferred using Maximum Likelihood (ML) with 10,000 bootstrap replicates to assess branch support. The resulting trees were visualized and annotated using FigTree v1.4.4 [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eProtein structure prediction was carried out using ColabFold [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The best models were aligned in PyMOL (Schr\u0026ouml;dinger, USA) and inspected for protein fold and active site conservation. The figures were prepared in UCSF ChimeraX [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Differential gene expression analysis\u003c/h2\u003e\u003cp\u003eDifferential gene expression (DGE) pairwise analysis was performed using the DESeq2 package [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. To identify genes whose expression patterns align with hypothesized functional roles and to address the core research questions, transcriptome pairs for comparison were selected (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) based on found production profiles of \u003cem\u003ecis\u003c/em\u003e-verbenol and verbenyl oleate across life stages, tissues, and sexes of \u003cem\u003eI. typographus\u003c/em\u003e ([\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]; see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). RNA-seq for various tissues were accessed again from BioProject accession number PRJNA679450 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], Bioproject PRJNA934749 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and newly generated RNA-seq data from the fat bodies of fed adult beetles from this study PRJNA1321731.\u003c/p\u003e\u003cp\u003eTo elucidate the presence of a male-specific gene coding for an enzyme that hydrolyses FA esters into \u003cem\u003ecis\u003c/em\u003e-verbenol, gene expression was expected to be specifically upregulated in the guts of fed or JH III-treated males. To support the hypothesis, we analyzed differential gene expression in the following pairwise comparisons: MFMg vs. FAMg; MFMg vs. MAMg; MJMg vs. FAMg; MJMg vs. MAMg. To identify the ester-forming gene responsible for synthesizing verbenyl fatty acid esters, candidate genes were expected to be overexpressed in the fat bodies of both sexes of immature beetles and adult males, but downregulated in the fat bodies of adult females. To investigate this, the following comparisons were made: FIFb vs. L1Wb; MIFb vs. L1Wb, and for downregulation: FFFb vs. FIFb. To identify ester-hydrolyzing enzymes active in newly emerged beetles, their genes were hypothesized to be upregulated in the fat bodies of both sexes. However, since RNAseq data from fat bodies of newly emerged beetles were not available, expression was inferred from comparisons involving immature and larval whole bodies: FIFb vs. L1Wb; MIFb vs. L1Wb. Alternatively, if the hydrolytic process occurred in the gut, comparisons were made between guts of immature and adult beetles: FIMg vs. FAMg; MIMg vs. FAMg.\u003c/p\u003e\u003cp\u003eFinally, to investigate the tissue-specific expression of the searched metabolic genes, we compared expression in midguts and fat bodies from individuals of the same developmental stage and sex, or between sexes. The full list of experimental groups used for DGE analysis is provided in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, with additional details in Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cem\u003ePairwise comparisons of experimental groups used for differential gene expression (DGE) of 26 selected enzymes across two tissues, various developmental stages, and sexes in\u003c/em\u003e Ips typographus.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLeading\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLeading\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eLeading\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale Fed Midgut (MFMg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMale Acetone Midgut (MAMg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMale Fed Fatbody (MFFb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFemale Fed Fatbody (FFFb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMale Fed Fatbody (MFFb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eMale Fed Midgut (MFMg)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale Fed Midgut (MFMg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFemale Acetone Midgut (FAMg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMale Fed Fatbody (MFFb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMale Immature Fatbody (MIFb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMale Immature Fatbody (MIFb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eMale Immature Midgut (MIMg)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale JHIII Midgut (MJMg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMale Acetone Midgut (MAMg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFemale Fed Fatbody (FFFb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFemale Immature Fatbody (FIFb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eFemale Immature Fatbody (FIFb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eFemale Immature Midgut (FIMg)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale JHIII Midgut (MJMg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFemale Acetone Midgut (FAMg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMale Fed Fatbody (MFFb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLarvae L1 Whole body (L1Wb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale Immature Midgut (MIMg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFemale Acetone Midgut (FAMg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFemale Fed Fatbody (FFFb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLarvae L1 Whole body (L1Wb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale JHIII Midgut (FJMg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFemale Acetone Midgut (FAMg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMale Immature Fatbody (MIFb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMale Acetone Midgut (MAMg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale Immature Midgut (FIMg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFemale Acetone Midgut (FAMg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFemale Immature Fatbody (FIFb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFemale Acetone Midgut (FAMg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMale Immature Fatbody (MIFb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLarvae L1 Whole body (L1Wb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFemale Immature Fatbody (FIFb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003evs.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLarvae L1 Whole body (L1Wb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eStatistical significance was determined using an adjusted p-value (padj). For each gene, the log2 fold change (log2FC) was calculated. Genes with log2FC\u0026thinsp;\u0026gt;\u0026thinsp;3.5 and a padj\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered differentially expressed.\u003c/p\u003e\u003cp\u003eA heatmap was constructed using Z-score normalized expression values to visualize expression profiles. Hierarchical clustering was based on Euclidean distance and average linkage. The heatmap was generated using the pheatmap package in R [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.6. cDNA synthesis and RT-qPCR of candidate genes\u003c/h2\u003e\u003cp\u003eBased on differential gene expression (DGE) analysis of the transcriptomic data from relevant comparison pairs (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Chap.\u0026nbsp;2.5), candidate ester-forming/hydrolysing genes that were significantly upregulated in at least two comparisons (adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and log₂ fold change\u0026thinsp;\u0026gt;\u0026thinsp;3.5) were selected. Furthermore, these genes were either upregulated or downregulated in specific comparison groups designed to address the core questions of this study (Chap.\u0026nbsp;2.5). From this analysis, seven carboxylesterase genes, Ityp-CE9, Ityp-CE10, Ityp-CE13, Ityp-CE15, Ityp-CE16, Ityp-CE20, Ityp-CE21, and one lipase gene, Ityp-Lip1, were selected for validation using reverse transcription quantitative PCR (RT-qPCR). Transcript levels of the selected candidate genes were quantified across developmental stages, between sexes, and tissues. Unsexed whole-body samples from larval instars (L1\u0026ndash;L3) and pupae were analyzed, along with dissected gut and fat body tissues from immature, newly emerged, and fed adults of both sexes.\u003c/p\u003e\u003cp\u003eTotal RNA from all relevant samples was isolated, quantified, and purified as previously described. One microgram of RNA was used as a template for first-strand cDNA synthesis using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems\u0026trade;, ThermoFisher Scientific, USA), following the manufacturer\u0026rsquo;s protocol. The reverse transcription reaction was carried out in a total volume of 20 \u0026micro;L, with three technical replicates per sample. These replicates were pooled following the reaction, and the resulting cDNA was stored at \u0026minus;\u0026thinsp;20\u0026deg;C until use.