Chemosensory Transmembrane Protein Families in the Coffee White Stemborer, Xylotrechus quadripes (Coleoptera: Cerambycidae)

Chemosensory Transmembrane Protein Families in the Coffee White Stemborer, Xylotrechus quadripes... Abstract The coffee white stemborer, Xylotrechus quadripes Chevrolat (Coleoptera: Cerambycidae), feeds primarily on Coffea arabica L. (Gentianales: Rubiaceae) with its egg, larva, and pupa being developed within the trunk. The detection of chemosensory-related cues linked to adult mating, host seeking, and recognition is driven by three chemoreceptor gene repertoires of odorant (ORs), gustatory (GRs), and ionotropic (IRs) receptors as well as sensory neuron membrane proteins (SNMPs). Yet, information on these genes involved in chemoreception is unavailable in X. quadripes and relatively poor in the cerambycid beetles. Here, we presented the identification of four chemosensory transmembrane proteins from the antennal transcriptome of X. quadripes, including 33 ORs, five GRs, 18 IRs, and four SNMPs. Phylogenetic analysis classified the ORs into groups 1, 2, 3, 7, and olfactory coreceptor (Orco), showing three potential candidates (OR13, OR17, and OR21) for the sensing of male sex pheromones. The IRs were clustered into 10 orthologous groups, with additional copies for IR41a, IR64a, and IR75 clades. Four SNMPs were distributed in four independent clades, possibly representing a complete set in this species. Expression profiles revealed that all the genes were highly expressed in antennae, suggesting their olfactory roles. In addition, most of the genes showed the expression in nonantennal tissues including thoraxes, abdomens, wings, and legs, suggesting their involvement in nonchemosensory functions. Of notice, a highly conserved coreceptor IR25a displayed male-biased expression in the antennae, as the first presence in the cerambycid beetles. This study has established reference resources for understanding the mechanisms underlying the interactions between/within this beetle and its host plants. Xylotrechus quadripes, odorant receptor, gustatory receptor, ionotropic receptor, sensory neuron membrane protein The interspecific interactions of insects and plants, as a basis underlying insect adaptation and speciation to various environmental contexts, have constantly received much attention in recent years. In nature, insects encounter an array of volatile stimuli derived from hosts and nonhosts to complete host seeking, predator avoidance, and oviposition site localization (Zhang and Schlyter 2004, Bruce et al. 2005). In parallel, another critical life event for insect survival and reproduction is mating as a primary intraspecific interaction associated with the reception and recognition of sex pheromones (Edward and Chapman 2011). These chemosensory cues produced by plants or conspecific partners are of particular importance in the searching of suitable hosts or correct mates. The insects have evolved several sensory proteins to sense thousands of semiochemicals involved in smell and taste. These chemosensory-related proteins comprise three receptor gene repertoires of odorant (ORs) (Clyne et al. 1999, Hallem and Carlson 2006), gustatory (GRs) (Clyne et al. 2000, Dunipace et al. 2001, Ling et al. 2014), and ionotropic (IRs) (Yao et al. 2005, Benton et al. 2009) receptors as well as sensory neuron membrane proteins (SNMPs) (Vogt et al. 2009, Pregitzer et al. 2014). They are present in olfactory (OSNs) or gustatory (GSNs) sensory neurons residing in hair-like structures (namely sensilla) primarily distributed in antennae, the principle chemosensory organ of insects. Both ORs and GRs of insects are in general seven transmembrane receptor proteins (Hallem et al. 2006), but Helicoverpa armigera GRs were predicted to have three to nine transmembrane domains (Xu et al. 2016). Among ORs (and GRs), relatively low amino acid identities are observed within a same species (Clyne et al. 1999, 2000; Hallem et al. 2006). Functional studies on these two types of chemoreceptors have been extensively conducted, where the ORs are tuned mainly to plant odorants and sex pheromones (Anderson et al. 2009, de Fouchier et al. 2017), and the GRs detect sugar and bitter tastants as well as carbon dioxide (Chyb et al. 2003, Jones et al. 2007, Slone et al. 2007, Lee et al. 2009). In comparison to extensive studies of ORs in Diptera and Lepidoptera, coleopteran OR functions have been restricted to three species of Tribolium castaneum (Engsontia et al. 2008), Megacyllene caryae (Mitchell et al. 2012), and Dendroctonus armandi (Zhang et al. 2016b). Of these, knockdown of the olfactory co-receptors (Orco) TcasOrco and DarmOrco from T. castaneum and D. armandi significantly decreased the responses to one aggregation pheromone and 11 host odorants, respectively (Engsontia et al. 2008, Zhang et al. 2016b). In addition to those, three candidate odorant receptors that were sensitive to three sex pheromones [OR3: (S)-2-methyl-1-butanol; OR5: 2-phenylethanol; and OR20: (2S,3R)-2,3-hexanediol] were identified in M. caryae, as the first ligand-specific ORs in the beetles functionally characterized (Mitchell et al. 2012). However, none of GR functions in Coleoptera have been reported to date. The third chemoreceptor family of IR belonging to a member of ionotropic glutamate receptors (iGluRs), was discovered from Drosophila species (Benton et al. 2009). Subsequently, a large number of IR genes have been identified from Diptera and other insect orders with a functional emphasis in Drosophila melanogaster (Abuin et al. 2011, Enjin et al. 2016, Ni et al. 2016). Sequence characteristics, expression profiles, and evolutionary analyses distinguished the Drosophila IRs into two subfamilies: antennal IRs (A-IRs) and divergent IRs (D-IRs) (Croset et al. 2010). With a combinatorial pattern of at least one stimuli-specific IR and one coreceptor IR, IRs respond to various types of ligands including general odorants, sex pheromones, and tastants (Abuin et al. 2011, Silbering et al. 2011). The A-IRs are highly conserved across insect species, and mainly bear olfactory roles related to the sensing of acids, amines, and other odorants. By comparison, the D-IRs with species- or genera-specific expansions are expressed in taste tissues with involvement of taste behaviors (Rytz et al. 2013, Koh et al. 2014, Stewart et al. 2015). Differing from three superfamilies of chemoreceptors, insect SNMPs have a small set of gene numbers, generally presenting two orthologs with copy number variations of each ortholog (Nichols and Vogt 2008, Vogt et al. 2009). For example, T. castaneum has six SNMPs with four SNMP1 copies, one SNMP2, and one SNMP3 (Dippel et al. 2016); each three SNMPs (two SNMP1s and one SNMP2) in Dendroctonus ponderosae and Ips typographus (Andersson et al. 2013), and three SNMPs (one SNMP1 and two SNMP2s) in Tomicus yunnanensis (Liu et al. 2018). To date, little information on SNMP functions is available in insects. With a few exceptions, studies have demonstrated that D. melanogaster SNMP1 is required for the detection of the sex pheromone cis-vaccinyl acetate (Benton et al. 2007, Jin et al. 2008). Additionally, Heliothis virescens SNMP1 could increase the sensitivity of pheromone stimulation (Pregitzer et al. 2014). However, in coleopteran species, functional studies of SNMPs remain to be lacking. The coffee white stemborer, Xylotrechus quadripes Chevrolat (Coleoptera: Cerambycidae), is the foremost destructive pest of Coffea arabica L. (Gentianales: Rubiaceae) that is planted widely in Asian countries including China and India due to its economic value and importance (Rhainds et al. 2002, Venkatesha and Dinesh 2012). This beetle has a relatively narrow range of host plants with a preference for C. arabica, and other alternative hosts contain Moraceae, Rubiaceae, Oleaceae, Anacardiaceae, and Lamiaceae families (Santosh et al. 2011, Venkatesha and Dinesh 2012). Three types of sex pheromone components (2S-hydroxy-3-decanone, 3-hydroxy-2-decanone and 2S,3S-dihydroxyoctane) and four other compounds (2-hydroxy-3-octanone, 2-phenylethanol, octanoic acid and 2,3-decanedione) have been identified from male beetles. In addition, the effects of male sex pheromones were evaluated in laboratory and field (Rhainds et al. 2001, Hall et al. 2006). Although many attempts (cultural, mechanical, chemical, biological control, and even resistant coffee cultivar methods) have been developed to control this beetle, the molecular mechanisms underlying the interactions between/within this beetle and its hosts have still been largely unexplored and poorly understood. Successful host or mate searching and recognition require a highly sophisticated chemosensory system of X. quadripes. Yet, little is known about olfactory- or taste-related genes in this beetle. This greatly restricts our ability to understand chemosensory mechanisms underlying the interactions between/within this beetle and its hosts, and to develop novel implemented control strategies in integrated pest management based on plant odorants and sex pheromones. Here, we sequenced and assembled an antennal transcriptome of adult X. quadripes by next-generation sequencing. Further, we identified and characterized four chemosensory-associated transmembrane protein families using phylogenetic and extensive expression profile analyses. This study has greatly implemented the information on molecular aspects of the interactions between/within this beetle and its hosts, and provides novel molecular targets for pest control. Materials and Methods Insects and Tissue Collection The infested coffee stems were cut in Ruili city, Yunnan Province, China. The harvested stems were brought and maintained in nylon net cages (1 m long × 1 m wide × 0.5 m high) at room temperature until X. quadripes eclosion. The emerged adults were sexed based on the heads and the last abdominal segments, and separated in individual cages. For transcriptome sequencing, about 200 antennae of 1–5 d old male and female adults between the seventh and ninth hour of the photophase were collected and mixed with a sex ratio of 1:1. For the analysis of expression profiles, various tissues including antennae, heads without antennae, thoraxes, abdomens, legs and wings of both sexes were collected, respectively. Then, collected tissues were immediately immerged in liquid nitrogen and stored at −80°C. Total RNA Extraction and First-Strand cDNA Synthesis Total RNA extraction was conducted from different tissues using TRIzol Reagent (Ambion, Life Technologies, Carlsbad, CA) according to the manufacturer’s protocol. RNA quality and integrity were examined using NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, San Jose, CA). Genomic DNA was first removed from RNA samples (1 µg) with genomic DNA Eraser at 42°C for 2 min. Next, first-strand complementary DNA (cDNA) synthesis was performed by PrimeScript RT Reagent Kit (TaKaRa, Dalian, Liaoning, China), following the procedure: 37°C for 15 min and 85°C for 5 s. Synthesized cDNA templates were stored at −20°C. Gene Identification, Sequence, and Phylogenetic Analysis The adult antennal transcriptome of X. quadripes was downloaded from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under the accession number of SRP143591. For the identification of candidate OR, GR, IR, and SNMP genes of X. quadripes, chemosensory-related transmembrane proteins from other cerambycid beetles, together with the genes of T. castaneum and D. melanogaster, were collected as queries to screen the standalone antennal transcriptome of X. quadripes using TBLASTN, with an E-value cutoff of e−5 and 20 maximum hits of each query. Further, identified genes were verified using BLASTX against the NCBI nonredundant protein sequence (nr) database. Sequence alignment and transmembrane domain prediction were conducted with MAFFT v7.308 (alignment parameters: scoring matrix of BLOSUM62 and gap open penalty of 1.53) (Katoh and Standley 2013) and TMHMM Server v2.0 (Krogh et al. 2001), respectively. In the OR tree, X. quadripes ORs with <150 amino acids (AAs) were excluded, and two XquaGRs (GR1 and GR2) were used as an outgroup to root the tree. The ORs were used from three other cerambycid beetles of Anoplophora chinensis, Anoplophora glabripennis, and M. caryae as well as a coleopteran model organism T. castaneum. In the GR tree, the GRs were used from three other coleopteran species of A. chinensis, A. glabripennis, and T. castaneum as well as D. melanogaster. In T. castaneum, we only selected the GRs: CO2 receptors, fructose receptors, sugar receptors, and 20 other GRs (10 GRs from each clade) previously classified into two independent clades (Dippel et al. 2016). In D. melanogaster, three GR subfamilies (CO2, fructose, and sugar GRs) and four known bitter receptors (GR32a, GR33a, GR66a, and GR93a) were included. For the tree set of IRs, the IRs were selected from the species of Brontispa longissima, D. ponderosae, I. typographus, T. castaneum, and D. melanogaster. In addition to those, iGluRs of T. castaneum and D. melanogaster were added to the phylogenetic analysis of IRs. The tree of SNMPs was built with SNMPs of 18 coleopteran species and D. melanogaster. Approximately maximum-likelihood