\u003c/p\u003e\u003cp\u003eGene-specific primers were designed using the PrimerQuest\u0026trade; Tool (Integrated DNA Technologies, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.idtdna.com\" target=\"_blank\"\u003ewww.idtdna.com\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.idtdna.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), based on the target gene sequences (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Primer parameters were optimized for a melting temperature of ~\u0026thinsp;60\u0026deg;C, GC content\u0026thinsp;~\u0026thinsp;55%, and primer length of ~\u0026thinsp;22 base pairs, and RT-qPCR reactions were performed using SYBR\u0026trade; Green Universal Master Mix (Applied Biosystems\u0026trade;, Thermo Fisher Scientific, USA). The cycling conditions on the real-time PCR system were set to initial denaturation at 95\u0026deg;C for 3 minutes, followed by 40 cycles of 95\u0026deg;C for 3 seconds and 60\u0026deg;C for 34 seconds, as described by [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e] and [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe ribosomal protein L6 (RPL6) gene was selected as the internal reference due to its stable expression across different life stages and between sexes [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. For each target gene and the reference gene, four biological replicates were analyzed, each with two technical replicates. The Ct cycle threshold (Ct) value for each biological replicate was calculated as the average of its technical replicates. Relative gene expression (transcript abundance) levels were calculated using the 2^(-ΔCt) method [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], where ΔCt corresponds to the difference between the Ct values of the target gene and the reference gene RPL6 (referred to as normalized expression). For reactions in which no amplification was detected across all four biological replicates, the Ct value was set to 40 (the maximum cycle number) to represent undetected expression [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Statistical analysis of verbenyl oleate and \u003cem\u003ecis\u003c/em\u003e-verbenol production and RT-qPCR expression data\u003c/h2\u003e\u003cp\u003eProduction of verbenyl oleate and \u003cem\u003ecis\u003c/em\u003e-verbenol across different life stages of \u003cem\u003eI. typographus\u003c/em\u003e, and following JHIII treatment, was analyzed based on data adapted from [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], respectively. Depending on the dataset, statistical evaluation was performed using a one-way analysis of variance (ANOVA) followed by Fisher\u0026rsquo;s least significant difference (LSD) test.\u003c/p\u003e\u003cp\u003eIndependent Student's t-tests were performed after qPCR to evaluate differences in gene expression between sexes. In addition, to assess intra-sexual variation across developmental stages, a one-way ANOVA followed by Tukey\u0026rsquo;s Honest Significant Difference (HSD) post-hoc test was used for multiple comparisons.\u003c/p\u003e\u003cp\u003eAll statistical analyses were performed using Microsoft Excel (Microsoft Corporation, 2018), with significance set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Statistical assumptions, including normality and homogeneity of variances, were checked prior to analysis. The \u003cem\u003ep\u003c/em\u003e-values derived from the \u003cem\u003et\u003c/em\u003e-tests and ANOVA were used to determine whether the observed differences were statistically significant. The null hypothesis assumed no significant differences in CE and LIP expression levels between the compared groups.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1 Production of verbenyl oleate and \u003cem\u003ecis\u003c/em\u003e-verbenol in different life stages, sexes, and tissues, and their induction by JHIII\u003c/p\u003e\u003cp\u003eTo elucidate the phenotypic context of final metabolite production and identify the life stages, sexes, and tissues in which the studied ester-forming/hydrolyzing genes are predicted to be upregulated, we examined the production patterns of verbenyl oleate and \u003cem\u003ecis\u003c/em\u003e-verbenol across different phenotypes and treatments in \u003cem\u003eI. typographus\u003c/em\u003e using GC-MS analysis ([\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B).\u003c/p\u003e\u003cp\u003eThe highest concentrations of verbenyl oleate, representing the pool of stored verbenyl esters, were detected in the fat bodies of immature beetles, irrespective of sex. After beetles emerged from their host tree, verbenyl oleate levels declined sharply, becoming undetectable in the fat bodies of females and decreasing to approximately 25% of the pre-emergence concentration in males. Verbenyl oleate was only detectable in adult males. In feeding males, the life stage associated with peak production of the aggregation pheromone \u003cem\u003ecis\u003c/em\u003e-verbenol, fat body levels of verbenyl oleate were further reduced, compared to newly emerged males.\u003c/p\u003e\u003cp\u003eThe highest concentrations of \u003cem\u003ecis\u003c/em\u003e-verbenol were observed in immature beetles of both sexes. However, after emergence, it was detected exclusively in males, with the highest levels found in feeding males, which are known to actively produce aggregation pheromones.\u003c/p\u003e\u003cp\u003eTo investigate the regulatory role of JH III in the biosynthesis of these compounds, hormone treatments were applied. JH III increased verbenyl oleate levels in the male fat body but caused a slight suppression in the male gut. Unexpectedly, JH III also induced verbenyl oleate production in females, in both the fat body and gut, although at lower concentrations than in males. For \u003cem\u003ecis\u003c/em\u003e-verbenol, significant induction was observed only in the guts of JH III-treated males, with no response in females. Additionally, a notable increase in \u003cem\u003ecis\u003c/em\u003e-verbenol content was observed in the fat bodies of feeding males, reinforcing their role as the primary pheromone-producing stage.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Transcriptome assembly\u003c/h2\u003e\u003cp\u003eTo establish a reliable database for identifying candidate ester-forming/hydrolyzing genes, we compiled and pooled relevant publicly available and newly generated transcriptomic datasets to assemble a comprehensive reference transcriptome. BUSCO analysis using the \u003cem\u003einsecta_odb10\u003c/em\u003e lineage dataset (1,367 orthologs) indicated a highly complete assembly. In transcriptome mode, 99.1% of Benchmarking Universal Single-Copy Orthologs (BUSCOs) were classified as complete (1,355/1,367), comprising 5.8% single-copy and 93.3% duplicated orthologs. Only 0.7% were fragmented, and 0.2% were missing.\u003c/p\u003e\u003cp\u003eTo further validate the assembly\u0026rsquo;s coding content, BUSCO analysis was performed in protein mode using TransDecoder-predicted ORFs. This revealed 96.9% complete BUSCOs (1,324 out of 1,367), including 14.3% single-copy and 82.6% duplicated genes. Only 1.8% were fragmented, and 1.3% were missing. These findings confirm that the transcriptome assembly is both structurally complete and rich in intact protein-coding sequences, affirming its reliability for downstream analyses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Identification and Phylogenetic Analysis of Esterase Orthologs.\u003c/h2\u003e\u003cp\u003eTo identify putative transcripts involved in ester formation and hydrolysis, we conducted a series of protein-protein local alignments using the predicted ORFs from our transcriptome assembly. These were compared against known ester-forming and hydrolyzing enzymes previously identified in bark beetles, other insect species, and functionally characterized enzymes from various taxa (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). based on following manual curation and gene annotation, we identified a total of 23 carboxylesterases, 2 lipases, one neurolactin-like, and one notum-like ortholog, all containing conserved catalytic triads comprising serine-histidine-glutamate/aspartate residues. The candidates are further reported as Ityp-CE1 to Ityp-CE24 and Ityp-LIP1, Ityp-LIP2, and Ityp-altCEA, Ityp-altCEB. The transcript sequence of Ityp-CE1-2 mapped with 100% homology into two distinct genomic loci represented by contigs JADDUH010000016.1 and JADDUH010000023.1 while keeping similar gene structure, thus pointing to local chimeric misassembly in draft genome GCA_016097725.1. Full sequence identifiers are provided in Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eSequences of candidate genes were filtered based on alignment length and homology, with redundant or partial entries excluded. Phylogenetic reconstruction using IQtree v2.2.2.6 placed the candidates into three main clades A-C, containing 33 previously characterized esterases from various taxa (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The putative \u003cem\u003eI. typographus\u003c/em\u003e esterases are represented by small genes, mostly ranging up to 5kb in size and sharing conserved structure with 9\u0026ndash;11 protein-coding exons interspersed with short introns (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). While the genes in clades A and B are structurally conserved with only a few exceptions, such as Ityp-CE10 and Ityp-CE11 containing longer intronic regions or Ityp-CE19 with only 8 protein-coding exons, the gene structures represented in clade C are more relaxed. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) All Ityp-CE genes encode proteins of more than 500 amino acids, which distinguishes them from putative lipases with size of roughly 330 residues. Smaller product sizes, Ityp-LIP1 and Ityp-LIP2, are given by a lower number of protein coding exons (7 and 6, respectively), and the genes also differ significantly in sequence homology (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B).\u003c/p\u003e\u003cp\u003eThe phylogenetic tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) demonstrates that many of the newly identified esterase candidates cluster into well-supported clades alongside known insect esterases, particularly those from closely related bark beetle species.\u003c/p\u003e\u003cp\u003eClade A contains eleven newly identified gene sequences from \u003cem\u003eI. typographus\u003c/em\u003e, including Ityp-CE9 and Ityp-CE10, which clustered closely with a group of previously characterized esterases from \u003cem\u003eDendroctonus armandi\u003c/em\u003e (AYN64424.1, AYN64425.1, AYN64427.1, AYN64429.1; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and \u003cem\u003eDendroctonus valens\u003c/em\u003e (UUB32789.1, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eClade B includes eight sequences and is subdivided into two well-supported branches. The first branch consists of five genes (Ityp-CE12 to Ityp-CE16) that cluster near \u003cem\u003eDendroctonus ponderosae\u003c/em\u003e esterase (AEE62728.1, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The second branch comprises Ityp-CE17 to Ityp-CE19, which cluster around \u003cem\u003eD. armandi\u003c/em\u003e esterase AYN64428.1 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eClade C contains the remaining five carboxylesterase sequences (Ityp-CE20 to Ityp-CE24). These sequences mostly fall outside of the main bark beetle carboxylesterase clusters, with the exception of two \u003cem\u003eD. armandi\u003c/em\u003e sequences and one from \u003cem\u003eTribolium castaneum\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), which cluster close to \u003cem\u003eItyp-CE20\u003c/em\u003e. This clade predominantly includes carboxylesterase-like sequences reported in more distantly related insect orders, including Hymenoptera, Lepidoptera, and Diptera. Notably, Ityp-CE22, Ityp-CE23, and Ityp-CE24 cluster near carboxylesterases from \u003cem\u003eSpodoptera\u003c/em\u003e spp., \u003cem\u003eLucilia spp.\u003c/em\u003e, and \u003cem\u003eCulex spp.\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eIn addition to carboxylesterases, we also identified two novel lipases, Ityp-LIP1 and Ityp-LIP2, based on sequence similarity to functionally characterized insect lipases. Ityp-LIP1 clustered most closely with lepidopteran lipases from \u003cem\u003eBombyx mori\u003c/em\u003e and \u003cem\u003eAntheraea pernyi\u003c/em\u003e, while Ityp-LIP2 grouped with hymenopteran lipases from \u003cem\u003eApis mellifera\u003c/em\u003e and \u003cem\u003eVespa basalis\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eLastly, we examined two additional genes via BLAST that resemble non-typical esterases. A neurolactin-like gene previously reported in \u003cem\u003eD. valens\u003c/em\u003e clustered closely with Ityp-altCEB, while a notum-like gene from \u003cem\u003eDrosophila melanogaster\u003c/em\u003e clustered with Ityp-altCEa (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eThus, based on sequence analyses and predicted function, Ityp-altCEA and Ityp-altCEB were excluded from further analyses as these two enzymes are most likely to act on protein or as membrane-associated proteins. Ityp-altCEA is a closely related sequence with features of NOTUM-like and palmitoleoyl-protein carboxylesterases, likely targeting lipid modifications on proteins. Ityp-altCEB shows similarity to neurotactin-like proteins and may adopt an α/β-hydrolase fold, but is predicted as a membrane-associated protein.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eA) Structure of 24 esterase-like (red arrow) and 2 lipase-like (orange arrow) candidate genes identified in I. typographus genome assembly GCA_016097725.1. Protein-coding exons are represented by black boxes, and genomic coordinates in contigs marked in italics are given by numbers surrounding the arrows. Candidate genes used for qPCR analyses are highlighted in dark color. B) Phylogenetic analysis of esterase orthologs illustrating relationships of I. typographus esterases to reference genes. A maximum-likelihood tree was reconstructed in IQ‐TREE v2.2.2.6 using the VT\u0026thinsp;+\u0026thinsp;G4 amino‐acid substitution model. Branch support was assessed with 10,000 nonparametric bootstrap replicates. Terminal labels indicate species abbreviations (e.g., Athal, Darm, Amel) and GenBank accession numbers. Colors represent taxonomic order; previously characterized enzymes are marked with red stars and described based on reported functions. Capital letters A-C represent three main clades of I. typographus carboxylesterase paralogs. The tree is presented in the form of a cladogram with transformed branches, and the scale bar represents the mean number of substitutions per site. Lipase B from the fungus\u003c/em\u003e Moesziomyces antarcticus \u003cem\u003ewas used as an outgroup protein.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eOn the level of predicted protein structure, both CE and LIP proteins show distinct architectures (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). They all share an 11-β-sheet core surrounded by α-helices, but the CE proteins are generally larger. All proteins contain a putative catalytic triad consisting of serine-histidine-glutamate/aspartate residues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Gene-Specific Expression Patterns\u003c/h2\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e3.4.1. NGS-based Gene Expression Analysis\u003c/h2\u003e\u003cp\u003eTo investigate gene expression dynamics across life stages, treatments, and sexes in \u003cem\u003eI. typographus\u003c/em\u003e, we performed gene expression analysis using both publicly available and newly generated RNA-seq datasets. To visualize expression patterns across all inspected tissues, development stages and sexes, we generated a heatmap representing TPM-normalized expression of identified candidate genes transformed to Z-scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Guided by these patterns and results from pairwise differential gene expression analyses according to Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (for complete results see Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), we identified several candidate esterases and lipases genes that may be involved in verbenyl ester hydrolysis and/or biosynthesis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCarboxylesterase-coding genes Ityp-CE9, Ityp-CE10, Ityp-CE13, Ityp-CE15, Ityp-CE20, and Ityp-CE21 were selected for further analyses based on their differential expression profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). All six showed significant upregulation in at least two comparisons (padj\u0026thinsp;\u0026lt;\u0026thinsp;0.05; log₂FC\u0026thinsp;\u0026gt;\u0026thinsp;3.5). In addition, two further genes were included: Ityp-LIP1, representing a different enzyme class, and Ityp-CE16, chosen for its distinct expression profile relative to the other candidates. Their expression patterns are as follows:\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cp\u003eItyp-CE9 exhibited a broad expression profile, with the highest expression detected in the fat bodies of immature beetles of both sexes. Expression was especially prominent in fat bodies of both sexes of immature beetles (Fig. 5). This tissue specificity was further supported by pairwise differential expression analysis, with significant upregulation in MIFb vs. MAMg (log₂FC = 5.29, padj = 1.36\u0026times;10\u003csup\u003e\u0026minus;15\u003c/sup\u003e) and in FIFb vs. FAMg (log₂FC = 3.71, padj = 3.56\u0026times;10\u003csup\u003e\u0026minus;14\u003c/sup\u003e). Notably, Ityp-CE9 expression was also induced in male midguts under feeding and JHIII treatment conditions. Its expression was significantly elevated in the MFMg vs. MAMg (log₂FC = 2.57, padj = 0.00008) and in the MJMg vs. MAMg (log₂FC = 1.75, padj = 0.00001). No comparable induction was observed in female midgut tissues (Fig. 6A, Tab. S3).\u003c/p\u003e\n\u003cp\u003eItyp-CE10 exhibited a similar expression trend to Ityp-CE9, although its overall transcript levels were lower. This gene showed the highest expression in fat bodies, particularly in immature beetles (Fig. 5). Significant upregulation was observed in FIFb vs. FAMg (log₂FC =5.44, padj = 5.16\u0026times;10\u003csup\u003e\u0026minus;23\u003c/sup\u003e) and MIFb vs. MAMg (log₂FC = 7.62, padj =2.09\u0026times;10\u003csup\u003e\u0026minus;22\u003c/sup\u003e). Furthermore, despite its lower expression levels there, Ityp-CE10 was also upregulated in the midguts of JHIII-treated males and immature females. Specifically, significant upregulation was detected in the MFMg (log₂FC = 2.68, padj = 0.03) and MJMg (log₂FC = 1.69, padj = 0.00003), compared to the MAMg control. Upregulation was also found in the midguts of immature beetles when compared with midguts of acetone-treated adults, FIMg vs. FAMg (log₂FC = 2.79, padj = 00003) and MIMg vs. FAMg (log₂FC = 3.38, padj = 6.99\u0026times;10\u003csup\u003e\u0026minus;8\u003c/sup\u003e) (Fig. 6B, Tab. S3).\u003c/p\u003e\n\u003cp\u003eItyp-CE13 was expressed broadly across tissues, with the highest levels observed in the immature fat bodies of both sexes (Fig. 5). After DGE, expression is demonstrably higher in the immature female fat bodies than midguts FIFb vs. FIMg (log₂FC = 2.68, padj = 0.0019), than in female acetone-treated midguts FIFb vs. FAMg (log₂FC = 2.67, padj = 1.15\u0026times;10\u003csup\u003e\u0026minus;9\u003c/sup\u003e), and as well the larvae FIFb vs. L1WB (log₂FC = 1.76, padj =1.26\u0026times;10\u003csup\u003e\u0026minus;7\u003c/sup\u003e). The lowest expression occurred in the male fed midguts, where it was strongly downregulated compared to the male acetone-treated midguts MFMg vs. MAMg (log₂FC = \u0026ndash;6.60, padj = 9.37\u0026times;10\u003csup\u003e\u0026minus;52\u003c/sup\u003e). Nonetheless, slight upregulation can be observed in the immature and JHIII-treated male midguts when compared to acetone-treated female midguts MIMg vs FAMg log₂FC = 1.98, padj =0.004) and MJMg vs FAMg log₂FC = 1.56, padj =0.00009), respectively (Fig.6C, Tab. S3).\u003c/p\u003e\n\u003cp\u003eItyp-CE15 showed the highest expression in the fat bodies of immature beetles (Fig. 5). This was demonstrated by significant upregulation in comparisons such as MIFb vs. L1Wb (log₂FC = 4.50, \u003cem\u003epadj\u003c/em\u003e = 0.001) and FIFb vs. L1Wb (log₂FC = 3.14, \u003cem\u003epadj\u003c/em\u003e = 0.004), as well as in other DGE comparisons where fat bodies of fed beetles were compared to control groups. No significant upregulation was observed in any midgut comparisons (Fig. 6D, Tab. S3).\u003c/p\u003e\n\u003cp\u003eUnlike\u0026nbsp;most of the analysed genes in this study, Ityp-CE16 was predominantly expressed in the midguts of beetles rather than in the fat bodies, with particularly high expression in the midguts of acetone-treated males and immature females (Fig. 5). This was evidenced by its significant downregulation in comparisons between fat bodies and midguts, for example, in MIFb vs. MIMg (log₂FC = -8.89, padj = 4.74\u0026times;10\u003csup\u003e\u0026minus;37\u003c/sup\u003e) and FIFb vs. FIMg (log₂FC = -10.5, padj =5.04\u0026times;10\u003csup\u003e\u0026minus;56\u003c/sup\u003e). A similar pattern of downregulation was observed across all comparison pairs where fat bodies (from both sexes and treatments) were compared with midguts (from both acetone-treated and fed beetles of both sexes). Moreover, Ityp-CE16 also showed elevated expression levels in the whole bodies of larvae, as shown in comparisons such as MIFb vs. L1Wb (log₂FC = -6.76, \u003cem\u003epadj\u003c/em\u003e = 2.71\u0026times;10\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003csup\u003e25\u003c/sup\u003e) and FIFb vs. L1Wb (log₂FC = -10.54, \u003cem\u003epadj\u003c/em\u003e = 9.31\u0026times;10\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003csup\u003e44\u003c/sup\u003e) (Fig. 6E, Tab. S3)\u003c/p\u003e\n\u003cp\u003eItyp-CE20 exhibited strong expression primarily in the midguts of acetone- and juvenile hormone-treated females, as well as in the fat bodies of feeding beetles of both sexes (Fig. 5). This was supported by its significant downregulation in the pairwise comparison FIFb vs. FAMg (log₂FC = -3.01, \u003cem\u003epadj\u003c/em\u003e = 8.18\u0026times;10\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003csup\u003e25\u003c/sup\u003e), and in all transcriptome comparisons where acetone-treated female midguts were used as the reference. Additionally, Ityp-CE20 was clearly expressed in the fat bodies of feeding beetles, as indicated by strong upregulation in comparisons such as FFFb vs. L1Wb (log₂FC = 5.86, \u003cem\u003epadj\u003c/em\u003e = 4.40\u0026times;10\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003csup\u003e17\u003c/sup\u003e) and MFFb vs. L1Wb (log₂FC = 5.78, \u003cem\u003epadj\u003c/em\u003e = 8.45\u0026times;10\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003csup\u003e12\u003c/sup\u003e). In contrast, its expression in larval tissues and in the midguts of juvenile or immature beetles remained consistently low (Fig. 6F, Tab. S3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eItyp-CE21\u003c/strong\u003e exhibited high expression levels in the midguts of immature beetles of both sexes, as well as in the fat bodies of feeding males (Fig. 5). This was demonstrated by significant upregulation in DGE comparisons such as \u003cstrong\u003eFIMg vs. FAMg\u003c/strong\u003e (log₂FC = 4.29, \u003cem\u003epadj\u003c/em\u003e = 3.54\u0026times;10\u003csup\u003e\u0026minus;14\u003c/sup\u003e) and \u003cstrong\u003eMIMg vs. FAMg\u003c/strong\u003e (log₂FC = 3.94, \u003cem\u003epadj\u003c/em\u003e = 2.74\u0026times;10\u003csup\u003e\u0026minus;22\u003c/sup\u003e). Conversely, when midguts of immature beetles were used as reference in other comparisons, \u003cstrong\u003eItyp-CE21\u003c/strong\u003e log₂FC was constantly negative, confirming elevated expression in this tissue. High expression in the fat bodies of fed males was further supported by the comparison \u003cstrong\u003eMFFb vs. MFMg\u003c/strong\u003e (log₂FC = 2.55, \u003cem\u003epadj\u003c/em\u003e = 5.51\u0026times;10\u003csup\u003e\u0026minus;19\u003c/sup\u003e)\u0026nbsp;(Fig. 6G,\u0026nbsp;Tab.\u0026nbsp;S3)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eItyp-LIP1\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eexhibited low overall expression across all tissues and treatments. The highest transcript levels were detected in the fat bodies of immature beetles and in the midguts of immature individuals (Fig. 5). This was supported by significant upregulation in pairwise comparisons \u003cstrong\u003eFIFb vs. FAMg\u003c/strong\u003e (log₂FC = 2.63, \u003cem\u003epadj\u003c/em\u003e = 0.0001) for fat bodies, and \u003cstrong\u003eFIMg vs. FAMg\u003c/strong\u003e (log₂FC = 3.61, \u003cem\u003epadj\u003c/em\u003e = 0.009), \u003cstrong\u003eMIMg vs. FAMg\u003c/strong\u003e (log₂FC = 3.91, \u003cem\u003epadj\u003c/em\u003e = 0.00001) for midguts (Fig. 6H, Tab. S3).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOverall, the majority of analyzed genes showed their highest expression in fat bodies, particularly in immature beetles (Ityp-CE9, -CE10, -CE13, -CE15, -CE20). Ityp-CE16 differed from this pattern by being predominantly expressed in midguts across sexes and treatments. Ityp-CE21 displayed stage-dependent expression, with strong activity in immature midguts of both sexes. This pattern is similar to the expression of Ityp-LIP1 with minor differences.\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003e\u0026nbsp;3.4.2. RT-qPCR of the selected candidates in different life stages, tissues, and sexes\u0026nbsp;\u003c/h4\u003e\n\u003cp\u003eTo further verify transcriptional dynamics of the eight selected candidate genes, we performed RT-qPCR analyses across multiple developmental stages and tissues of \u003cem\u003eI. typographus\u003c/em\u003e. Expression was examined in whole bodies of larval and pupal stages, as well as in the gut and fat body tissues of adult beetles from both sexes (Fig. 7). Supporting data, including Ct values and results of statistical analyses, are provided in Table S4.\u003c/p\u003e\n\u003cp\u003eRT-qPCR expression profile of Ityp-CE9 in whole-body samples of juvenile beetle life stages was similar across all larval stages, followed by a marked decline in pupae (Fig. 7A1). In fat body tissue, expression patterns paralleled those in the gut, with generally similar levels between sexes at all stages. Although the emerged and fed males showed higher values than females, these differences were not statistically significant. Across life stages, expression was highest in immature beetles, dropped in emerged beetles, and rose again in fed beetles, with a trend toward higher levels in fed males (Fig. 7A2). In gut tissue, immature males exhibited slightly lower expression than females, but the difference was not significant. In emerged and fed adults, expression levels were not sex-specific (Fig. 6A3).