phylogenetic tree was constructed using FastTree version 2.1.5 with default settings (Price et al. 2010). The tree was viewed and edited by FigTree v1.4.3. The accession numbers of the proteins used in the trees were listed in Supplementary Table S1. Expression Profile of Candidate Chemosensory Genes The primers (Supplementary Table S2) of candidate chemosensory genes were designed by Beacon Designer 8.13 (PREMIER Biosoft International, Palo Alto, CA). The amplified product sizes were at least 360 bp, and thus the genes encoding <120 AAs were excluded from the analysis of expression profiles. Reverse transcription PCR was used to determine the expression of chemosensory genes in different tissues of both sexes. A total volume of 25 µl were established, containing 2.5 µl of 10 × PCR Buffer (Mg2+ plus), 0.2 mM of deoxyribonucleoside triphosphate (dNTP) Mixture (each 2.5 mM), 0.5 µM of each primer, and 0.15 µl of recombinant Thermus aquaticus (rTaq) DNA polymerase (5 U/µl) (TaKaRa, Dalian, Liaoning, China). PCR reaction was performed using the following procedure: an initial denaturation at 94°C for 3 min, 35 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 40 s, and final extension for 5 min at 72°C. PCR products were analyzed using 1.2% (w/v) agrose gel. Negative control was set using sterile water as the template. Ribosomal protein L10 (RPL10) gene was used as a reference gene to control the quality and quantity of cDNA templates. For most of chemosensory genes, two biological replicates were performed in some tissues. Results Candidate Odorant Receptors From the sequenced antennal transcriptome, 33 transcripts encoding ORs were identified including a highly conserved Orco across insects (Vosshall and Hansson 2011). These genes were named de-novo using the Arabic numerals from 1 to 32 outside Orco. Of these 33 ORs, 12 genes (OR1-3, OR5-12, and Orco) were predicted to have complete open reading frames (ORFs) varying from 345 to 480 AAs in length. The remaining ORs were fragments, of which OR29 showed the shortest sequence encoding 52 AAs and the longest cDNA sequence encoded 344 AAs for OR14. Among 12 full-length ORs, Orco shared extremely high identities to its orthologs from the longhorned beetles of A. chinensis (91%), A. glabripennis (91%), M. caryae (95%), and even noncerambycid species T. castaneum (85%). The remaining nine ORs exhibited moderate AA identities (46–79%) to the ORs from M. caryae with the exception that XquaOR9 and XquaOR11 shared 39% and 43% identities to AchiOR19 and AglaOR38, respectively. However, an extremely low identity (mean value = 14%) was observed among 12 full-length XquaORs (Table 1 and Supplementary Additional File 1). Our phylogenetic analysis, based on OR protein sequences of four cerambycid beetles and T. castaneum, revealed seven groups of coleopteran ORs in addition to Orco group. Five groups (1, 2, 3, 7, and Orco) comprised at least one OR from X. quadripes, with eight candidates for groups 2 or 7. In particular, group 7 was phylogenetically formed only by OR members of cerambycid beetles. On the other hand, three orthologs of McarOR3, OR5, and OR20 in M. caryae known functional ligands (Mitchell et al. 2012) were detected in X. quadripes, i.e., XquaOR13, OR21, and OR17. Notably, we found that six members of ORs (AglaOR3, AchiOR20, McarOR41PAR, McarOR44, McarOR50PAR, and TcasOR275FIX) had high sequence divergence and did not show clear phylogenetic relationships to each other or with other groups. It appears that they may be considered to be outgroup(s) of groups 4, 5, and 6 in T. cantaneum (Fig. 1A). Fig. 1. View largeDownload slide View largeDownload slide Candidate odorant receptor gene family in X. quadripes. (A) Maximum-likelihood tree of ORs from four cerambycid beetles and T. castaneum. Seven groups (1–7) are defined following the previous classification of ORs. Bootstrap values with <0.80 are shown in circles. XquaGR1 and GR2 are used as the outgroup to root the tree. Three ORs of M. caryae (OR3, OR5, and OR20) known functional ligands are indicated with their respective corresponding orthologs in X. quadripes. (B) Expression profile of 28 X. quadripes ORs. A reference gene, RPL10, is used to check the quality and quantity of cDNA templates. A, antenna; H, head without antennae; T, thorax; Ab, abdomen; L, leg, W, wing and NC, negative control using sterile water as the template. Fig. 1. View largeDownload slide View largeDownload slide Candidate odorant receptor gene family in X. quadripes. (A) Maximum-likelihood tree of ORs from four cerambycid beetles and T. castaneum. Seven groups (1–7) are defined following the previous classification of ORs. Bootstrap values with <0.80 are shown in circles. XquaGR1 and GR2 are used as the outgroup to root the tree. Three ORs of M. caryae (OR3, OR5, and OR20) known functional ligands are indicated with their respective corresponding orthologs in X. quadripes. (B) Expression profile of 28 X. quadripes ORs. A reference gene, RPL10, is used to check the quality and quantity of cDNA templates. A, antenna; H, head without antennae; T, thorax; Ab, abdomen; L, leg, W, wing and NC, negative control using sterile water as the template. To determine sex- and tissue-specific expression of identified ORs as well as reconstruct their transcripts, 28 ORs were selected. The expression of all OR genes was enriched in the antennae with no sexual difference. Some ORs showed broad expression in all tested tissues like OR1, OR6, OR16, OR20, OR24, and Orco. In addition to the antennae, several ORs were detected in another important chemosensory organ of legs at considerable levels, including OR1, OR6, OR11, OR12, OR16, and Orco. In nonchemosensory tissues, over half of ORs were detected, some of which showed obviously high expression in thoraxes (OR6, OR11, and OR16) and abdomens (OR13, OR16, OR18, and OR20) (Fig. 1B). Candidate Gustatory Receptors Five GR transcripts were found from the transcriptome, namely GR1-5. Of these, GR1 and GR2 were full-length sequences with 38% identity to each other, and three other GRs (GR3-5) were partial sequences, encoding 134, 79, and 113 AAs, respectively (Table 1 and Supplementary Additional File 1). The GR phylogenetic tree indicated that XquaGR5 was clustered into fructose receptors, and thus was likely to be a candidate for the detection of fructose in X. quadripes. The remaining four XquaGRs may be candidate bitter receptors. None of CO2 and sugar GRs was found from this transcriptome (Fig. 2A). Expression profile analysis indicated that XquaGR1, GR2, and GR3 were broadly expressed in all tested tissues of both sexes (Fig. 2B). Table 1. Blast results of candidate chemosensory membrane proteins in X. quadripes Gene  ORF (AA)  Full length  NCBI blast hit (references/name/species)  E value  Identity (%)  Odorant receptor (OR)   ORco  480  Yes  XP_018568191.1 odorant receptor coreceptor [Anoplophora glabripennis]  0.0  91   OR1  419  Yes  XP_023020469.1 odorant receptor 94b-like [Leptinotarsa decemlineata]  3e−147  50   OR2  357  Yes  XP_015832942.1 PREDICTED: odorant receptor Or1-like [Tribolium castaneum]  7e−74  35   OR3  345  Yes  XP_018578867.1 odorant receptor 49b-like [Anoplophora glabripennis]  1e−115  52   OR4  317  No  XP_018577142.1 odorant receptor 4-like [Anoplophora glabripennis]  2e−90  45   OR5  419  Yes  XP_018577142.1 odorant receptor 4-like [Anoplophora glabripennis]  3e−122  43   OR6  384  Yes  AUF73037.1 odorant receptor [Anoplophora chinensis]  2e−75  39   OR7  361  Yes  XP_023309849.1 odorant receptor 49b-like [Anoplophora glabripennis]  4e−59  36   OR8  422  Yes  ALR72579.1 odorant receptor OR36 [Colaphellus bowringi]  2e−113  44   OR9  404  Yes  AUF73022.1 odorant receptor [Anoplophora chinensis]  7e−49  30   OR10  388  Yes  AUF73043.1 odorant receptor [Anoplophora chinensis]  1e−51  27   OR11  387  Yes  XP_018567969.1 odorant receptor Or2-like [Anoplophora glabripennis]  3e−118  43   OR12  397  Yes  ALR72575.1 odorant receptor OR32 [Colaphellus bowringi]  8e−50  31   OR13  175  No  XP_018564808.1 odorant receptor Or1-like [Anoplophora glabripennis]  2e−74  66   OR14  344  No  XP_023026692.1 odorant receptor 49b-like [Leptinotarsa decemlineata]  2e−159  63   OR15  151  No  XP_018567067.1 odorant receptor Or2-like [Anoplophora glabripennis]  2e−14  31   OR16  176  No  AUF73030.1 odorant receptor [Anoplophora chinensis]  2e−54  48   OR17  230  No  ALR72568.1 odorant receptor OR24 [Colaphellus bowringi]  5e−82  50   OR18  151  No  AIX97138.1 olfactory receptor 3, partial [Rhyzopertha dominica]  2e−19  30   OR19  186  No  ALR72565.1 odorant receptor OR20 [Colaphellus bowringi]  9e−30  35   OR20  242  No  XP_023310030.1 odorant receptor Or1-like [Anoplophora glabripennis]  7e−116  72   OR21  322  No  ALR72579.1 odorant receptor OR36 [Colaphellus bowringi]  1e−77  43   OR22  283  No  XP_022917715.1 odorant receptor 49b-like [Onthophagus taurus]  9e−17  29   OR23  180  No  XP_023311541.1 odorant receptor 22b-like [Anoplophora glabripennis]  4e−16  32   OR24  242  No  APC94230.1 odorant receptor 18 [Pyrrhalta maculicollis]  8e−82  49   OR25  152  No  XP_023023560.1 odorant receptor 47b-like [Leptinotarsa decemlineata]  4e−25  37   OR26  174  No  AUF73044.1 odorant receptor [Anoplophora chinensis]  7e−44  40   OR27  241  No  XP_023019277.1 odorant receptor 4-like [Leptinotarsa decemlineata]  9e−31  30   OR28  102  No  XP_023313103.1 odorant receptor 43a-like isoform X2 [Anoplophora glabripennis]  6e−41  65   OR29  52  No  AUF73036.1 odorant receptor [Anoplophora chinensis]  1e−18  76   OR30  103  No  AUF73020.1 odorant receptor [Anoplophora chinensis]  3e−39  63   OR31  102  No  XP_014363329.1 PREDICTED: odorant receptor Or1-like [Papilio machaon]  4e−17  44   OR32  96  No  XP_018575789.2 putative odorant receptor 71a [Anoplophora glabripennis]  7e−27  51  Ionotropic glutamate receptor (iGluR) and ionotropic receptor (IR)   iGluR1  930  Yes  XP_018563905.1 glutamate receptor ionotropic, kainate 2-like [Anoplophora glabripennis]  0.0  63   iGluR2  223  No  XP_018569924.1 glutamate receptor ionotropic, kainate 2-like [Anoplophora glabripennis]  5e−102  66   iGluR3  645  No  XP_023312109.1 glutamate receptor ionotropic, kainate 2-like isoform X4 [Anoplophora glabripennis]  0.0  92   iGluR4  670  No  XP_018570638.1 glutamate receptor ionotropic, kainate 2-like isoform X1 [Anoplophora glabripennis]  0.0  59   iGluR5  561  No  XP_023024797.1 glutamate receptor ionotropic, kainate 2-like [Leptinotarsa decemlineata]  9e−136  40   iGluR6  758  No  XP_018563904.1 glutamate receptor ionotropic, kainate 2-like isoform X2 [Anoplophora glabripennis]  0.0  59   iGluR7  611  No  XP_018570638.1 glutamate receptor ionotropic, kainate 2-like isoform X1 [Anoplophora glabripennis]  0.0  71   iGluR8  248  No  XP_018570639.1 glutamate receptor ionotropic, kainate 1-like isoform X2 [Anoplophora glabripennis]  2e−103  65   IR1  170  No  AUF73085.1 ionotropic receptor [Anoplophora chinensis]  2e−42  48   IR2  200  No  AST36363.1 putative ionotropic receptor IR75q.1 [Cydia fagiglandana]  8e−15  30   IR8a  664  No  ALR72538.1 ionotropic receptor 8a [Colaphellus bowringi]  2e−159  78   IR21a  158  No  XP_023313061.1 ionotropic receptor 21a-like [Anoplophora glabripennis]  4e−75  72   IR25a  935  Yes  XP_018574744.1 ionotropic receptor 25a [Anoplophora glabripennis]  0.0  85   IR40a  65  No  XP_023310509.1 ionotropic receptor 40a [Anoplophora glabripennis]  5e−33  91   IR41a.1  635  Yes  AKC58587.1 chemosensory ionotropic receptor 41a [Anomala corpulenta]  2e−132  37   IR41a.2  232  No  AKC58587.1 chemosensory ionotropic receptor 41a [Anomala corpulenta]  2e−32  34   IR41a.3  240  No  NP_001345663.1 ionotropic receptor 41a [Aedes aegypti]  3e−39  37   IR64a.1  392  No  XP_023311151.1 glutamate receptor 2-like [Anoplophora glabripennis]  0.0  66   IR64a.2  90  No  XP_018571126.1 glutamate receptor 2-like [Anoplophora glabripennis]  4e−34  62   IR68a  193  No  XP_018568048.1 glutamate receptor ionotropic, kainate 5 [Anoplophora glabripennis]  2e−88  59   IR75q  623  Yes  APC94348.1 ionotropic receptor 5, partial [Pyrrhalta aenescens]  4e−125  36   IR75q.1  601  Yes  APC94348.1 ionotropic receptor 5, partial [Pyrrhalta aenescens]  5e−137  38   IR75q.2  427  No  ANQ46495.1 ionotropic receptor 3 [Phyllotreta striolata]  0.0  62   IR75s  601  Yes  XP_018562688.2 probable glutamate receptor [Anoplophora glabripennis]  0.0  70   IR76b  561  Yes  XP_018568700.1 glutamate receptor ionotropic, delta-1 [Anoplophora glabripennis]  0.0  65   IR93a  705  No  XP_018576792.1 ionotropic receptor 93a isoform X1 [Anoplophora glabripennis]  0.0  67  Gustatory receptor (GR)   GR1  339  Yes  XP_018565150.1 G-protein coupled receptor moody-like isoform X1 [Anoplophora glabripennis]  0.0  85   GR2  335  Yes  AKC58582.1 gustatory receptor 5 [Anomala corpulenta]  4e−157  68   GR3  134  No  ALR72527.1 gustatory receptor 1, partial [Colaphellus bowringi]  2e−29  44   GR4  79  No  XP_023310039.1 putative gustatory receptor 2a [Anoplophora glabripennis]  5e−19  51   GR5  113  No  AUF73059.1 gustatory receptor, partial [Anoplophora chinensis]  7e−33  57  Sensory neuron membrane protein (SNMP)   SNMP1a  521  Yes  ALR72542.1 sensory neuron membrane protein SNMP1a [Colaphellus bowringi]  0.0  65   SNMP1b  531  Yes  ALR72543.1 sensory neuron membrane protein SNMP1b [Colaphellus bowringi]  0.0  59   SNMP2a  520  Yes  XP_018566911.1 sensory neuron membrane protein 2 [Anoplophora glabripennis]  0.0  63   SNMP2b  505  Yes  XP_018566912.1 sensory neuron membrane protein 2-like [Anoplophora glabripennis]  0.0  67  Gene  ORF (AA)  Full length  NCBI blast hit (references/name/species)  E value  Identity (%)  Odorant receptor (OR)   ORco  480  Yes  XP_018568191.1 odorant receptor coreceptor [Anoplophora glabripennis]  0.0  91   OR1  419  Yes  XP_023020469.1 odorant receptor 94b-like [Leptinotarsa decemlineata]  3e−147  50   OR2  357  Yes  XP_015832942.1 PREDICTED: odorant receptor Or1-like [Tribolium castaneum]  7e−74  35   OR3  345  Yes  XP_018578867.1 odorant receptor 49b-like [Anoplophora glabripennis]  1e−115  52   OR4  317  No  XP_018577142.1 odorant receptor 4-like [Anoplophora glabripennis]  2e−90  45   OR5  419  Yes  XP_018577142.1 odorant receptor 4-like [Anoplophora glabripennis]  3e−122  43   OR6  384  Yes  AUF73037.1 odorant receptor [Anoplophora chinensis]  2e−75  39   OR7  361  Yes  XP_023309849.1 odorant receptor 49b-like [Anoplophora glabripennis]  4e−59  36   OR8  422  Yes  ALR72579.1 odorant receptor OR36 [Colaphellus bowringi]  2e−113  44   OR9  404  Yes  AUF73022.1 odorant receptor [Anoplophora chinensis]  7e−49  30   OR10  388  Yes  AUF73043.1 odorant receptor [Anoplophora chinensis]  1e−51  27   OR11  387  Yes  XP_018567969.1 odorant receptor Or2-like [Anoplophora glabripennis]  3e−118  43   OR12  397  Yes  ALR72575.1 odorant receptor OR32 [Colaphellus bowringi]  8e−50  31   OR13  175  No  XP_018564808.1 odorant receptor Or1-like [Anoplophora glabripennis]  2e−74  66   OR14  344  No  XP_023026692.1 odorant receptor 49b-like [Leptinotarsa decemlineata]  2e−159  63   OR15  151  No  XP_018567067.1 odorant receptor Or2-like [Anoplophora glabripennis]  2e−14  31   OR16  176  No  AUF73030.1 odorant receptor [Anoplophora chinensis]  2e−54  48   OR17  230  No  ALR72568.1 odorant receptor OR24 [Colaphellus bowringi]  5e−82  50   OR18  151  No  AIX97138.1 olfactory receptor 3, partial [Rhyzopertha dominica]  2e−19  30   OR19  186  No  ALR72565.1 odorant receptor OR20 [Colaphellus bowringi]  9e−30  35   OR20  242  No  XP_023310030.1 odorant receptor Or1-like [Anoplophora glabripennis]  7e−116  72   OR21  322  No  ALR72579.1 odorant receptor OR36 [Colaphellus bowringi]  1e−77  43   OR22  283  No  XP_022917715.1 odorant receptor 49b-like [Onthophagus taurus]  9e−17  29   OR23  180  No  XP_023311541.1 odorant receptor 22b-like [Anoplophora glabripennis]  4e−16  32   OR24  242  No  APC94230.1 odorant receptor 18 [Pyrrhalta maculicollis]  8e−82  49   OR25  152  No  XP_023023560.1 odorant receptor 47b-like [Leptinotarsa decemlineata]  4e−25  37   OR26  174  No  AUF73044.1 odorant receptor [Anoplophora chinensis]  7e−44  40   OR27  241  No  XP_023019277.1 odorant receptor 4-like [Leptinotarsa decemlineata]  9e−31  30   OR28  102  No  XP_023313103.1 odorant receptor 43a-like isoform X2 [Anoplophora glabripennis]  6e−41  65   OR29  52  No  AUF73036.1 odorant receptor [Anoplophora chinensis]  1e−18  76   OR30  103  No  AUF73020.1 odorant receptor [Anoplophora chinensis]  3e−39  63   OR31  102  No  XP_014363329.1 PREDICTED: odorant receptor Or1-like [Papilio machaon]  4e−17  44   OR32  96  No  XP_018575789.2 putative odorant receptor 71a [Anoplophora glabripennis]  7e−27  51  Ionotropic glutamate receptor (iGluR) and ionotropic receptor (IR)   iGluR1  930  Yes  XP_018563905.1 glutamate receptor ionotropic, kainate 2-like [Anoplophora glabripennis]  0.0  63   iGluR2  223  No  XP_018569924.1 glutamate receptor ionotropic, kainate 2-like [Anoplophora glabripennis]  5e−102  66   iGluR3  645  No  XP_023312109.1 glutamate receptor ionotropic, kainate 2-like isoform X4 [Anoplophora glabripennis]  0.0  92   iGluR4  670  No  XP_018570638.1 glutamate receptor ionotropic, kainate 2-like isoform X1 [Anoplophora glabripennis]  0.0  59   iGluR5  561  No  XP_023024797.1 glutamate receptor ionotropic, kainate 2-like [Leptinotarsa decemlineata]  9e−136  40   iGluR6  758  No  XP_018563904.1 glutamate receptor ionotropic, kainate 2-like isoform X2 [Anoplophora glabripennis]  0.0  59   iGluR7  611  No  XP_018570638.1 glutamate receptor ionotropic, kainate 2-like isoform X1 [Anoplophora glabripennis]  0.0  71   iGluR8  248  No  XP_018570639.1 glutamate receptor ionotropic, kainate 1-like isoform X2 [Anoplophora glabripennis]  2e−103  65   IR1  170  No  AUF73085.1 ionotropic receptor [Anoplophora chinensis]  2e−42  48   IR2  200  No  AST36363.1 putative ionotropic receptor IR75q.1 [Cydia fagiglandana]  8e−15  30   IR8a  664  No  ALR72538.1 ionotropic receptor 8a [Colaphellus bowringi]  2e−159  78   IR21a  158  No  XP_023313061.1 ionotropic receptor 21a-like [Anoplophora glabripennis]  4e−75  72   IR25a  935  Yes  XP_018574744.1 ionotropic receptor 25a [Anoplophora glabripennis]  0.0  85   IR40a  65  No  XP_023310509.1 ionotropic receptor 40a [Anoplophora glabripennis]  5e−33  91   IR41a.1  635  Yes  AKC58587.1 chemosensory ionotropic receptor 41a [Anomala corpulenta]  2e−132  37   IR41a.2  232  No  AKC58587.1 chemosensory ionotropic receptor 41a [Anomala corpulenta]  2e−32  34   IR41a.3  240  No  NP_001345663.1 ionotropic receptor 41a [Aedes aegypti]  3e−39  37   IR64a.1  392  No  XP_023311151.1 glutamate receptor 2-like [Anoplophora glabripennis]  0.0  66   IR64a.2  90  No  XP_018571126.1 glutamate receptor 2-like [Anoplophora glabripennis]  4e−34  62   IR68a  193  No  XP_018568048.1 glutamate receptor ionotropic, kainate 5 [Anoplophora glabripennis]  2e−88  59   IR75q  623  Yes  APC94348.1 ionotropic receptor 5, partial [Pyrrhalta aenescens]  4e−125  36   IR75q.1  601  Yes  APC94348.1 ionotropic receptor 5, partial [Pyrrhalta aenescens]  5e−137  38   IR75q.2  427  No  ANQ46495.1 ionotropic receptor 3 [Phyllotreta striolata]  0.0  62   IR75s  601  Yes  XP_018562688.2 probable glutamate receptor [Anoplophora glabripennis]  0.0  70   IR76b  561  Yes  XP_018568700.1 glutamate receptor ionotropic, delta-1 [Anoplophora glabripennis]  0.0  65   IR93a  705  No  XP_018576792.1 ionotropic receptor 93a isoform X1 [Anoplophora glabripennis]  0.0  67  Gustatory receptor (GR)   GR1  339  Yes  XP_018565150.1 G-protein coupled receptor moody-like isoform X1 [Anoplophora glabripennis]  0.0  85   GR2  335  Yes  AKC58582.1 gustatory receptor 5 [Anomala corpulenta]  4e−157  68   GR3  134  No  ALR72527.1 gustatory receptor 1, partial [Colaphellus bowringi]  2e−29  44   GR4  79  No  XP_023310039.1 putative gustatory receptor 2a [Anoplophora glabripennis]  5e−19  51   GR5  113  No  AUF73059.1 gustatory receptor, partial [Anoplophora chinensis]  7e−33  57  Sensory neuron membrane protein (SNMP)   SNMP1a  521  Yes  ALR72542.1 sensory neuron membrane protein SNMP1a [Colaphellus bowringi]  0.0  65   SNMP1b  531  Yes  ALR72543.1 sensory neuron membrane protein SNMP1b [Colaphellus bowringi]  0.0  59   SNMP2a  520  Yes  XP_018566911.1 sensory neuron membrane protein 2 [Anoplophora glabripennis]  0.0  63   SNMP2b  505  Yes  XP_018566912.1 sensory neuron membrane protein 2-like [Anoplophora glabripennis]  0.0  67  View Large Table 1. Blast results of candidate chemosensory membrane proteins in X. quadripes Gene  ORF (AA)  Full length  NCBI blast hit (references/name/species)  E value  Identity (%)  Odorant receptor (OR)   ORco  480  Yes  XP_018568191.1 odorant receptor coreceptor [Anoplophora glabripennis]  0.0  91   OR1  419  Yes  XP_023020469.1 odorant receptor 94b-like [Leptinotarsa decemlineata]  3e−147  50   OR2  357  Yes  XP_015832942.1 PREDICTED: odorant receptor Or1-like [Tribolium castaneum]  7e−74  35   OR3  345  Yes  XP_018578867.1 odorant receptor 49b-like [Anoplophora glabripennis]  1e−115  52   OR4  317  No  XP_018577142.1 odorant receptor 4-like [Anoplophora glabripennis]  2e−90  45   OR5  419  Yes  XP_018577142.1 odorant receptor 4-like [Anoplophora glabripennis]  3e−122  43   OR6  384  Yes  AUF73037.1 odorant receptor [Anoplophora chinensis]  2e−75  39   OR7  361  Yes  XP_023309849.1 odorant receptor 49b-like [Anoplophora glabripennis]  4e−59  36   OR8  422  Yes  ALR72579.1 odorant receptor OR36 [Colaphellus bowringi]  2e−113  44   OR9  404  Yes  AUF73022.1 odorant receptor [Anoplophora chinensis]  7e−49  30   OR10  388  Yes  AUF73043.1 odorant receptor [Anoplophora chinensis]  1e−51  27   OR11  387  Yes  XP_018567969.1 odorant receptor Or2-like [Anoplophora glabripennis]  3e−118  43   OR12  397  Yes  ALR72575.1 odorant receptor OR32 [Colaphellus bowringi]  8e−50  31   OR13  175  No  XP_018564808.1 odorant receptor Or1-like [Anoplophora glabripennis]  2e−74  66   OR14  344  No  XP_023026692.1 odorant receptor 49b-like [Leptinotarsa decemlineata]  2e−159  63   OR15  151  No  XP_018567067.1 odorant receptor Or2-like [Anoplophora glabripennis]  2e−14  31   OR16  176  No  AUF73030.1 odorant receptor [Anoplophora chinensis]  2e−54  48   OR17  230  No  ALR72568.1 odorant receptor OR24 [Colaphellus bowringi]  5e−82  50   OR18  151  No  AIX97138.1 olfactory receptor 3, partial [Rhyzopertha dominica]  2e−19  30   OR19  186  No  ALR72565.1 odorant receptor OR20 [Colaphellus bowringi]  9e−30  35   OR20  242  No  XP_023310030.1 odorant receptor Or1-like [Anoplophora glabripennis]  7e−116  72   OR21  322  No  ALR72579.1 odorant receptor OR36 [Colaphellus bowringi]  1e−77  43   OR22  283  No  XP_022917715.1 odorant receptor 49b-like [Onthophagus taurus]  9e−17  29   OR23  180  No  XP_023311541.1 odorant receptor 22b-like [Anoplophora glabripennis]  4e−16  32   OR24  242  No  APC94230.1 odorant receptor 18 [Pyrrhalta maculicollis]  8e−82  49   OR25  152  No  XP_023023560.1 odorant receptor 47b-like [Leptinotarsa decemlineata]  4e−25  37   OR26  174  No  AUF73044.1 odorant receptor [Anoplophora chinensis]  7e−44  40   OR27  241  No  XP_023019277.1 odorant receptor 4-like [Leptinotarsa decemlineata]  9e−31  30   OR28  102  No  XP_023313103.1 odorant receptor 43a-like isoform X2 [Anoplophora glabripennis]  6e−41  65   OR29  52  No  AUF73036.1 odorant receptor [Anoplophora chinensis]  1e−18  76   OR30  103  No  AUF73020.1 odorant receptor [Anoplophora chinensis]  3e−39  63   OR31  102  No  XP_014363329.1 PREDICTED: odorant receptor Or1-like [Papilio machaon]  4e−17  44   OR32  96  No  XP_018575789.2 putative odorant receptor 71a [Anoplophora glabripennis]  7e−27  51  Ionotropic glutamate receptor (iGluR) and ionotropic receptor (IR)   iGluR1  930  Yes  XP_018563905.1 glutamate receptor ionotropic, kainate 2-like [Anoplophora glabripennis]  0.0  63   iGluR2  223  No  XP_018569924.1 glutamate receptor ionotropic, kainate 2-like [Anoplophora glabripennis]  5e−102  66   iGluR3  645  No  XP_023312109.1 glutamate receptor ionotropic, kainate 2-like isoform X4 [Anoplophora glabripennis]  0.0  92   iGluR4  670  No  XP_018570638.1 glutamate receptor ionotropic, kainate 2-like isoform X1 [Anoplophora glabripennis]  0.0  59   iGluR5  561  No  XP_023024797.1 glutamate receptor ionotropic, kainate 2-like [Leptinotarsa decemlineata]  9e−136  40   iGluR6  758  No  XP_018563904.1 glutamate receptor ionotropic, kainate 2-like isoform X2 [Anoplophora glabripennis]  0.0  59   iGluR7  611  No  XP_018570638.1 glutamate receptor ionotropic, kainate 2-like isoform X1 [Anoplophora glabripennis]  0.0  71   iGluR8  248  No  XP_018570639.1 glutamate receptor ionotropic, kainate 1-like isoform X2 [Anoplophora glabripennis]  2e−103  65   IR1  170  No  AUF73085.1 ionotropic receptor [Anoplophora chinensis]  2e−42  48   IR2  200  No  AST36363.1 putative ionotropic receptor IR75q.1 [Cydia fagiglandana]  8e−15  30   IR8a  664  No  ALR72538.1 ionotropic receptor 8a [Colaphellus bowringi]  2e−159  78   IR21a  158  No  XP_023313061.1 ionotropic receptor 21a-like [Anoplophora glabripennis]  4e−75  72   IR25a  935  Yes  XP_018574744.1 ionotropic receptor 25a [Anoplophora glabripennis]  0.0  85   IR40a  65  No  XP_023310509.1 ionotropic receptor 40a [Anoplophora glabripennis]  5e−33  91   IR41a.1  635  Yes  AKC58587.1 chemosensory ionotropic receptor 41a [Anomala corpulenta]  2e−132  37   IR41a.2  232  No  AKC58587.1 chemosensory ionotropic receptor 41a [Anomala corpulenta]  2e−32  34   IR41a.3  240  No  NP_001345663.1 ionotropic receptor 41a [Aedes aegypti]  3e−39  37   IR64a.1  392  No  XP_023311151.1 glutamate receptor 2-like [Anoplophora glabripennis]  0.0  66   IR64a.2  90  No  XP_018571126.1 glutamate receptor 2-like [Anoplophora glabripennis]  4e−34  62   IR68a  193  No  XP_018568048.1 glutamate receptor ionotropic, kainate 5 [Anoplophora glabripennis]  2e−88  59   IR75q  623  Yes  APC94348.1 ionotropic receptor 5, partial [Pyrrhalta aenescens]  4e−125  36   IR75q.1  601  Yes  APC94348.1 ionotropic receptor 5, partial [Pyrrhalta aenescens]  5e−137  38   IR75q.2  427  No  ANQ46495.1 ionotropic receptor 3 [Phyllotreta striolata]  0.0  62   IR75s  601  Yes  XP_018562688.2 probable glutamate receptor [Anoplophora glabripennis]  0.0  70   IR76b  561  Yes  XP_018568700.1 glutamate receptor ionotropic, delta-1 [Anoplophora glabripennis]  0.0  65   IR93a  705  No  XP_018576792.1 ionotropic receptor 93a isoform X1 [Anoplophora glabripennis]  0.0  67  Gustatory receptor (GR)   GR1  339  Yes  XP_018565150.1 G-protein coupled receptor moody-like isoform X1 [Anoplophora glabripennis]  0.0  85   GR2  335  Yes  AKC58582.1 gustatory receptor 5 [Anomala corpulenta]  4e−157  68   GR3  134  No  ALR72527.1 gustatory receptor 1, partial [Colaphellus bowringi]  2e−29  44   GR4  79  No  XP_023310039.1 putative gustatory receptor 2a [Anoplophora glabripennis]  5e−19  51   GR5  113  No  AUF73059.1 gustatory receptor, partial [Anoplophora chinensis]  7e−33  57  Sensory neuron membrane protein (SNMP)   SNMP1a  521  Yes  ALR72542.1 sensory neuron membrane protein SNMP1a [Colaphellus bowringi]  0.0  65   SNMP1b  531  Yes  ALR72543.1 sensory neuron membrane protein SNMP1b [Colaphellus bowringi]  0.0  59   SNMP2a  520  Yes  XP_018566911.1 sensory neuron membrane protein 2 [Anoplophora glabripennis]  0.0  63   SNMP2b  505  Yes  XP_018566912.1 sensory neuron membrane protein 2-like [Anoplophora glabripennis]  0.0  67  Gene  ORF (AA)  Full length  NCBI blast hit (references/name/species)  E value  Identity (%)  Odorant receptor (OR)   ORco  480  Yes  XP_018568191.1 odorant receptor coreceptor [Anoplophora glabripennis]  0.0  91   OR1  419  Yes  XP_023020469.1 odorant receptor 94b-like [Leptinotarsa decemlineata]  3e−147  50   OR2  357  Yes  XP_015832942.1 PREDICTED: odorant receptor Or1-like [Tribolium castaneum]  7e−74  35   OR3  345  Yes  XP_018578867.1 odorant receptor 49b-like [Anoplophora glabripennis]  1e−115  52   OR4  317  No  XP_018577142.1 odorant receptor 4-like [Anoplophora glabripennis]  2e−90  45   OR5  419  Yes  XP_018577142.1 odorant receptor 4-like [Anoplophora glabripennis]  3e−122  43   OR6  384  Yes  AUF73037.1 odorant receptor [Anoplophora chinensis]  2e−75  39   OR7  361  Yes  XP_023309849.1 odorant receptor 49b-like [Anoplophora glabripennis]  4e−59  36   OR8  422  Yes  ALR72579.1 odorant receptor OR36 [Colaphellus bowringi]  2e−113  44   OR9  404  Yes  AUF73022.1 odorant receptor [Anoplophora chinensis]  7e−49  30   OR10  388  Yes  AUF73043.1 odorant receptor [Anoplophora chinensis]  1e−51  27   OR11  387  Yes  XP_018567969.1 odorant receptor Or2-like [Anoplophora glabripennis]  3e−118  43   OR12  397  Yes  ALR72575.1 odorant receptor OR32 [Colaphellus bowringi]  8e−50  31   OR13  175  No  XP_018564808.1 odorant receptor Or1-like [Anoplophora glabripennis]  2e−74  66   OR14  344  No  XP_023026692.1 odorant receptor 49b-like [Leptinotarsa decemlineata]  2e−159  63   OR15  151  No  XP_018567067.1 odorant receptor Or2-like [Anoplophora glabripennis]  2e−14  31   OR16  176  No  AUF73030.1 odorant receptor [Anoplophora chinensis]  2e−54  48   OR17  230  No  ALR72568.1 odorant receptor OR24 [Colaphellus bowringi]  5e−82  50   OR18  151  No  AIX97138.1 olfactory receptor 3, partial [Rhyzopertha dominica]  2e−19  30   OR19  186  No  ALR72565.1 odorant receptor OR20 [Colaphellus bowringi]  9e−30  35   OR20  242  No  XP_023310030.1 odorant receptor Or1-like [Anoplophora glabripennis]  7e−116  72   OR21  322  No  ALR72579.1 odorant receptor OR36 [Colaphellus bowringi]  1e−77  43   OR22  283  No  XP_022917715.1 odorant receptor 49b-like [Onthophagus taurus]  9e−17  29   OR23  180  No  XP_023311541.1 odorant receptor 22b-like [Anoplophora glabripennis]  4e−16  32   OR24  242  No  APC94230.1 odorant receptor 18 [Pyrrhalta maculicollis]  8e−82  49   OR25  152  No  XP_023023560.1 odorant receptor 47b-like [Leptinotarsa decemlineata]  4e−25  37   OR26  174  No  AUF73044.1 odorant receptor [Anoplophora chinensis]  7e−44  40   OR27  241  No  XP_023019277.1 odorant receptor 4-like [Leptinotarsa decemlineata]  9e−31  30   OR28  102  No  XP_023313103.1 odorant receptor 43a-like isoform X2 [Anoplophora glabripennis]  6e−41  65   OR29  52  No  AUF73036.1 odorant receptor [Anoplophora chinensis]  1e−18  76   OR30  103  No  AUF73020.1 odorant receptor [Anoplophora chinensis]  3e−39  63   OR31  102  No  XP_014363329.1 PREDICTED: odorant receptor Or1-like [Papilio machaon]  4e−17  44   OR32  96  No  XP_018575789.2 putative odorant receptor 71a [Anoplophora glabripennis]  7e−27  51  Ionotropic glutamate receptor (iGluR) and ionotropic receptor (IR)   iGluR1  930  Yes  XP_018563905.1 glutamate receptor ionotropic, kainate 2-like [Anoplophora glabripennis]  0.0  63   iGluR2  223  No  XP_018569924.1 glutamate receptor ionotropic, kainate 2-like [Anoplophora glabripennis]  5e−102  66   iGluR3  645  No  XP_023312109.1 glutamate receptor ionotropic, kainate 2-like isoform X4 [Anoplophora glabripennis]  0.0  92   iGluR4  670  No  XP_018570638.1 glutamate receptor ionotropic, kainate 2-like isoform X1 [Anoplophora glabripennis]  0.0  59   iGluR5  561  No  XP_023024797.1 glutamate receptor ionotropic, kainate 2-like [Leptinotarsa decemlineata]  9e−136  40   iGluR6  758  No  XP_018563904.1 glutamate receptor ionotropic, kainate 2-like isoform X2 [Anoplophora glabripennis]  0.0  59   iGluR7  611  No  XP_018570638.1 glutamate receptor ionotropic, kainate 2-like isoform X1 [Anoplophora glabripennis]  0.0  71   iGluR8  248  No  XP_018570639.1 glutamate receptor ionotropic, kainate 1-like isoform X2 [Anoplophora glabripennis]  2e−103  65   IR1  170  No  AUF73085.1 ionotropic receptor [Anoplophora chinensis]  2e−42  48   IR2  200  No  AST36363.1 putative ionotropic receptor IR75q.1 [Cydia fagiglandana]  8e−15  30   IR8a  664  No  ALR72538.1 ionotropic receptor 8a [Colaphellus bowringi]  2e−159  78   IR21a  158  No  XP_023313061.1 ionotropic receptor 21a-like [Anoplophora glabripennis]  4e−75  72   IR25a  935  Yes  XP_018574744.1 ionotropic receptor 25a [Anoplophora glabripennis]  0.0  85   IR40a  65  No  XP_023310509.1 ionotropic receptor 40a [Anoplophora glabripennis]  5e−33  91   IR41a.1  635  Yes  AKC58587.1 chemosensory ionotropic receptor 41a [Anomala corpulenta]  2e−132  37   IR41a.2  232  No  AKC58587.1 chemosensory ionotropic receptor 41a [Anomala corpulenta]  2e−32  34   IR41a.3  240  No  NP_001345663.1 ionotropic receptor 41a [Aedes aegypti]  3e−39  37   IR64a.1  392  No  XP_023311151.1 glutamate receptor 2-like [Anoplophora glabripennis]  0.0  66   IR64a.2  90  No  XP_018571126.1 glutamate receptor 2-like [Anoplophora glabripennis]  4e−34  62   IR68a  193  No  XP_018568048.1 glutamate receptor ionotropic, kainate 5 [Anoplophora glabripennis]  2e−88  59   IR75q  623  Yes  APC94348.1 ionotropic receptor 5, partial [Pyrrhalta aenescens]  4e−125  36   IR75q.1  601  Yes  APC94348.1 ionotropic receptor 5, partial [Pyrrhalta aenescens]  5e−137  38   IR75q.2  427  No  ANQ46495.1 ionotropic receptor 3 [Phyllotreta striolata]  0.0  62   IR75s  601  Yes  XP_018562688.2 probable glutamate receptor [Anoplophora glabripennis]  0.0  70   IR76b  561  Yes  XP_018568700.1 glutamate receptor ionotropic, delta-1 [Anoplophora glabripennis]  0.0  65   IR93a  705  No  XP_018576792.1 ionotropic receptor 93a isoform X1 [Anoplophora glabripennis]  0.0  67  Gustatory receptor (GR)   GR1  339  Yes  XP_018565150.1 G-protein coupled receptor moody-like isoform X1 [Anoplophora glabripennis]  0.0  85   GR2  335  Yes  AKC58582.1 gustatory receptor 5 [Anomala corpulenta]  4e−157  68   GR3  134  No  ALR72527.1 gustatory receptor 1, partial [Colaphellus bowringi]  2e−29  44   GR4  79  No  XP_023310039.1 putative gustatory receptor 2a [Anoplophora glabripennis]  5e−19  51   GR5  113  No  AUF73059.1 gustatory receptor, partial [Anoplophora chinensis]  7e−33  57  Sensory neuron membrane protein (SNMP)   SNMP1a  521  Yes  ALR72542.1 sensory neuron membrane protein SNMP1a [Colaphellus bowringi]  0.0  65   SNMP1b  531  Yes  ALR72543.1 sensory neuron membrane protein SNMP1b [Colaphellus bowringi]  0.0  59   SNMP2a  520  Yes  XP_018566911.1 sensory neuron membrane protein 2 [Anoplophora glabripennis]  0.0  63   SNMP2b  505  Yes  XP_018566912.1 sensory neuron membrane protein 2-like [Anoplophora glabripennis]  0.0  67  View Large Fig. 2. View largeDownload slide Candidate gustatory receptor gene family in X. quadripes. (A) Maximum-likelihood tree of GRs from Coleoptera and D. melanogaster. CO2, fructose, and sugar receptors are highlighted. Bootstrap values with <0.80 are shown in circles. Achi, A. chinensis; Agla, A. glabripennis; Dmel, D. melanogaster and Tcas, T. castaneum. (B) Expression profile of three X. quadripes GRs. Information on the quality and quantity of the reference gene and tissue abbreviations is shown in Fig. 1. Fig. 2. View largeDownload slide Candidate gustatory receptor gene family in X. quadripes. (A) Maximum-likelihood tree of GRs from Coleoptera and D. melanogaster. CO2, fructose, and sugar receptors are highlighted. Bootstrap values with <0.80 are shown in circles. Achi, A. chinensis; Agla, A. glabripennis; Dmel, D. melanogaster and Tcas, T. castaneum. (B) Expression profile of three X. quadripes GRs. Information on the quality and quantity of the reference gene and tissue abbreviations is shown in Fig. 1. Candidate Ionotropic Receptors We identified a total of 18 IRs and eight iGluRs from the transcriptome. The nomenclature of IRs were followed the conventions of D. melanogaster and T. castaneum based on the phylogeny and orthology. Two candidate IRs were likely to be members of the D-IRs subfamily and were named as IR1 and IR2. iGluRs were named with the Arabic numerals from 1 to 8. Six IRs (IR25a, IR41a.1, IR75q, IR75q.1, IR75s, and IR76b) and one iGluR (iGluR1) exhibited full-length ORFs. Of them, IR25a shared the longest sequence encoding 935 AAs, while IR76b showed the shortest sequence encoding 561 AAs. Sequence alignment analysis indicated that both XquaIR75q and XquaIR75q.1 exhibited a high identity (58%). A conserved coreceptor XquaIR25a had the highest identity (85%) to AglaIR25a (Table 1 and Supplementary Additional File 1). Phylogenetic analysis clustered the XquaIRs into 10 orthologous groups, consisting of IR8a, IR25a, IR21a, IR40a, IR41a, IR64a, IR68a, IR75, IR76b, and IR93a. Two other IRs of IR1 and IR2 were grouped into the D-IRs subfamily of T. castaneum and D. melanogaster. As expected, two co-receptors of IR8a and IR25a were close to the iGluR family in phylogeny. Three A-IRs lineage-expansions in X. quadripes were observed with two copies in IR64a, three in IR41a, and four in IR75 (Fig. 3A). Fig. 3. View largeDownload slide View largeDownload slide Candidate ionotropic receptor gene family in X. quadripes. (A) Maximum-likelihood tree of IRs and iGluRs from Coleoptera and D. melanogaster. Based on the phylogeny and sequence orthology of IRs, various groups are divided with highlighted clades of co-receptors and lineage-expansions. Newly identified XquaiGluRs and IRs are labeled with red. Bootstrap values with <0.80 are shown in circles. Other iGluR subfamilies are used as the outgroup to root the tree. Blon, B. longissima; Dpon, D. ponderosae; Ityp, I. typographus; Tcas, T. castaneum; Xqua, X. quadripes and Dmel, D. melanogaster. (B) Expression profile of eight X. quadripes iGluRs. (C) Expression profile of 15 X. quadripes IRs. Information on the quality and quantity of the reference gene and tissue abbreviations is shown in Fig. 1. Fig. 3. View largeDownload slide View largeDownload slide Candidate ionotropic receptor gene family in X. quadripes. (A) Maximum-likelihood tree of IRs and iGluRs from Coleoptera and D. melanogaster. Based on the phylogeny and sequence orthology of IRs, various groups are divided with highlighted clades of co-receptors and lineage-expansions. Newly identified XquaiGluRs and IRs are labeled with red. Bootstrap values with <0.80 are shown in circles. Other iGluR subfamilies are used as the outgroup to root the tree. Blon, B. longissima; Dpon, D. ponderosae; Ityp, I. typographus; Tcas, T. castaneum; Xqua, X. quadripes and Dmel, D. melanogaster. (B) Expression profile of eight X. quadripes iGluRs. (C) Expression profile of 15 X. quadripes IRs. Information on the quality and quantity of the reference gene and tissue abbreviations is shown in Fig. 1. In the expression analysis of iGluRs, five genes (iGluR4-8) were present in all tissues and the remaining three iGluRs (iGluR1-3) were detected in at least four tissue types of male and female adults (Fig. 3B). Similarly, most of IR genes including three candidate co-receptors IR8a, IR25a and IR76b were also expressed widely in all tested tissues with antennal-predominant levels. Intriguingly, some genes displayed sex-differential expression. For example, both IR1 and IR2 had female-biased expression, while IR21a and IR25a expression was more abundant in male antennae relative to that of females. Except three co-receptors, some IRs (e.g., three copies of IR41a, IR75s, and IR93a) were also present in nonchemosensory tissues such as heads without antennae, thoraxes, or abdomens (Fig. 3C). Candidate SNMPs Based on the transcriptome, we found four SNMPs, namely SNMP1a, SNMP1b, SNMP2a, and SNMP2b, based on the phylogeny and NCBI Blast results. These genes encoded similar ORF lengths varying from 505 to 531 AAs. They were full-length cDNAs and shared moderate protein identities to SNMPs of other beetles (65% and 59% identities between XquaSNMP1a/CbowSNMP1a and XquaSNMP1b/CbowSNMP1b, respectively; 63% and 67% between XquaSNMP2a/AglaSNMP2 and XquaSNMP2b/AglaSNMP2-like). However, the identities among four XquaSNMPs were relatively low, varying from 22% to 40% (mean value = 29%) (Table 1 and Supplementary Additional File 1). Based on primarily the protein sequences of coleopteran