\u003c/p\u003e\n\u003cp\u003eItyp-CE10 was expressed in samples from whole bodies of juvenile beetles relatively constantly across larval stages L1 to L3 before increasing significantly in L3 and then sharply decreasing in pupae (Fig. 7B1). In fat body tissue, immature beetles showed the highest expression levels in both sexes, with values significantly greater than those of emerged and fed individuals. Emerged adults displayed reduced expression, while fed beetles showed a modest increase. Expression was generally similar between males and females within each stage, although immature males exhibited slightly higher levels than females (\u003cem\u003ep\u003c/em\u003e= 0.04) (Fig. 7B2). In gut tissue, expression was again highest in immature adults of both sexes, significantly exceeding that of emerged and fed stages. Emerged and fed individuals exhibited reduced expression with no major sex-specific differences (Fig. 7B3).\u003c/p\u003e\n\u003cp\u003eRT-qPCR expression level of Ityp-CE13 in whole bodies of young beetles increased steadily from L1 to L3 larvae, then dropped sharply to near-zero in pupae (Fig.7C1), resembling patterns seen in Ityp-CE9 (Fig. 6A1), Ityp-CE10 (Fig. 7B1). In fat body tissue, expression rose again in immature beetles of both sexes, although significantly more in males (\u003cem\u003ep\u003c/em\u003e= 0.018). In newly emerged adults, levels dropped sharply, but females still expressed significantly more than males (\u003cem\u003ep\u003c/em\u003e= 0.0005). In fed adults, expression increased again, with higher levels in males than females (Fig. 7B2), again resembling expression of Ityp-CE9 (Fig. 7A2), Ityp-CE10 (Fig. 7B2). In gut tissue, Ityp-CE13 was highly expressed in immature beetles of both sexes, with no significant sex difference. Expression declined in newly emerged adults and remained low in fed beetles, with a non-significant decrease in fed males (Fig. 7C3).\u003c/p\u003e\n\u003cp\u003eIn whole-body samples, Ityp-CE15 expression was notably high only in L3 larvae, with very low levels in L1, L2, and pupae (Fig. 7D1). In fat body tissue, expression was generally higher than in gut tissue, with a trend toward higher levels in males. The only significant sex-based difference occurred in the immature stage with higher expression in male tissue (\u003cem\u003ep\u003c/em\u003e= 0.016). Across life stages, expression increased from immature to emerged adults, then declined after feeding, significantly so in females (Fig. 7D2). In gut tissue, immature females expressed more than immature males (\u003cem\u003ep\u003c/em\u003e= 0.035). Female expression declined progressively from immature to emerged to fed adults, whereas males peaked at the emerged stage (Fig. 7D3).\u003c/p\u003e\n\u003cp\u003eExpression of Ityp-CE16 in juvenile beetles increased from L1 to L3, followed by a sharp decline to near-zero levels in pupae (Fig. 6E1), resembling patterns seen in Ityp-CE9 (Fig. 7A1), Ityp-CE10 (Fig. 7B1), and Ityp-CE13 (Fig. 7C1). In fat body tissue, expression remained low at all three adult stages, with a significant male bias in newly emerged adults (\u003cem\u003ep\u003c/em\u003e= 0.023), consistent with the profile of the above genes (Fig. 7E2). In gut tissues, Ityp-CE16 was the most highly expressed among all tissues and showed exceptionally high transcript levels compared to other genes in the beetle midgut (Fig. 7E3). In females, expression peaked in immature beetles, dropped sharply in newly emerged adults, and rose again in fed adults, a trend also seen in Ityp-CE9 (Fig. 7A3), Ityp-CE10 (Fig. 7B3), and Ityp-CE13 (Fig. 7C3). In males, expression was similarly high in immature beetles, decreased in newly emerged adults (less sharply than in females), and, unlike females, declined further after feeding. This profile, though at much higher absolute levels, resembled that of Ityp-CE13 (Fig. 7E3).\u003c/p\u003e\n\u003cp\u003eItyp-CE20 was expressed in the whole bodies of young beetles, mostly in L1 larvae and lower in L2, L3, and pupae (Fig. 7F1). In fat body tissue, expression was equally low in immature beetles of both sexes, but increased several-fold in newly emerged females, reaching significantly higher levels than in males (\u003cem\u003ep\u003c/em\u003e=0.016). In fed adults, expression dropped again, equalizing between sexes (Fig. 7F2). In gut tissue, females showed a similar profile: expression increased sharply in newly emerged adults, reaching levels several-fold higher than in males (\u003cem\u003ep\u003c/em\u003e= 0.050), but was sex-equal and lower in immature and fed beetles (Fig. 7F3).\u003c/p\u003e\n\u003cp\u003eThe last from carboxylesterases \u0026nbsp;Ityp-CE21 expression in whole bodies of young beetles was uniformly low across larval stages (L1-L3) but rose sharply in pupae (Fig. 7G1), although high variability among pupae rendered the difference non-significant. \u0026nbsp;In fat body tissue, clear sex-specific differences were present at all stages. Immature females expressed more than males (\u003cem\u003ep\u003c/em\u003e= 0.029), while in emerged (\u003cem\u003ep\u003c/em\u003e= 0.032) and fed adults (\u003cem\u003ep\u003c/em\u003e= 0.026), expression was higher in males. In females, expression peaked in the immature stage; in males, it peaked in the emerged stage (Fig. 7G2). In gut tissue, overall expression was very low. The only significant sex-specific difference occurred in the immature stage (\u003cem\u003ep\u003c/em\u003e= 0.0003), with higher expression in males. In males, expression remained consistent across stages; in females, it was low in immature and emerged beetles, with a slight increase after feeding (Fig. 7G3).\u003c/p\u003e\n\u003cp\u003eSimilarly to the transcriptome data, the expression profile of Ityp-Lip1 was consistently lower than that of the other carboxylesterases analyzed in this study. In whole bodies of larvae and pupae, Ityp-Lip1 expression was below the detection limit of the method used (Fig. 7H1). In the fat body tissue, expression was higher than in other samples, and peaked in both sexes during the emerged stage. Furthermore, in fed adults, expression in the fat body was significantly higher in males (p= 0.035) (Fig. 7H2). In the gut tissues, overall expression levels were lower than in the fat bodies, and expression in the guts of feeding females and immature males was below the detection limit. The highest expression could again be observed in the guts of the emerged stage of both sexes. A significant sex-specific difference was observed in the guts of the immature stage, where females exhibited higher expression levels than males (\u003cem\u003ep\u003c/em\u003e= 0.011) (Fig. 7H3).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003ch3\u003e4.1. Production profiles of verbenyl oleate and \u003cem\u003ecis\u003c/em\u003e-verbenol as indicators of the genetic basis of their biosynthesis\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eThe highest verbenyl oleate concentrations were found in the fat bodies of immature beetles of both sexes that had not yet left the bark and did not require pheromonal communication [3]. This may show that, at this stage, verbenyl oleate may be produced as a detoxification product of \u0026alpha;-pinene ingested during maturation feeding of juvenile beetles (larvae, pupae, immature). Based on this, we initially investigated immature beetles for enzymes catalyzing verbenyl ester formation.\u003c/p\u003e\n\u003cp\u003eA sharp decline in verbenyl oleate content after emergence in both sexes, though males retained or produced small amounts [3], led us to examine newly emerged beetles for ester-hydrolyzing enzymes that could release stored energy.\u003c/p\u003e\n\u003cp\u003eIn feeding males, the key stage for \u003cem\u003ecis\u003c/em\u003e-verbenol pheromone production, fat body concentrations of verbenyl oleate were lower than in newly emerged males. This suggests partial hydrolysis of stored esters to release free \u003cem\u003ecis\u003c/em\u003e-verbenol for pheromone production [3]. This interpretation was supported by the effect of JH III, which stimulated \u003cem\u003ecis\u003c/em\u003e-verbenol production in the gut of emerged males while reducing verbenyl oleate levels in the same tissue [7]. These findings indicate ester cleavage as a source of pheromonal \u003cem\u003ecis\u003c/em\u003e-verbenol and guided our search for an adult male-specific hydrolyzing enzyme. Changes in \u003cem\u003ecis\u003c/em\u003e-verbenol levels and residual verbenyl oleates in the gut suggest that both esterification and hydrolysis may occur there, alongside \u0026alpha;-pinene detoxification by hydroxylation. However, the exact tissue specificity of these processes remains unresolved.