SNMPs, we constructed the maximum-likelihood tree. Four XquaSNMPs were classified into two large groups of SNMP1 and SNMP2, which were further divided into four relatively independent clades of SNMP1-I, SNMP1-II, SNMP2-I, and SNMP2-II (Fig. 4A). Expression profile analysis revealed that SNMP1a was broadly expressed in tested tissues; SNMP1b expression was significantly enriched in antennae; SNMP2a also showed a relatively broad distribution with an obvious predominance in female wings and SNMP2b transcripts were detected in all tested tissues except heads without antennae (Fig. 4B). Fig. 4. View largeDownload slide Candidate sensory neuron membrane protein gene family in X. quadripes. (A) Maximum-likelihood tree of SNMPs from Coleoptera and D. melanogaster. Newly identified XquaSNMPs are labeled with red. Bootstrap values with <0.80 are shown in circles. In Coleoptera, four SNMP groups are distinguished according to the phylogenetic analysis. In the CD36 group, EMP-B, Epithelial Membrane Protein, isoform B; Crq-A, Croquemort isoform A and Pes, Peste. Achi, A. chinensis; Agla, A. glabripennis; Acor, Anomala corpulenta; Aqua, Ambrostoma quadriimpressum; Atum, Aethina tumida; Blon, Brontispa longissima; Cbow, Colaphellus bowringi; Dhel, Dastarcus helophoroides; Dmel, D. melanogaster; Dpon, D. ponderosae; Dval, D. valens; Ityp, I. typographus; Malt, Monochamus alternatus; Paen, Pyrrhalta aenescens; Pmac, Pyrrhalta maculicollis; Pstr, Phyllotreta striolata; Tcas, T. castaneum; Tmol, Tenebrio molitor, Tyun, T. yunnanensis and Xqua, X. quadripes. (B) Expression profile of four X. quadripes SNMPs. Information on the quality and quantity of the reference gene and tissue abbreviations are shown in Fig. 1. Fig. 4. View largeDownload slide Candidate sensory neuron membrane protein gene family in X. quadripes. (A) Maximum-likelihood tree of SNMPs from Coleoptera and D. melanogaster. Newly identified XquaSNMPs are labeled with red. Bootstrap values with <0.80 are shown in circles. In Coleoptera, four SNMP groups are distinguished according to the phylogenetic analysis. In the CD36 group, EMP-B, Epithelial Membrane Protein, isoform B; Crq-A, Croquemort isoform A and Pes, Peste. Achi, A. chinensis; Agla, A. glabripennis; Acor, Anomala corpulenta; Aqua, Ambrostoma quadriimpressum; Atum, Aethina tumida; Blon, Brontispa longissima; Cbow, Colaphellus bowringi; Dhel, Dastarcus helophoroides; Dmel, D. melanogaster; Dpon, D. ponderosae; Dval, D. valens; Ityp, I. typographus; Malt, Monochamus alternatus; Paen, Pyrrhalta aenescens; Pmac, Pyrrhalta maculicollis; Pstr, Phyllotreta striolata; Tcas, T. castaneum; Tmol, Tenebrio molitor, Tyun, T. yunnanensis and Xqua, X. quadripes. (B) Expression profile of four X. quadripes SNMPs. Information on the quality and quantity of the reference gene and tissue abbreviations are shown in Fig. 1. Discussion Most of insects, including the coffee white stemborer, use external environmental chemical cues produced by plants and animals to locate and recognize hosts and partners. Our current work has identified and characterized four crucial chemosensory transmembrane protein families of ORs, GRs, IRs, and SNMPs, serving as the perception and recognition of chemical cues. As preliminary work of down-stream functional performance, this study on gene identification, phylogeny and expression profile is of particular significance for further functional studies and will allow for target experiments to identify potential molecular targets for the control of this pest. ORs primarily detect plant odorants and sex pheromones. They have been widely functioned in the orders of Diptera and Lepidoptera (Hallem and Carlson 2006, Zhang et al. 2014, de Fouchier et al. 2017), but very little is known in the cerambycid beetles. Our study identified a total of 33 OR candidates including a conserved Orco across insects (Vosshall and Hansson 2011). In comparison to three other cerambycid beetles of M. caryae (57 ORs) (Mitchell et al. 2012), A. glabripennis (131 ORs) (McKenna et al. 2016) and A. chinensis (44 ORs) (Wang et al. 2017), this coffee white stemborer has a relatively small sets of ORs, but more than nine in Monochamus alternatus (Wang et al. 2014). Meanwhile, this number is less than 341 in T. castaneum (Engsontia et al. 2008), 43 in I. typographus and 49 in D. ponderosae (Andersson et al. 2013), nearly equal to 34 in Ambrostoma quadriimpressum (Wang et al. 2016), and more than 22 in D. valens (Gu et al. 2015), 26 in Pyrrhalta aenescens, and 22 in Pyrrhalta maculicollis (Zhang et al. 2016a). The difference in gene numbers indicates that some IRs remain to be identified in X. quadripes, largely supported by results of our phylogenetic analysis as well as the fact that the identification is based solely on the transcriptome analysis rather than genomic data. For example, we did not detect the members of groups 4 and 5 in this species, but A. glabripennis ORs were present in the two groups (McKenna et al. 2016). To date, limited functional information on coleopteran ORs is available, including two conserved co-receptors Orco from D. armandi (Zhang et al. 2016b) and T. castaneum (Engsontia et al. 2008). As stimulus-specific ORs in the beetles, only three candidate pheromone receptors in M. caryae (McarOR3, OR5, and OR20) have been functionally characterized. They are respectively sensitive to (S)-2-methyl-1-butanol, 2-phenylethanol and (2S,3R)-2,3-hexanediol, sex pheromones of male beetles (Mitchell et al. 2012). Our study shows that XquaOR13, OR21, and OR17 are sisters to McarOR3, OR5, and OR20, respectively. Notably, 2-phenylethanol was also detected in male X. quadripes, and hence may be one of odorant candidates for XquaOR21. Additionally, some ketone analogs like 2S-hydroxy-3-decanone, 3-hydroxy-2-decanone and 2-hydroxy-3-octanone have been found from male X. quadripes (Hall et al. 2006). Coupled with antennal-enriched expression of three XquaORs, we suggest that they may respond to male-released compounds including sex pheromones. Expression profiling analysis reveals that most of ORs are specifically or highly expressed in antennae of both sexes, supporting their presence in the antennae and olfactory roles. Similar results have been reported in several other coleopteran species like Colaphellus bowringi (Li et al. 2015b), Anomala corpulenta (Li et al. 2015a), Holotrichia oblita (Li et al. 2017), and Phyllotreta striolata (Wu et al. 2016). In other tissues, OR expression indicates their functional diversities associated with gustatory and nonchemosensory functions (Kang and Koo 2012). Based on the analyses of sequence alignments and phylogenetic tree, 16 conserved XquaIRs show orthology to IRs of other coleopteran species and D. melanogaster, and thus are classified into the A-IRs subfamily (Croset et al. 2010). Notably, three orthologous groups of IR41a, IR64a, and IR75 generally possess multiple copies in X. quadripes, other Coleoptera and Diptera (Rytz et al. 2013). T. castaneum has two copies of IR41a, three of IR64a, and three of IR75 (Croset et al. 2010). One of IR41a and eight of IR75 are identified in A. glabripennis, but no IR64a ortholog is found (McKenna et al. 2016). One, one, and four copies are present in IR41a, IR64a, and IR75 of D. melanogaster, respectively (Benton et al. 2009). Fifteen, one, and eleven of IR41a, IR64a, and IR75 are detected in A. aegypti, respectively (Croset et al. 2010, Rytz et al. 2013). Of the 18 XquaIRs identified, two IRs (IR1 and IR2) show no clear orthology to any receptors in coleopteran species or D. melanogaster, possibly as candidates for the species-specific D-IRs subfamily. In the analysis of expression profile, three broadly expressed co-receptors (IR8a, IR25a, and IR76b) provide evidence that at least a coreceptor IR is required for stimulus-specific IR functions (Abuin et al. 2011, Silbering et al. 2011). Of notice, XquaIR25a expression appears to be male-biased in antennae, consistent with EaffIR25a expression in the copepod Eurytemora affinis (Eyun et al. 2017) but in contrast to female-biased expression of H. oblita HoblIR25a (Li et al. 2017). The two other co-receptors (IR8a and IR76b) in X. quadripes display no differential expression between the sexes, similar to Drosophila species (Shiao et al. 2015), T. castaneum (Dippel et al. 2016), and H. melpomene (van Schooten et al. 2016). The remaining 12 IRs exhibit additional expression except the antennae, indicating their functional diversities in this beetle associated with olfactory, gustatory, and nonchemosensory functions. In comparison to other coleopteran species (220 GRs in T. castaneum and 234 in A. glabripennis) (Tribolium Genome Sequencing Consortium 2008, McKenna et al. 2016), our current study identified only five GRs from the antennal transcriptome of X. quadripes. This is possibly related to low or no expression of GRs in antennae. As indicated by previous studies of antennal transcriptome analyses from coleopteran and noncoleopteran species, GRs are typically expressed at low levels in the antennae (Liu et al. 2014, 2015; Wang et al. 2014; Li et al. 2015b; Hu et al. 2016). In T. castaneum, six copies of SNMPs have been identified with four SNMP1s, one SNMP2, and one SNMP3 (Dippel et al. 2016). Apart from that, other coleopteran species possess one to four SNMPs including two in A. glabripennis (Hu et al. 2016), three in A. chinensis (Wang et al. 2017), and one in M. alternatus (Wang et al. 2014). Four SNMPs in X. quadripes were identified and clustered in four groups, together with previous studies (Nichols and Vogt 2008, Vogt et al. 2009), possibly representing a complete set in this species. Of these four XquaSNMPs, SNMP1b has more specific expression in antennae possibly involved in olfaction. As expected, its orthologous SNMP1 has been demonstrated to be essential for the sensing of sex pheromones in Diptera (D. melanogaster) and Lepidoptera (H. virescens) (Benton et al. 2007, Jin et al. 2008, Pregitzer et al. 2014). Considering the fact that XquaSNMP1b has a close relationship with DmelSNMP1 in phylogeny, it is inferred that this gene may have a similar function linked to the detection of sex pheromones. Adults of the cerambycid beetles have important life activities involved in chemosensory behaviors such as mate recognition and host searching. The information on the identification and characterization of chemosensory-associated genes is of particular significance for the control of this pest. In this study, we have identified 60 novel candidates for the sensing of semiochemicals through the antennal transcriptome. In particular, three ORs (OR13, OR17, and OR21) are likely to be strong candidate pheromone receptors that detect male-produced sex pheromones in X. quadripes. The extensive expression map of these genes gives an implication in olfactory, gustatory, and nonsensory functions, and more importantly offers reference data for further functional studies in this beetle. Supplementary Material Supplementary data are available at Environmental Entomology online. Acknowledgments We are grateful to Mr. Shuai-Shuai Ji, Miss Meng-Li Zhou, and Miss Hui Wu for collecting the tissues of X. quadripes. This work was supported by the Youth Project of Applied Basic Research of Yunnan Province (2017FD101), the National Natural Science Foundation of China (31601647), and the National Undergraduate Training Program of Innovation and Entrepreneurship (201710677006). References Abuin, L., B. Bargeton, M. H. Ulbrich, E. Y. Isacoff, S. Kellenberger, and R. Benton. 2011. 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Chemosensory Transmembrane Protein Families in the Coffee White Stemborer, Xylotrechus quadripes (Coleoptera: Cerambycidae)

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Entomological Society of America
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© The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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0046-225X
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1938-2936
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10.1093/ee/nvy076
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Abstract

Abstract The coffee white stemborer, Xylotrechus quadripes Chevrolat (Coleoptera: Cerambycidae), feeds primarily on Coffea arabica L. (Gentianales: Rubiaceae) with its egg, larva, and pupa being developed within the trunk. The detection of chemosensory-related cues linked to adult mating, host seeking, and recognition is driven by three chemoreceptor gene repertoires of odorant (ORs), gustatory (GRs), and ionotropic (IRs) receptors as well as sensory neuron membrane proteins (SNMPs). Yet, information on these genes involved in chemoreception is unavailable in X. quadripes and relatively poor in the cerambycid beetles. Here, we presented the identification of four chemosensory transmembrane proteins from the antennal transcriptome of X. quadripes, including 33 ORs, five GRs, 18 IRs, and four SNMPs. Phylogenetic analysis classified the ORs into groups 1, 2, 3, 7, and olfactory coreceptor (Orco), showing three potential candidates (OR13, OR17, and OR21) for the sensing of male sex pheromones. The IRs were clustered into 10 orthologous groups, with additional copies for IR41a, IR64a, and IR75 clades. Four SNMPs were distributed in four independent clades, possibly representing a complete set in this species. Expression profiles revealed that all the genes were highly expressed in antennae, suggesting their olfactory roles. In addition, most of the genes showed the expression in nonantennal tissues including thoraxes, abdomens, wings, and legs, suggesting their involvement in nonchemosensory functions. Of notice, a highly conserved coreceptor IR25a displayed male-biased expression in the antennae, as the first presence in the cerambycid beetles. This study has established reference resources for understanding the mechanisms underlying the interactions between/within this beetle and its host plants. Xylotrechus quadripes, odorant receptor, gustatory receptor, ionotropic receptor, sensory neuron membrane protein The interspecific interactions of insects and plants, as a basis underlying insect adaptation and speciation to various environmental contexts, have constantly received much attention in recent years. In nature, insects encounter an array of volatile stimuli derived from hosts and nonhosts to complete host seeking, predator avoidance, and oviposition site localization (Zhang and Schlyter 2004, Bruce et al. 2005). In parallel, another critical life event for insect survival and reproduction is mating as a primary intraspecific interaction associated with the reception and recognition of sex pheromones (Edward and Chapman 2011). These chemosensory cues produced by plants or conspecific partners are of particular importance in the searching of suitable hosts or correct mates. The insects have evolved several sensory proteins to sense thousands of semiochemicals involved in smell and taste. These chemosensory-related proteins comprise three receptor gene repertoires of odorant (ORs) (Clyne et al. 1999, Hallem and Carlson 2006), gustatory (GRs) (Clyne et al. 2000, Dunipace et al. 2001, Ling et al. 2014), and ionotropic (IRs) (Yao et al. 2005, Benton et al. 2009) receptors as well as sensory neuron membrane proteins (SNMPs) (Vogt et al. 2009, Pregitzer et al. 2014). They are present in olfactory (OSNs) or gustatory (GSNs) sensory neurons residing in hair-like structures (namely sensilla) primarily distributed in antennae, the principle chemosensory organ of insects. Both ORs and GRs of insects are in general seven transmembrane receptor proteins (Hallem et al. 2006), but Helicoverpa armigera GRs were predicted to have three to nine transmembrane domains (Xu et al. 2016). Among ORs (and GRs), relatively low amino acid identities are observed within a same species (Clyne et al. 1999, 2000; Hallem et al. 2006). Functional studies on these two types of chemoreceptors have been extensively conducted, where the ORs are tuned mainly to plant odorants and sex pheromones (Anderson et al. 2009, de Fouchier et al. 2017), and the GRs detect sugar and bitter tastants as well as carbon dioxide (Chyb et al. 2003, Jones et al. 2007, Slone et al. 2007, Lee et al. 2009). In comparison to extensive studies of ORs in Diptera and Lepidoptera, coleopteran OR functions have been restricted to three species of Tribolium castaneum (Engsontia et al. 2008), Megacyllene caryae (Mitchell et al. 2012), and Dendroctonus armandi (Zhang et al. 2016b). Of these, knockdown of the olfactory co-receptors (Orco) TcasOrco and DarmOrco from T. castaneum and D. armandi significantly decreased the responses to one aggregation pheromone and 11 host odorants, respectively (Engsontia et al. 2008, Zhang et al. 2016b). In addition to those, three candidate odorant receptors that were sensitive to three sex pheromones [OR3: (S)-2-methyl-1-butanol; OR5: 2-phenylethanol; and OR20: (2S,3R)-2,3-hexanediol] were identified in M. caryae, as the first ligand-specific ORs in the beetles functionally characterized (Mitchell et al. 2012). However, none of GR functions in Coleoptera have been reported to date. The third chemoreceptor family of IR belonging to a member of ionotropic glutamate receptors (iGluRs), was discovered from Drosophila species (Benton et al. 2009). Subsequently, a large number of IR genes have been identified from Diptera and other insect orders with a functional emphasis in Drosophila melanogaster (Abuin et al. 2011, Enjin et al. 2016, Ni et al. 2016). Sequence characteristics, expression profiles, and evolutionary analyses distinguished the Drosophila IRs into two subfamilies: antennal IRs (A-IRs) and divergent IRs (D-IRs) (Croset et al. 2010). With a combinatorial pattern of at least one stimuli-specific IR and one coreceptor IR, IRs respond to various types of ligands including general odorants, sex pheromones, and tastants (Abuin et al. 2011, Silbering et al. 2011). The A-IRs are highly conserved across insect species, and mainly bear olfactory roles related to the sensing of acids, amines, and other odorants. By comparison, the D-IRs with species- or genera-specific expansions are expressed in taste tissues with involvement of taste behaviors (Rytz et al. 2013, Koh et al. 2014, Stewart et al. 2015). Differing from three superfamilies of chemoreceptors, insect SNMPs have a small set of gene numbers, generally presenting two orthologs with copy number variations of each ortholog (Nichols and Vogt 2008, Vogt et al. 2009). For example, T. castaneum has six SNMPs with four SNMP1 copies, one SNMP2, and one SNMP3 (Dippel et al. 2016); each three SNMPs (two SNMP1s and one SNMP2) in Dendroctonus ponderosae and Ips typographus (Andersson et al. 2013), and three SNMPs (one SNMP1 and two SNMP2s) in Tomicus yunnanensis (Liu et al. 2018). To date, little information on SNMP functions is available in insects. With a few exceptions, studies have demonstrated that D. melanogaster SNMP1 is required for the detection of the sex pheromone cis-vaccinyl acetate (Benton et al. 2007, Jin et al. 2008). Additionally, Heliothis virescens SNMP1 could increase the sensitivity of pheromone stimulation (Pregitzer et al. 2014). However, in coleopteran species, functional studies of SNMPs remain to be lacking. The coffee white stemborer, Xylotrechus quadripes Chevrolat (Coleoptera: Cerambycidae), is the foremost destructive pest of Coffea arabica L. (Gentianales: Rubiaceae) that is planted widely in Asian countries including China and India due to its economic value and importance (Rhainds et al. 2002, Venkatesha and Dinesh 2012). This beetle has a relatively narrow range of host plants with a preference for C. arabica, and other alternative hosts contain Moraceae, Rubiaceae, Oleaceae, Anacardiaceae, and Lamiaceae families (Santosh et al. 2011, Venkatesha and Dinesh 2012). Three types of sex pheromone components (2S-hydroxy-3-decanone, 3-hydroxy-2-decanone and 2S,3S-dihydroxyoctane) and four other compounds (2-hydroxy-3-octanone, 2-phenylethanol, octanoic acid and 2,3-decanedione) have been identified from male beetles. In addition, the effects of male sex pheromones were evaluated in laboratory and field (Rhainds et al. 2001, Hall et al. 2006). Although many attempts (cultural, mechanical, chemical, biological control, and even resistant coffee cultivar methods) have been developed to control this beetle, the molecular mechanisms underlying the interactions between/within this beetle and its hosts have still been largely unexplored and poorly understood. Successful host or mate searching and recognition require a highly sophisticated chemosensory system of X. quadripes. Yet, little is known about olfactory- or taste-related genes in this beetle. This greatly restricts our ability to understand chemosensory mechanisms underlying the interactions between/within this beetle and its hosts, and to develop novel implemented control strategies in integrated pest management based on plant odorants and sex pheromones. Here, we sequenced and assembled an antennal transcriptome of adult X. quadripes by next-generation sequencing. Further, we identified and characterized four chemosensory-associated transmembrane protein families using phylogenetic and extensive expression profile analyses. This study has greatly implemented the information on molecular aspects of the interactions between/within this beetle and its hosts, and provides novel molecular targets for pest control. Materials and Methods Insects and Tissue Collection The infested coffee stems were cut in Ruili city, Yunnan Province, China. The harvested stems were brought and maintained in nylon net cages (1 m long × 1 m wide × 0.5 m high) at room temperature until X. quadripes eclosion. The emerged adults were sexed based on the heads and the last abdominal segments, and separated in individual cages. For transcriptome sequencing, about 200 antennae of 1–5 d old male and female adults between the seventh and ninth hour of the photophase were collected and mixed with a sex ratio of 1:1. For the analysis of expression profiles, various tissues including antennae, heads without antennae, thoraxes, abdomens, legs and wings of both sexes were collected, respectively. Then, collected tissues were immediately immerged in liquid nitrogen and stored at −80°C. Total RNA Extraction and First-Strand cDNA Synthesis Total RNA extraction was conducted from different tissues using TRIzol Reagent (Ambion, Life Technologies, Carlsbad, CA) according to the manufacturer’s protocol. RNA quality and integrity were examined using NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, San Jose, CA). Genomic DNA was first removed from RNA samples (1 µg) with genomic DNA Eraser at 42°C for 2 min. Next, first-strand complementary DNA (cDNA) synthesis was performed by PrimeScript RT Reagent Kit (TaKaRa, Dalian, Liaoning, China), following the procedure: 37°C for 15 min and 85°C for 5 s. Synthesized cDNA templates were stored at −20°C. Gene Identification, Sequence, and Phylogenetic Analysis The adult antennal transcriptome of X. quadripes was downloaded from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under the accession number of SRP143591. For the identification of candidate OR, GR, IR, and SNMP genes of X. quadripes, chemosensory-related transmembrane proteins from other cerambycid beetles, together with the genes of T. castaneum and D. melanogaster, were collected as queries to screen the standalone antennal transcriptome of X. quadripes using TBLASTN, with an E-value cutoff of e−5 and 20 maximum hits of each query. Further, identified genes were verified using BLASTX against the NCBI nonredundant protein sequence (nr) database. Sequence alignment and transmembrane domain prediction were conducted with MAFFT v7.308 (alignment parameters: scoring matrix of BLOSUM62 and gap open penalty of 1.53) (Katoh and Standley 2013) and TMHMM Server v2.0 (Krogh et al. 2001), respectively. In the OR tree, X. quadripes ORs with <150 amino acids (AAs) were excluded, and two XquaGRs (GR1 and GR2) were used as an outgroup to root the tree. The ORs were used from three other cerambycid beetles of Anoplophora chinensis, Anoplophora glabripennis, and M. caryae as well as a coleopteran model organism T. castaneum. In the GR tree, the GRs were used from three other coleopteran species of A. chinensis, A. glabripennis, and T. castaneum as well as D. melanogaster. In T. castaneum, we only selected the GRs: CO2 receptors, fructose receptors, sugar receptors, and 20 other GRs (10 GRs from each clade) previously classified into two independent clades (Dippel et al. 2016). In D. melanogaster, three GR subfamilies (CO2, fructose, and sugar GRs) and four known bitter receptors (GR32a, GR33a, GR66a, and GR93a) were included. For the tree set of IRs, the IRs were selected from the species of Brontispa longissima, D. ponderosae, I. typographus, T. castaneum, and D. melanogaster. In addition to those, iGluRs of T. castaneum and D. melanogaster were added to the phylogenetic analysis of IRs. The tree of SNMPs was built with SNMPs of 18 coleopteran species and D. melanogaster. Approximately maximum-likelihood phylogenetic tree was constructed using FastTree version 2.1.5 with default settings (Price et al. 2010). The tree was viewed and edited by FigTree v1.4.3. The accession numbers of the proteins used in the trees were listed in Supplementary Table S1. Expression Profile of Candidate Chemosensory Genes The primers (Supplementary Table S2) of candidate chemosensory genes were designed by Beacon Designer 8.13 (PREMIER Biosoft International, Palo Alto, CA). The amplified product sizes were at least 360 bp, and thus the genes encoding <120 AAs were excluded from the analysis of expression profiles. Reverse transcription PCR was used to determine the expression of chemosensory genes in different tissues of both sexes. A total volume of 25 µl were established, containing 2.5 µl of 10 × PCR Buffer (Mg2+ plus), 0.2 mM of deoxyribonucleoside triphosphate (dNTP) Mixture (each 2.5 mM), 0.5 µM of each primer, and 0.15 µl of recombinant Thermus aquaticus (rTaq) DNA polymerase (5 U/µl) (TaKaRa, Dalian, Liaoning, China). PCR reaction was performed using the following procedure: an initial denaturation at 94°C for 3 min, 35 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 40 s, and final extension for 5 min at 72°C. PCR products were analyzed using 1.2% (w/v) agrose gel. Negative control was set