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003e4.2 Selection of candidate genes\u003c/h3\u003e\n\u003cp\u003eTo disentangle the enzyme classes involved in the metabolism of monoterpenyl fatty acid esters in \u003cem\u003eI. typographus\u003c/em\u003e, we focused our gene-level analysis on known insect ester-forming and -hydrolysing enzymes, primarily carboxylesterases, along with lipases and acyltransferases to a lesser extent. To broaden the scope and capture potentially uncharacterized or divergent enzyme families, we expanded our homology-based searches to include reference sequences from phylogenetically distant organisms, such as fish, plants, and humans. This approach allowed us to identify 27 putative ester-modifying genes in \u003cem\u003eI. typographus\u003c/em\u003e, all encoding enzymes with a catalytic triad. Most were carboxylesterases (23), with two lipases and two other distinct enzymes, but no acyltransferases met the similarity criteria. All identified genes were closely related in sequence to those from bark beetles or other insects. The 27 candidate genes were ubiquitously expressed across sexes, developmental stages, tissues, and in response to juvenile hormone (JH) treatment. The criteria established for pairwise comparison of gene expression across different transcriptomes led to the selection of eight candidate genes for further investigation. These included seven carboxylesterases, Ityp-CE9,10,13,15,16,20,21, and one lipase, Ityp-Lip1.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003e4.3 Description and putative functions of selected esters-forming/hydrolyzing genes\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003e\u003cstrong\u003eItyp-CE9, Ityp-CE10:\u003c/strong\u003e Ityp-CE9 and Ityp-CE10, both located in clade A of the phylogenetic tree, differ in gene structure: Ityp-CE9 contains 9 exons, while Ityp-CE10 has 11 exons with longer intronic regions, suggesting an evolutionarily older origin [70, 71]. Both genes showed their highest expression in the fat bodies of immature beetles of both sexes and lower expression in the midguts. Pairwise comparisons revealed notable upregulation of expression in the guts of fed and JH III-treated males, compared to both control and newly emerged males. This expression pattern aligns with observed metabolic changes: elevated levels of \u003cem\u003ecis\u003c/em\u003e-verbenol and reduced levels of verbenyl oleate in the guts of fed and JH III-treated males (Fig. 2A\u0026ndash;B; [3, 7]). Ityp-CE9 and Ityp-CE10 clustered phylogenetically with carboxylesterases from \u003cem\u003eDendroctonus armandi\u003c/em\u003e (AYN64424.1, AYN64425.1, AYN64427.1, AYN64429.1; Tab. 1, Fig. 3B) and \u003cem\u003eDendroctonus valens\u003c/em\u003e (UUB32789.1; Tab. 1, Fig. 3B), genes suggested to be involved in detoxification. Notably, Ityp-CE9 is closely related to Darm-AYN64429.1, whose function in monoterpene detoxification was particularly highlighted when \u003cem\u003eD. armandi\u003c/em\u003e was exposed to \u0026alpha;-pinene [15]. RT-qPCR profiling of Ityp-CE9 and Ityp-CE10 across life stages, sexes, and tissues (midgut and fat body) in \u003cem\u003eI. typographus\u003c/em\u003e revealed highly similar expression patterns, indicating a correlation between their expression and feeding activity in beetles. Although both genes showed higher expression in the midguts and fat bodies of adult males than females, this difference was not always statistically significant. According to this, the trend suggests that these enzymes may be involved in male-specific hydrolysis of verbenyl esters to release \u003cem\u003ecis\u003c/em\u003e-verbenol in the gut. However, no strong overexpression in the gut of feeding males was observed. While it remains possible that male-specific hydrolysis could occur in the fat bodies, it is more plausible that these enzymes are involved in the biosynthesis of monoterpenyl FA esters, likely as part of a detoxification mechanism active during juvenile beetle stages in response to monoterpene exposure. In adults, the male-biased expression could reflect a sex-specific metabolic function, as these monoterpenyl FA esters are absent in females\u0026apos; fat bodies.\u003c/p\u003e\n\u003cp\u003eSince \u003cstrong\u003eItyp-CE9\u003c/strong\u003e and \u003cstrong\u003eItyp-CE10\u003c/strong\u003e, initially promising for alternative \u003cem\u003ecis\u003c/em\u003e-verbenol production [3], appear instead to function in general detoxification, we propose a new hypothesis: rather than searching for a hydrolytic enzyme in fed male midguts, the key may lie in identifying an \u003cstrong\u003eester-forming gene\u003c/strong\u003e that is \u003cstrong\u003edownregulated during feeding\u003c/strong\u003e. This gene would normally convert \u003cem\u003ecis\u003c/em\u003e-verbenol into ester-bound forms, and its downregulation would reduce ester formation, allowing free \u003cem\u003ecis\u003c/em\u003e-verbenol to accumulate in guts of feeding males for pheromone use. Gene expression data point to Ityp-CE13 and later discussed Ityp-CE15, especially Ityp-CE16, as promising candidates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eItyp-CE13:\u003c/strong\u003e Carboxylesterase Ityp-CE13 clustered in clade B, closely with Dpon-AEE62728.1 from \u003cem\u003eDendroctonus ponderosae\u003c/em\u003e, a gene previously implicated in the biosynthesis of \u003cem\u003etrans\u003c/em\u003e-verbenol, likely through the formation or hydrolysis of verbenyl esters [6, 11]. The gene contains 11 exons and has a relatively short intronic region. In transcriptome comparisons, Ityp-CE13 showed an expression profile similar to Ityp-CE9 and Ityp-CE10, with the highest levels in the fat bodies of immature beetles of both sexes and detectable expression in the midguts of acetone-treated and JHIII-treated males. A notable exception was the strong downregulation observed in the midguts of feeding males. qPCR confirmed a similar expression pattern to that of the previously described genes Ityp-CE9 and Ityp-CE10: expression peaked in the fat bodies of immature beetles, declined after emergence, and rose again in fed beetles. Midgut expression followed the same general pattern, again with the exception of markedly low levels in fed male midguts. This sex-specific suppression of Ityp-CE13 after feeding in adult male gut is unexpected and may be linked to pheromone production, for example, by switching off further detoxification of \u003cem\u003ecis\u003c/em\u003e-verbenol to its esters to increase the availability of \u003cem\u003ecis\u003c/em\u003e-verbenol for pheromone communication in males. However, the underlying mechanism remains unclear.\u003c/p\u003e\n\u003cp\u003eA possible compelling role for \u003cstrong\u003eItyp-CE9\u003c/strong\u003e, \u003cstrong\u003eItyp-CE10\u003c/strong\u003e, and \u003cstrong\u003eItyp-CE13\u003c/strong\u003e may lie in the \u003cstrong\u003ebiosynthesis of verbenyl esters\u003c/strong\u003e during the \u003cstrong\u003eimmature stages\u003c/strong\u003e, when these compounds are the most abundant. Supporting this, all three carboxylesterases showed \u003cstrong\u003eelevated expression in the fat body\u003c/strong\u003e of both sexes at this developmental phase.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eItyp-CE15:\u003c/strong\u003e Even though carboxylesterase Ityp-CE15 has a similar length and the same number of exons (11) as Ityp-CE13, and clusters closely with it in clade B, also near Dpon-AEE62728, it exhibits distinct expression profiles compared to Ityp-CE13, both in DGE analysis and qPCR. Among the selected carboxylesterases analyzed here so far, Ityp-CE15 consistently exhibited lower overall expression in the compared transcriptomes. Pairwise DGE comparisons indicated that Ityp-CE15 is sex-neutral, strongly fat body\u0026ndash;specific, and developmentally enriched in immature beetles, with no detectable feeding or hormonal responsiveness. qPCR showed the highest expression in fat bodies, similar to that observed for Ityp-CE13, but notably, Ityp-CE15 displayed higher expression in the fat body of newly emerged beetles compared to immature individuals. This temporal pattern supports its candidacy as a hydrolytic esterase potentially involved in cleaving monoterpenyl FA esters during the developmental transition from the energy-acquiring, feeding immature stage to the non-feeding, emerged adult stage, when beetles must locate a new host tree and initiate mating. The male-biased expression further suggests a sex-specific metabolic role, potentially associated with the reduced FA ester content observed in adult females. These findings make the Ityp-CE15 a candidate for carboxylesterase catalysing the hydrolysis of verbenyl esters in newly emerged beetles, potentially to mobilise stored energy reserves, but also its downregulation in the feeding male gut makes it possible to be involved in \u003cem\u003ecis\u003c/em\u003e-verbenol enrichment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eItyp-CE16:\u003c/strong\u003e Another carboxyl esterase gene, Ityp-CE16, also clusters within clade B, positioned near Ityp-CE13, Ityp-CE15, and Dpond AEE62728. Its gene structure is relatively compact, with short intronic regions and only nine exons. Among all Ityp-CE/LIP genes analyzed in this study, Ityp-CE16 was the only one to exhibit significantly higher expression in midgut tissues of both sexes, in contrast to the fat-body or mixed-tissue enrichment observed for most other genes. The generally robust expression peaked in the midguts of immature and acetone-treated beetles of both sexes, as shown by pairwise comparisons, and was unaffected by JHIII treatment. RT-qPCR results confirmed that midgut expression in both immature and adult beetles was approximately an order of magnitude higher than in the corresponding fat bodies, with slightly higher levels in emerged males than in females. Although Ityp-CE16 is midgut-specific, its expression pattern does not correspond with the production curves of \u003cem\u003ecis\u003c/em\u003e-verbenol or verbenyl oleate in fed males or after JHIII treatment, making it an unlikely candidate for the male gut-specific gene with verbenyl esters hydrolytic function, but still may be involved in\u003cem\u003e\u0026nbsp;cis\u003c/em\u003e-verbenol enrichment as discussed above.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eItyp-CE20:\u003c/strong\u003e Ityp-CE20 clusters within the distant clade C, alongside carboxyl-like esterases from various insect orders. Its closest relative sequence is a putative carboxylesterase from \u003cem\u003eD. armandi\u003c/em\u003e Darm-AYN64426.1 [15], with nearby branches including a juvenile hormone esterase from \u003cem\u003eTribolium castaneum\u003c/em\u003e\u0026nbsp; Tcas-NM_001193294.1 [21]. On the sister branch was clustering venom carboxylesterases from \u003cem\u003eBombus ignitus\u003c/em\u003e Bign-MW699017.1 [20], esterase 6 from \u003cem\u003eD. melanogaster\u003c/em\u003e Dmel-ALV82133.1, and carboxylesterase from \u003cem\u003eEpiphyas postvittana\u003c/em\u003e Epos-JAI18199.1\u0026nbsp;[47, 48]. The gene contains moderately long intronic regions and consists of 10 exons. Transcriptome data revealed that Ityp-CE20 is most abundantly expressed in the midguts of both immature and adult beetles, with particularly high levels in the midguts of acetone-treated females, considered equivalent to emerged females. Elevated expression was also observed in the fat bodies of feeding beetles. RT-qPCR analysis confirmed that expression peaks in the fat bodies and midguts of emerging females, exceeding levels in males and other life stages. This sex- and stage-specific increase in expression among females may be linked to preparation for future reproduction, possibly by enhancing energy reserves, initiating female-specific metabolic pathways, or processes associated with juvenile hormone biosynthesis. From the perspective of verbenyl ester metabolism, its involvement appears unlikely, as neither verbenyl esters nor \u003cem\u003ecis\u003c/em\u003e-verbenol were detected in emerged females\u0026nbsp;[3].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eItyp-CE21:\u003c/strong\u003e Ityp-CE21 also clusters with carboxyl-like esterases in clade C and groups near Darm-AYN64423.1, a putative carboxylesterase from \u003cem\u003eD. armandi\u003c/em\u003e [15], and is closely related to a venom carboxylesterase from Apis mellifera [19]. The gene comprises 10 exons and features exceptionally long intronic regions, second only to Ityp-CE10 among the carboxylesterases analysed, which may indicate an older evolutionary origin [70, 71]. Although expression varied between sexes and life stages in immature and adult beetles, with a general bias toward males, notable upregulation was observed only during the pupal stage. This expression pattern suggests a possible role in developmental processes, potentially related to hormonal regulation or lipid metabolism during the metamorphosis from pupa to pre-adult, within endocrine-associated metabolic pathways, rather than in direct detoxification or pheromone biosynthesis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eItyp-Lip1:\u003c/strong\u003e The only selected lipase, Ityp-LIP1, was phylogenetically grouped within a monophyletic branch of reference lipases that is distinct from carboxylesterases. Lipase has a different gene structure and a different shape of binding site than studied carboxylesterases. It has 7 exons and long intronic regions, similar to Ityp-CE10 and Ityp-CE21. \u0026nbsp;It clustered most closely with lepidopteran lipases from \u003cem\u003eBombyx mori\u003c/em\u003e and \u003cem\u003eAntheraea pernyi\u003c/em\u003e [37, 38]. Expression in gut tissues and whole-body samples was generally low. Transcriptome data showed significant upregulation of Ityp-LIP1 in the immature beetles, but RT-qPCR demonstrated that Ityp-LIP1 expression is highest in the fat bodies of emerged beetles of both sexes. However, the stage- and sex-specific expression of Ityp-Lip1, though relatively modest, suggests it may play a specialized role, possibly related to the reduced fatty acid ester content observed in adult females, similar to the function proposed for Ityp-CE15.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue location of verbenyl FA esters forming/hydrolysis:\u003c/strong\u003e The precise localization of verbenyl fatty acid ester formation or hydrolysis remains an open question. While previous studies, e.g., [15], [16], [35], [72], focused on carboxylesterase roles in gut detoxification, our findings support a broader hypothesis: detoxification of monoterpenols and hydrolysis of verbenyl esters may also occur in the fat body, as all genes were expressed in both tissues. However, caution is warranted, since the anatomical proximity of gut and fat body may lead to tissue cross-contamination during dissection, possibly explaining low-level expression in gut samples [3].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFuture perspectives:\u003c/strong\u003e Despite extensive analyses, none of the eight candidate genes could be conclusively linked to a specific enzymatically catalyzed step in the metabolism of verbenyl esters, presumed precursors in an alternative biosynthetic pathway for pheromonal \u003cem\u003ecis\u003c/em\u003e-verbenol, leaving key mechanistic questions unresolved.\u003c/p\u003e\n\u003cp\u003eTo better understand this understudied and technically challenging enzyme group, central to bark beetle metabolism and pheromone biosynthesis, \u003cstrong\u003ecomplementary investigative approaches\u003c/strong\u003e are essential. Future work should prioritize \u003cstrong\u003efunctional characterization\u003c/strong\u003e of top candidate genes, starting with \u003cstrong\u003ecloning into bacterial expression vectors\u003c/strong\u003e and conducting \u003cstrong\u003eenzymatic activity assays\u003c/strong\u003e using verbenyl esters as substrates, with acylglycerols and wax esters as controls [73\u0026ndash;75]. These findings should be validated through \u003cstrong\u003eRNAi-mediated gene knockdowns\u003c/strong\u003e to assess in vivo function [76]. Together, these approaches will clarify the functional diversity and biological roles of ester-forming and -hydrolyzing enzymes, not only in \u003cem\u003eI. typographus\u003c/em\u003e but also across other insect species.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eAlthough ester-forming and ester-breaking enzymes are believed to play key roles in many metabolic processes, they are still poorly understood in insects. Comprehensive gene sequencing and functional studies for these enzymes are rare. In this study, we used both phylogenetic analysis and differential gene expression data to discover 27 previously uncharacterized genes in the bark \u003cem\u003ebeetle I. typographus\u003c/em\u003e that likely code for ester-forming or -hydrolyzing enzymes. These include 23 carboxylesterases, two (phospho)lipases, one notum-like gene, and one neurolactin-like gene.\u003c/p\u003e\n\u003cp\u003eFrom this group, eight genes were selected for more detailed expression analysis. These were seven carboxylesterases (Ityp-CE9, CE10, CE13, CE15, CE16, CE20, CE21) and one lipase (Ityp-Lip1). Their potential roles were investigated with a focus on identifying genes possibly involved in the metabolism of verbenyl FA esters—suspected intermediate compounds in the biosynthesis of the pheromone \u003cem\u003ecis\u003c/em\u003e-verbenol. To support this, we compared gene expression across different beetle life stages and forms (phenotypes) that vary in their verbenyl oleate and \u003cem\u003ecis\u003c/em\u003e-verbenol production.\u003c/p\u003e\n\u003cp\u003eWhile the functional involvement of these eight candidates in pheromone metabolism remains hypothetical, their expression patterns provide a valuable foundation for further study. Future functional assays and gene silencing experiments will be essential to validate their roles. Furthermore, given the central importance of aggregation pheromones in \u003cem\u003eI. typographus\u003c/em\u003e behaviour, elucidating the genetic underpinnings of pheromonal \u003cem\u003ecis\u003c/em\u003e-verbenol biosynthesis may support the development of targeted pest management strategies that modulate beetle populations rather than eradicate them, thereby helping to maintain ecological balance.