using sterile water as the template. Ribosomal protein L10 (RPL10) gene was used as a reference gene to control the quality and quantity of cDNA templates. For most of chemosensory genes, two biological replicates were performed in some tissues. Results Candidate Odorant Receptors From the sequenced antennal transcriptome, 33 transcripts encoding ORs were identified including a highly conserved Orco across insects (Vosshall and Hansson 2011). These genes were named de-novo using the Arabic numerals from 1 to 32 outside Orco. Of these 33 ORs, 12 genes (OR1-3, OR5-12, and Orco) were predicted to have complete open reading frames (ORFs) varying from 345 to 480 AAs in length. The remaining ORs were fragments, of which OR29 showed the shortest sequence encoding 52 AAs and the longest cDNA sequence encoded 344 AAs for OR14. Among 12 full-length ORs, Orco shared extremely high identities to its orthologs from the longhorned beetles of A. chinensis (91%), A. glabripennis (91%), M. caryae (95%), and even noncerambycid species T. castaneum (85%). The remaining nine ORs exhibited moderate AA identities (46–79%) to the ORs from M. caryae with the exception that XquaOR9 and XquaOR11 shared 39% and 43% identities to AchiOR19 and AglaOR38, respectively. However, an extremely low identity (mean value = 14%) was observed among 12 full-length XquaORs (Table 1 and Supplementary Additional File 1). Our phylogenetic analysis, based on OR protein sequences of four cerambycid beetles and T. castaneum, revealed seven groups of coleopteran ORs in addition to Orco group. Five groups (1, 2, 3, 7, and Orco) comprised at least one OR from X. quadripes, with eight candidates for groups 2 or 7. In particular, group 7 was phylogenetically formed only by OR members of cerambycid beetles. On the other hand, three orthologs of McarOR3, OR5, and OR20 in M. caryae known functional ligands (Mitchell et al. 2012) were detected in X. quadripes, i.e., XquaOR13, OR21, and OR17. Notably, we found that six members of ORs (AglaOR3, AchiOR20, McarOR41PAR, McarOR44, McarOR50PAR, and TcasOR275FIX) had high sequence divergence and did not show clear phylogenetic relationships to each other or with other groups. It appears that they may be considered to be outgroup(s) of groups 4, 5, and 6 in T. cantaneum (Fig. 1A). Fig. 1. View largeDownload slide View largeDownload slide Candidate odorant receptor gene family in X. quadripes. (A) Maximum-likelihood tree of ORs from four cerambycid beetles and T. castaneum. Seven groups (1–7) are defined following the previous classification of ORs. Bootstrap values with <0.80 are shown in circles. XquaGR1 and GR2 are used as the outgroup to root the tree. Three ORs of M. caryae (OR3, OR5, and OR20) known functional ligands are indicated with their respective corresponding orthologs in X. quadripes. (B) Expression profile of 28 X. quadripes ORs. A reference gene, RPL10, is used to check the quality and quantity of cDNA templates. A, antenna; H, head without antennae; T, thorax; Ab, abdomen; L, leg, W, wing and NC, negative control using sterile water as the template. Fig. 1. View largeDownload slide View largeDownload slide Candidate odorant receptor gene family in X. quadripes. (A) Maximum-likelihood tree of ORs from four cerambycid beetles and T. castaneum. Seven groups (1–7) are defined following the previous classification of ORs. Bootstrap values with <0.80 are shown in circles. XquaGR1 and GR2 are used as the outgroup to root the tree. Three ORs of M. caryae (OR3, OR5, and OR20) known functional ligands are indicated with their respective corresponding orthologs in X. quadripes. (B) Expression profile of 28 X. quadripes ORs. A reference gene, RPL10, is used to check the quality and quantity of cDNA templates. A, antenna; H, head without antennae; T, thorax; Ab, abdomen; L, leg, W, wing and NC, negative control using sterile water as the template. To determine sex- and tissue-specific expression of identified ORs as well as reconstruct their transcripts, 28 ORs were selected. The expression of all OR genes was enriched in the antennae with no sexual difference. Some ORs showed broad expression in all tested tissues like OR1, OR6, OR16, OR20, OR24, and Orco. In addition to the antennae, several ORs were detected in another important chemosensory organ of legs at considerable levels, including OR1, OR6, OR11, OR12, OR16, and Orco. In nonchemosensory tissues, over half of ORs were detected, some of which showed obviously high expression in thoraxes (OR6, OR11, and OR16) and abdomens (OR13, OR16, OR18, and OR20) (Fig. 1B). Candidate Gustatory Receptors Five GR transcripts were found from the transcriptome, namely GR1-5. Of these, GR1 and GR2 were full-length sequences with 38% identity to each other, and three other GRs (GR3-5) were partial sequences, encoding 134, 79, and 113 AAs, respectively (Table 1 and Supplementary Additional File 1). The GR phylogenetic tree indicated that XquaGR5 was clustered into fructose receptors, and thus was likely to be a candidate for the detection of fructose in X. quadripes. The remaining four XquaGRs may be candidate bitter receptors. None of CO2 and sugar GRs was found from this transcriptome (Fig. 2A). Expression profile analysis indicated that XquaGR1, GR2, and GR3 were broadly expressed in all tested tissues of both sexes (Fig. 2B). Table 1. Blast results of candidate chemosensory membrane proteins in X. quadripes Gene  ORF (AA)  Full length  NCBI blast hit (references/name/species)  E value  Identity (%)  Odorant receptor (OR)   ORco  480  Yes  XP_018568191.1 odorant receptor coreceptor [Anoplophora glabripennis]  0.0  91   OR1  419  Yes  XP_023020469.1 odorant receptor 94b-like [Leptinotarsa decemlineata]  3e−147  50   OR2  357  Yes  XP_015832942.1 PREDICTED: odorant receptor Or1-like [Tribolium castaneum]  7e−74  35   OR3  345  Yes  XP_018578867.1 odorant receptor 49b-like [Anoplophora glabripennis]  1e−115  52   OR4  317  No  XP_018577142.1 odorant receptor 4-like [Anoplophora glabripennis]  2e−90  45   OR5  419  Yes  XP_018577142.1 odorant receptor 4-like [Anoplophora glabripennis]  3e−122  43   OR6  384  Yes  AUF73037.1 odorant receptor [Anoplophora chinensis]  2e−75  39   OR7  361  Yes  XP_023309849.1 odorant receptor 49b-like [Anoplophora glabripennis]  4e−59  36   OR8  422  Yes  ALR72579.1 odorant receptor OR36 [Colaphellus bowringi]  2e−113  44   OR9  404  Yes  AUF73022.1 odorant receptor [Anoplophora chinensis]  7e−49  30   OR10  388  Yes  AUF73043.1 odorant receptor [Anoplophora chinensis]  1e−51  27   OR11  387  Yes  XP_018567969.1 odorant receptor Or2-like [Anoplophora glabripennis]  3e−118  43   OR12  397  Yes  ALR72575.1 odorant receptor OR32 [Colaphellus bowringi]  8e−50  31   OR13  175  No  XP_018564808.1 odorant receptor Or1-like [Anoplophora glabripennis]  2e−74  66   OR14  344  No  XP_023026692.1 odorant receptor 49b-like [Leptinotarsa decemlineata]  2e−159  63   OR15  151  No  XP_018567067.1 odorant receptor Or2-like [Anoplophora glabripennis]  2e−14  31   OR16  176  No  AUF73030.1 odorant receptor [Anoplophora chinensis]  2e−54  48   OR17  230  No  ALR72568.1 odorant receptor OR24 [Colaphellus bowringi]  5e−82  50   OR18  151  No  AIX97138.1 olfactory receptor 3, partial [Rhyzopertha dominica]  2e−19  30   OR19  186  No  ALR72565.1 odorant receptor OR20 [Colaphellus bowringi]  9e−30  35   OR20  242  No  XP_023310030.1 odorant receptor Or1-like [Anoplophora glabripennis]  7e−116  72   OR21  322  No  ALR72579.1 odorant receptor OR36 [Colaphellus bowringi]  1e−77  43   OR22  283  No  XP_022917715.1 odorant receptor 49b-like [Onthophagus taurus]  9e−17  29   OR23  180  No  XP_023311541.1 odorant receptor 22b-like [Anoplophora glabripennis]  4e−16  32   OR24  242  No  APC94230.1 odorant receptor 18 [Pyrrhalta maculicollis]  8e−82  49   OR25  152  No  XP_023023560.1 odorant receptor 47b-like [Leptinotarsa decemlineata]  4e−25  37   OR26  174  No  AUF73044.1 odorant receptor [Anoplophora chinensis]  7e−44  40   OR27  241  No  XP_023019277.1 odorant receptor 4-like [Leptinotarsa decemlineata]  9e−31  30   OR28  102  No  XP_023313103.1 odorant receptor 43a-like isoform X2 [Anoplophora glabripennis]  6e−41  65   OR29  52  No  AUF73036.1 odorant receptor [Anoplophora chinensis]  1e−18  76   OR30  103  No  AUF73020.1 odorant receptor [Anoplophora chinensis]  3e−39  63   OR31  102  No  XP_014363329.1 PREDICTED: odorant receptor Or1-like [Papilio machaon]  4e−17  44   OR32  96  No  XP_018575789.2 putative odorant receptor 71a [Anoplophora glabripennis]  7e−27  51  Ionotropic glutamate receptor (iGluR) and ionotropic receptor (IR)   iGluR1  930  Yes  XP_018563905.1 glutamate receptor ionotropic, kainate 2-like [Anoplophora glabripennis]  0.0  63   iGluR2  223  No  XP_018569924.1 glutamate receptor ionotropic, kainate 2-like [Anoplophora glabripennis]  5e−102  66   iGluR3  645  No  XP_023312109.1 glutamate receptor ionotropic, kainate 2-like isoform X4 [Anoplophora glabripennis]  0.0  92   iGluR4  670  No  XP_018570638.1 glutamate receptor ionotropic, kainate 2-like isoform X1 [Anoplophora glabripennis]  0.0  59   iGluR5  561  No  XP_023024797.1 glutamate receptor ionotropic, kainate 2-like [Leptinotarsa decemlineata]  9e−136  40   iGluR6  758  No  XP_018563904.1 glutamate receptor ionotropic, kainate 2-like isoform X2 [Anoplophora glabripennis]  0.0  59   iGluR7  611  No  XP_018570638.1 glutamate receptor ionotropic, kainate 2-like isoform X1 [Anoplophora glabripennis]  0.0  71   iGluR8  248  No  XP_018570639.1 glutamate receptor ionotropic, kainate 1-like isoform X2 [Anoplophora glabripennis]  2e−103  65   IR1  170  No  AUF73085.1 ionotropic receptor [Anoplophora chinensis]  2e−42  48   IR2  200  No  AST36363.1 putative ionotropic receptor IR75q.1 [Cydia fagiglandana]  8e−15  30   IR8a  664  No  ALR72538.1 ionotropic receptor 8a [Colaphellus bowringi]  2e−159  78   IR21a  158  No  XP_023313061.1 ionotropic receptor 21a-like [Anoplophora glabripennis]  4e−75  72   IR25a  935  Yes  XP_018574744.1 ionotropic receptor 25a [Anoplophora glabripennis]  0.0  85   IR40a  65  No  XP_023310509.1 ionotropic receptor 40a [Anoplophora glabripennis]  5e−33  91   IR41a.1  635  Yes  AKC58587.1 chemosensory ionotropic receptor 41a [Anomala corpulenta]  2e−132  37   IR41a.2  232  No  AKC58587.1 chemosensory ionotropic receptor 41a [Anomala corpulenta]  2e−32  34   IR41a.3  240  No  NP_001345663.1 ionotropic receptor 41a [Aedes aegypti]  3e−39  37   IR64a.1  392  No  XP_023311151.1 glutamate receptor 2-like [Anoplophora glabripennis]  0.0  66   IR64a.2  90  No  XP_018571126.1 glutamate receptor 2-like [Anoplophora glabripennis]  4e−34  62   IR68a  193  No  XP_018568048.1 glutamate receptor ionotropic, kainate 5 [Anoplophora glabripennis]  2e−88  59   IR75q  623  Yes  APC94348.1 ionotropic receptor 5, partial [Pyrrhalta aenescens]  4e−125  36   IR75q.1  601  Yes  APC94348.1 ionotropic receptor 5, partial [Pyrrhalta aenescens]  5e−137  38   IR75q.2  427  No  ANQ46495.1 ionotropic receptor 3 [Phyllotreta striolata]  0.0  62   IR75s  601  Yes  XP_018562688.2 probable glutamate receptor [Anoplophora glabripennis]  0.0  70   IR76b  561  Yes  XP_018568700.1 glutamate receptor ionotropic, delta-1 [Anoplophora glabripennis]  0.0  65   IR93a  705  No  XP_018576792.1 ionotropic receptor 93a isoform X1 [Anoplophora glabripennis]  0.0  67  Gustatory receptor (GR)   GR1  339  Yes  XP_018565150.1 G-protein coupled receptor moody-like isoform X1 [Anoplophora glabripennis]  0.0  85   GR2  335  Yes  AKC58582.1 gustatory receptor 5 [Anomala corpulenta]  4e−157  68   GR3  134  No  ALR72527.1 gustatory receptor 1, partial [Colaphellus bowringi]  2e−29  44   GR4  79  No  XP_023310039.1 putative gustatory receptor 2a [Anoplophora glabripennis]  5e−19  51   GR5  113  No  AUF73059.1 gustatory receptor, partial [Anoplophora chinensis]  7e−33  57  Sensory neuron membrane protein (SNMP)   SNMP1a  521  Yes  ALR72542.1 sensory neuron membrane protein SNMP1a [Colaphellus bowringi]  0.0  65   SNMP1b  531  Yes  ALR72543.1 sensory neuron membrane protein SNMP1b [Colaphellus bowringi]  0.0  59   SNMP2a  520  Yes  XP_018566911.1 sensory neuron membrane protein 2 [Anoplophora glabripennis]  0.0  63   SNMP2b  505  Yes  XP_018566912.1 sensory neuron membrane protein 2-like [Anoplophora glabripennis]  0.0  67  Gene  ORF (AA)  Full length  NCBI blast hit (references/name/species)  E value  Identity (%)  Odorant receptor (OR)   ORco  480  Yes  XP_018568191.1 odorant receptor coreceptor [Anoplophora glabripennis]  0.0  91   OR1  419  Yes  XP_023020469.1 odorant receptor 94b-like [Leptinotarsa decemlineata]  3e−147  50   OR2  357  Yes  XP_015832942.1 PREDICTED: odorant receptor Or1-like [Tribolium castaneum]  7e−74  35   OR3  345  Yes  XP_018578867.1 odorant receptor 49b-like [Anoplophora glabripennis]  1e−115  52   OR4  317  No  XP_018577142.1 odorant receptor 4-like [Anoplophora glabripennis]  2e−90  45   OR5  419  Yes  XP_018577142.1 odorant receptor 4-like [Anoplophora glabripennis]  3e−122  43   OR6  384  Yes  AUF73037.1 odorant receptor [Anoplophora chinensis]  2e−75  39   OR7  361  Yes  XP_023309849.1 odorant receptor 49b-like [Anoplophora glabripennis]  4e−59  36   OR8  422  Yes  ALR72579.1 odorant receptor OR36 [Colaphellus bowringi]  2e−113  44   OR9  404  Yes  AUF73022.1 