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eMFMg\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eMale Fed Midgut\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eMIMg\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eMale Immature Midgut\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eFIMg\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eFemale Immature Midgut\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eL1WB\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eLarvae1 Whole Body\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eMJMg\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eMale JHIII Midgut\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eMAMg\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eMale Acetone Midgut\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eFJMg\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eFemale JHIII Midgut\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eFAMg\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eFemale Acetone Midgut\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eMIFb\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eMale Immature Fat Body\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eFIFb\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eFemale Immature Fat Body\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eMFFb\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eMale Fed Fat Body\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eFFFb\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eFemale Fed Fat Body.\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthics approval\u003c/h2\u003e\u003cp\u003eNo specific permits were required. All experiments were conducted on insects (\u003cem\u003eIps typographus\u003c/em\u003e) that are neither endangered nor protected species within the European Union. According to EU Directive 2010/63/EU on the protection of animals used for scientific purposes, research involving invertebrates such as insects does not require formal ethical approval.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003cp\u003eAll authors consented to their participation in this research.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eAll authors consent to the publication of this manuscript.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eJaroslav Str\u0026aacute;dal, Rajarajan Ramakrishnan and Anna Jirošov\u0026aacute; were funded by the funding agency Czech Science Foundation GACR 23\u0026ndash;07916 S, Czech Republic. Jaroslav Str\u0026aacute;dal was funded by the student Internal Grant Commission [IGA_A_32_24, JAROSLAV STRADAL] at the Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, Prague, Czech Republic. Ondřej Lukšan, computational resources were provided by the e-INFRA CZ project (ID:90254), supported by the Ministry of Education, Youth and Sports of the Czech Republic and High Performance Computing Service Group at IOCB, CAS.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJSt: Conceptualization, beetle rearing, collection, treatments, dissections, genomic analyses, method development, statistical analyses, manuscript drafting, and writing. RR: Treatments, metabolomic data analysis, method development, genomic analyses, manuscript drafting. OL: Genomic analyses, method development, bioinformatic data processing, manuscript drafting, and writing. MT: Conceptualization, genomic, transcriptomic and phylogenetic analyses, method development, manuscript drafting, and writing. JSy: Beetle rearing, collection, treatments, manuscript drafting. SM: Genomic analyses, method development, manuscript editing. IP: Conceptualization, manuscript editing and review, supervision. AJ: Conceptualization, study design, metabolomic data analysis, formal analysis, manuscript writing, editing, review, supervision.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Forest CZU enterprise for providing biological material.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files. Publicly available datasets used were sourced from NCBI Bioprojects under accession numbers PRJNA679450, PRJNA934749 and PRJNA1321731. Newly identified putative esterase sequences are available under accession numbers PX172087, PX172088, PX172089, PX172090, PX172091, PX172092, PX172093, PX172094, PX172095, PX172096, PX172097, PX172098, PX172099, PX172100, PX172101, PX172102, PX172103, PX172104, PX172105, PX172106, PX172107, PX172108, PX172109, PX172110, PX172111, PX172112, PX172113, PX172114 and the new *Ips typographus* transcriptome assembly as well as predicted protein structures are available at [https://doi.org/10.5281/zenodo.17063688](https:/doi.org/10.5281/zenodo.17063688) [77].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHl\u0026aacute;sny T, Zimov\u0026aacute; S, Merganičov\u0026aacute; K, Štěp\u0026aacute;nek P, Modlinger R, Turč\u0026aacute;ni M. Devastating outbreak of bark beetles in the Czech Republic: Drivers, impacts, and management implications. 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Zenodo; 2025. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5281/zenodo.17063688\u003c/span\u003e\u003cspan address=\"10.5281/zenodo.17063688\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"European spruce bark beetle, carboxylesterase, lipase, pheromone biosynthesis, transcriptomics, coleoptera, lipid metabolism, fatty acyl esters, detoxification","lastPublishedDoi":"10.21203/rs.3.rs-7583299/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7583299/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e\u003cp\u003eThe bark beetle \u003cem\u003eIps typographus\u003c/em\u003e (Coleoptera: Curculionidae: Scolytinae) is a major pest of spruce trees in Central Europe. Its ecological success is mediated by a male-produced aggregation pheromone, which includes the monoterpene \u003cem\u003ecis\u003c/em\u003e-verbenol. \u003cem\u003eCis\u003c/em\u003e-verbenol is biosynthesized from host-derived α-pinene, but can also be released through enzymatic cleavage of verbenyl-fatty acyl esters, which are initially produced by young beetles during maturation feeding and stored in their fat bodies. The main objective of this study was to identify the rarely studied ester-forming and hydrolyzing enzymes in \u003cem\u003eI. typographus\u003c/em\u003e, and to suggest their possible roles in beetle metabolism.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e\u003cp\u003eBy blasting reference gene set against a newly assembled \u003cem\u003eI. typographus\u003c/em\u003e transcriptome and performing phylogenetic analyses, we identified 27 novel ester-modifying genes: 23 carboxylesterases, two (phospho)lipases, one notum-like gene, and one neurolactin-like gene. Full gene structures were described. Based on GC-MS measured production profiles of verbenyl oleate and \u003cem\u003ecis\u003c/em\u003e-verbenol across beetle life stages and phenotypes, transcriptome pairs were selected for differential expression analysis. Eight genes were chosen for detailed RT-qPCR expression profiling across sexes, developmental stages, and tissues. Based on these findings, we propose possible roles of genes encoding enzymes in verbenyl-fatty acyl ester metabolism or broader lipid metabolic processes in bark beetles. However, functional validation through enzyme assays and gene silencing will be necessary to confirm their specific roles.\u003c/p\u003e\u003ch2\u003eConclusion:\u003c/h2\u003e\u003cp\u003eAlthough the functions of these candidate genes remain hypothetical, the identification and structural description of 27 new ester-modifying enzymes provide important insight into this poorly characterized enzyme group in insects. Furthermore, understanding the genetic basis of \u003cem\u003ecis\u003c/em\u003e-verbenol biosynthesis in \u003cem\u003eI. typographus\u003c/em\u003e may support the development of novel, pheromone-based pest management strategies.\u003c/p\u003e","manuscriptTitle":"Screening of Ester-Forming and Hydrolyzing Enzymes Linked to Pheromone Production in Ips typographus (Linnaeus, 1758)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-03 20:33:01","doi":"10.21203/rs.3.rs-7583299/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-09T09:15:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-04T11:39:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326352288701936286871985053177259866251","date":"2025-11-07T15:40:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-30T14:01:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"311470041458895146782923667867091216777","date":"2025-10-13T12:56:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-21T20:54:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-11T10:50:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-11T10:49:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2025-09-10T13:09:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"54ce9dce-dceb-4735-abd3-9f78c481c48d","owner":[],"postedDate":"October 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:30:15+00:00","versionOfRecord":{"articleIdentity":"rs-7583299","link":"https://doi.org/10.1186/s12864-026-12773-0","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2026-03-24 16:10:24","publishedOnDateReadable":"March 24th, 2026"},"versionCreatedAt":"2025-10-03 20:33:01","video":"","vorDoi":"10.1186/s12864-026-12773-0","vorDoiUrl":"https://doi.org/10.1186/s12864-026-12773-0","workflowStages":[]},"version":"v1","identity":"rs-7583299","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7583299","identity":"rs-7583299","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}