odorant receptor [Anoplophora chinensis]  7e−49  30   OR10  388  Yes  AUF73043.1 odorant receptor [Anoplophora chinensis]  1e−51  27   OR11  387  Yes  XP_018567969.1 odorant receptor Or2-like [Anoplophora glabripennis]  3e−118  43   OR12  397  Yes  ALR72575.1 odorant receptor OR32 [Colaphellus bowringi]  8e−50  31   OR13  175  No  XP_018564808.1 odorant receptor Or1-like [Anoplophora glabripennis]  2e−74  66   OR14  344  No  XP_023026692.1 odorant receptor 49b-like [Leptinotarsa decemlineata]  2e−159  63   OR15  151  No  XP_018567067.1 odorant receptor Or2-like [Anoplophora glabripennis]  2e−14  31   OR16  176  No  AUF73030.1 odorant receptor [Anoplophora chinensis]  2e−54  48   OR17  230  No  ALR72568.1 odorant receptor OR24 [Colaphellus bowringi]  5e−82  50   OR18  151  No  AIX97138.1 olfactory receptor 3, partial [Rhyzopertha dominica]  2e−19  30   OR19  186  No  ALR72565.1 odorant receptor OR20 [Colaphellus bowringi]  9e−30  35   OR20  242  No  XP_023310030.1 odorant receptor Or1-like [Anoplophora glabripennis]  7e−116  72   OR21  322  No  ALR72579.1 odorant receptor OR36 [Colaphellus bowringi]  1e−77  43   OR22  283  No  XP_022917715.1 odorant receptor 49b-like [Onthophagus taurus]  9e−17  29   OR23  180  No  XP_023311541.1 odorant receptor 22b-like [Anoplophora glabripennis]  4e−16  32   OR24  242  No  APC94230.1 odorant receptor 18 [Pyrrhalta maculicollis]  8e−82  49   OR25  152  No  XP_023023560.1 odorant receptor 47b-like [Leptinotarsa decemlineata]  4e−25  37   OR26  174  No  AUF73044.1 odorant receptor [Anoplophora chinensis]  7e−44  40   OR27  241  No  XP_023019277.1 odorant receptor 4-like [Leptinotarsa decemlineata]  9e−31  30   OR28  102  No  XP_023313103.1 odorant receptor 43a-like isoform X2 [Anoplophora glabripennis]  6e−41  65   OR29  52  No  AUF73036.1 odorant receptor [Anoplophora chinensis]  1e−18  76   OR30  103  No  AUF73020.1 odorant receptor [Anoplophora chinensis]  3e−39  63   OR31  102  No  XP_014363329.1 PREDICTED: odorant receptor Or1-like [Papilio machaon]  4e−17  44   OR32  96  No  XP_018575789.2 putative odorant receptor 71a [Anoplophora glabripennis]  7e−27  51  Ionotropic glutamate receptor (iGluR) and ionotropic receptor (IR)   iGluR1  930  Yes  XP_018563905.1 glutamate receptor ionotropic, kainate 2-like [Anoplophora glabripennis]  0.0  63   iGluR2  223  No  XP_018569924.1 glutamate receptor ionotropic, kainate 2-like [Anoplophora glabripennis]  5e−102  66   iGluR3  645  No  XP_023312109.1 glutamate receptor ionotropic, kainate 2-like isoform X4 [Anoplophora glabripennis]  0.0  92   iGluR4  670  No  XP_018570638.1 glutamate receptor ionotropic, kainate 2-like isoform X1 [Anoplophora glabripennis]  0.0  59   iGluR5  561  No  XP_023024797.1 glutamate receptor ionotropic, kainate 2-like [Leptinotarsa decemlineata]  9e−136  40   iGluR6  758  No  XP_018563904.1 glutamate receptor ionotropic, kainate 2-like isoform X2 [Anoplophora glabripennis]  0.0  59   iGluR7  611  No  XP_018570638.1 glutamate receptor ionotropic, kainate 2-like isoform X1 [Anoplophora glabripennis]  0.0  71   iGluR8  248  No  XP_018570639.1 glutamate receptor ionotropic, kainate 1-like isoform X2 [Anoplophora glabripennis]  2e−103  65   IR1  170  No  AUF73085.1 ionotropic receptor [Anoplophora chinensis]  2e−42  48   IR2  200  No  AST36363.1 putative ionotropic receptor IR75q.1 [Cydia fagiglandana]  8e−15  30   IR8a  664  No  ALR72538.1 ionotropic receptor 8a [Colaphellus bowringi]  2e−159  78   IR21a  158  No  XP_023313061.1 ionotropic receptor 21a-like [Anoplophora glabripennis]  4e−75  72   IR25a  935  Yes  XP_018574744.1 ionotropic receptor 25a [Anoplophora glabripennis]  0.0  85   IR40a  65  No  XP_023310509.1 ionotropic receptor 40a [Anoplophora glabripennis]  5e−33  91   IR41a.1  635  Yes  AKC58587.1 chemosensory ionotropic receptor 41a [Anomala corpulenta]  2e−132  37   IR41a.2  232  No  AKC58587.1 chemosensory ionotropic receptor 41a [Anomala corpulenta]  2e−32  34   IR41a.3  240  No  NP_001345663.1 ionotropic receptor 41a [Aedes aegypti]  3e−39  37   IR64a.1  392  No  XP_023311151.1 glutamate receptor 2-like [Anoplophora glabripennis]  0.0  66   IR64a.2  90  No  XP_018571126.1 glutamate receptor 2-like [Anoplophora glabripennis]  4e−34  62   IR68a  193  No  XP_018568048.1 glutamate receptor ionotropic, kainate 5 [Anoplophora glabripennis]  2e−88  59   IR75q  623  Yes  APC94348.1 ionotropic receptor 5, partial [Pyrrhalta aenescens]  4e−125  36   IR75q.1  601  Yes  APC94348.1 ionotropic receptor 5, partial [Pyrrhalta aenescens]  5e−137  38   IR75q.2  427  No  ANQ46495.1 ionotropic receptor 3 [Phyllotreta striolata]  0.0  62   IR75s  601  Yes  XP_018562688.2 probable glutamate receptor [Anoplophora glabripennis]  0.0  70   IR76b  561  Yes  XP_018568700.1 glutamate receptor ionotropic, delta-1 [Anoplophora glabripennis]  0.0  65   IR93a  705  No  XP_018576792.1 ionotropic receptor 93a isoform X1 [Anoplophora glabripennis]  0.0  67  Gustatory receptor (GR)   GR1  339  Yes  XP_018565150.1 G-protein coupled receptor moody-like isoform X1 [Anoplophora glabripennis]  0.0  85   GR2  335  Yes  AKC58582.1 gustatory receptor 5 [Anomala corpulenta]  4e−157  68   GR3  134  No  ALR72527.1 gustatory receptor 1, partial [Colaphellus bowringi]  2e−29  44   GR4  79  No  XP_023310039.1 putative gustatory receptor 2a [Anoplophora glabripennis]  5e−19  51   GR5  113  No  AUF73059.1 gustatory receptor, partial [Anoplophora chinensis]  7e−33  57  Sensory neuron membrane protein (SNMP)   SNMP1a  521  Yes  ALR72542.1 sensory neuron membrane protein SNMP1a [Colaphellus bowringi]  0.0  65   SNMP1b  531  Yes  ALR72543.1 sensory neuron membrane protein SNMP1b [Colaphellus bowringi]  0.0  59   SNMP2a  520  Yes  XP_018566911.1 sensory neuron membrane protein 2 [Anoplophora glabripennis]  0.0  63   SNMP2b  505  Yes  XP_018566912.1 sensory neuron membrane protein 2-like [Anoplophora glabripennis]  0.0  67  View Large Table 1. Blast results of candidate chemosensory membrane proteins in X. quadripes Gene  ORF (AA)  Full length  NCBI blast hit (references/name/species)  E value  Identity (%)  Odorant receptor (OR)   ORco  480  Yes  XP_018568191.1 odorant receptor coreceptor [Anoplophora glabripennis]  0.0  91   OR1  419  Yes  XP_023020469.1 odorant receptor 94b-like [Leptinotarsa decemlineata]  3e−147  50   OR2  357  Yes  XP_015832942.1 PREDICTED: odorant receptor Or1-like [Tribolium castaneum]  7e−74  35   OR3  345  Yes  XP_018578867.1 odorant receptor 49b-like [Anoplophora glabripennis]  1e−115  52   OR4  317  No  XP_018577142.1 odorant receptor 4-like [Anoplophora glabripennis]  2e−90  45   OR5  419  Yes  XP_018577142.1 odorant receptor 4-like [Anoplophora glabripennis]  3e−122  43   OR6  384  Yes  AUF73037.1 odorant receptor [Anoplophora chinensis]  2e−75  39   OR7  361  Yes  XP_023309849.1 odorant receptor 49b-like [Anoplophora glabripennis]  4e−59  36   OR8  422  Yes  ALR72579.1 odorant receptor OR36 [Colaphellus bowringi]  2e−113  44   OR9  404  Yes  AUF73022.1 odorant receptor [Anoplophora chinensis]  7e−49  30   OR10  388  Yes  AUF73043.1 odorant receptor [Anoplophora chinensis]  1e−51  27   OR11  387  Yes  XP_018567969.1 odorant receptor Or2-like [Anoplophora glabripennis]  3e−118  43   OR12  397  Yes  ALR72575.1 odorant receptor OR32 [Colaphellus bowringi]  8e−50  31   OR13  175  No  XP_018564808.1 odorant receptor Or1-like [Anoplophora glabripennis]  2e−74  66   OR14  344  No  XP_023026692.1 odorant receptor 49b-like [Leptinotarsa decemlineata]  2e−159  63   OR15  151  No  XP_018567067.1 odorant receptor Or2-like [Anoplophora glabripennis]  2e−14  31   OR16  176  No  AUF73030.1 odorant receptor [Anoplophora chinensis]  2e−54  48   OR17  230  No  ALR72568.1 odorant receptor OR24 [Colaphellus bowringi]  5e−82  50   OR18  151  No  AIX97138.1 olfactory receptor 3, partial [Rhyzopertha dominica]  2e−19  30   OR19  186  No  ALR72565.1 odorant receptor OR20 [Colaphellus bowringi]  9e−30  35   OR20  242  No  XP_023310030.1 odorant receptor Or1-like [Anoplophora glabripennis]  7e−116  72   OR21  322  No  ALR72579.1 odorant receptor OR36 [Colaphellus bowringi]  1e−77  43   OR22  283  No  XP_022917715.1 odorant receptor 49b-like [Onthophagus taurus]  9e−17  29   OR23  180  No  XP_023311541.1 odorant receptor 22b-like [Anoplophora glabripennis]  4e−16  32   OR24  242  No  APC94230.1 odorant receptor 18 [Pyrrhalta maculicollis]  8e−82  49   OR25  152  No  XP_023023560.1 odorant receptor 47b-like [Leptinotarsa decemlineata]  4e−25  37   OR26  174  No  AUF73044.1 odorant receptor [Anoplophora chinensis]  7e−44  40   OR27  241  No  XP_023019277.1 odorant receptor 4-like [Leptinotarsa decemlineata]  9e−31  30   OR28  102  No  XP_023313103.1 odorant receptor 43a-like isoform X2 [Anoplophora glabripennis]  6e−41  65   OR29  52  No  AUF73036.1 odorant receptor [Anoplophora chinensis]  1e−18  76   OR30  103  No  AUF73020.1 odorant receptor [Anoplophora chinensis]  3e−39  63   OR31  102  No  XP_014363329.1 PREDICTED: odorant receptor Or1-like [Papilio machaon]  4e−17  44   OR32  96  No  XP_018575789.2 putative odorant receptor 71a [Anoplophora glabripennis]  7e−27  51  Ionotropic glutamate receptor (iGluR) and ionotropic receptor (IR)   iGluR1  930  Yes  XP_018563905.1 glutamate receptor ionotropic, kainate 2-like [Anoplophora glabripennis]  0.0  63   iGluR2  223  No  XP_018569924.1 glutamate receptor ionotropic, kainate 2-like [Anoplophora glabripennis]  5e−102  66   iGluR3  645  No  XP_023312109.1 glutamate receptor ionotropic, kainate 2-like isoform X4 [Anoplophora glabripennis]  0.0  92   iGluR4  670  No  XP_018570638.1 glutamate receptor ionotropic, kainate 2-like isoform X1 [Anoplophora glabripennis]  0.0  59   iGluR5  561  No  XP_023024797.1 glutamate receptor ionotropic, kainate 2-like [Leptinotarsa decemlineata]  9e−136  40   iGluR6  758  No  XP_018563904.1 glutamate receptor ionotropic, kainate 2-like isoform X2 [Anoplophora glabripennis]  0.0  59   iGluR7  611  No  XP_018570638.1 glutamate receptor ionotropic, kainate 2-like isoform X1 [Anoplophora glabripennis]  0.0  71   iGluR8  248  No  XP_018570639.1 glutamate receptor ionotropic, kainate 1-like isoform X2 [Anoplophora glabripennis]  2e−103  65   IR1  170  No  AUF73085.1 ionotropic receptor [Anoplophora chinensis]  2e−42  48   IR2  200  No  AST36363.1 putative ionotropic receptor IR75q.1 [Cydia fagiglandana]  8e−15  30   IR8a  664  No  ALR72538.1 ionotropic receptor 8a [Colaphellus bowringi]  2e−159  78   IR21a  158  No  XP_023313061.1 ionotropic receptor 21a-like [Anoplophora glabripennis]  4e−75  72   IR25a  935  Yes  XP_018574744.1 ionotropic receptor 25a [Anoplophora glabripennis]  0.0  85   IR40a  65  No  XP_023310509.1 ionotropic receptor 40a [Anoplophora glabripennis]  5e−33  91   IR41a.1  635  Yes  AKC58587.1 chemosensory ionotropic receptor 41a [Anomala corpulenta]  2e−132  37   IR41a.2  232  No  AKC58587.1 chemosensory ionotropic receptor 41a [Anomala corpulenta]  2e−32  34   IR41a.3  240  No  NP_001345663.1 ionotropic receptor 41a [Aedes aegypti]  3e−39  37   IR64a.1  392  No  XP_023311151.1 glutamate receptor 2-like [Anoplophora glabripennis]  0.0  66   IR64a.2  90  No  XP_018571126.1 glutamate receptor 2-like [Anoplophora glabripennis]  4e−34  62   IR68a  193  No  XP_018568048.1 glutamate receptor ionotropic, kainate 5 [Anoplophora glabripennis]  2e−88  59   IR75q  623  Yes  APC94348.1 ionotropic receptor 5, partial [Pyrrhalta aenescens]  4e−125  36   IR75q.1  601  Yes  APC94348.1 ionotropic receptor 5, partial [Pyrrhalta aenescens]  5e−137  38   IR75q.2  427  No  ANQ46495.1 ionotropic receptor 3 [Phyllotreta striolata]  0.0  62   IR75s  601  Yes  XP_018562688.2 probable glutamate receptor [Anoplophora glabripennis]  0.0  70   IR76b  561  Yes  XP_018568700.1 glutamate receptor ionotropic, delta-1 [Anoplophora glabripennis]  0.0  65   IR93a  705  No  XP_018576792.1 ionotropic receptor 93a isoform X1 [Anoplophora glabripennis]  0.0  67  Gustatory receptor (GR)   GR1  339  Yes  XP_018565150.1 G-protein coupled receptor moody-like isoform X1 [Anoplophora glabripennis]  0.0  85   GR2  335  Yes  AKC58582.1 gustatory receptor 5 [Anomala corpulenta]  4e−157  68   GR3  134  No  ALR72527.1 gustatory receptor 1, partial [Colaphellus bowringi]  2e−29  44   GR4  79  No  XP_023310039.1 putative gustatory receptor 2a [Anoplophora glabripennis]  5e−19  51   GR5  113  No  AUF73059.1 gustatory receptor, partial [Anoplophora chinensis]  7e−33  57  Sensory neuron membrane protein (SNMP)   SNMP1a  521  Yes  ALR72542.1 sensory neuron membrane protein SNMP1a [Colaphellus bowringi]  0.0  65   SNMP1b  531  Yes  ALR72543.1 sensory neuron membrane protein SNMP1b [Colaphellus bowringi]  0.0  59   SNMP2a  520  Yes  XP_018566911.1 sensory neuron membrane protein 2 [Anoplophora glabripennis]  0.0  63   SNMP2b  505  Yes  XP_018566912.1 sensory neuron membrane protein 2-like [Anoplophora glabripennis]  0.0  67  Gene  ORF (AA)  Full length  NCBI blast hit (references/name/species)  E value  Identity (%)  Odorant receptor (OR)   ORco  480  Yes  XP_018568191.1 odorant receptor coreceptor [Anoplophora glabripennis]  0.0  91   OR1  419  Yes  XP_023020469.1 odorant receptor 94b-like [Leptinotarsa decemlineata]  3e−147  50   OR2  357  Yes  XP_015832942.1 PREDICTED: odorant receptor Or1-like [Tribolium castaneum]  7e−74  35   OR3  345  Yes  XP_018578867.1 odorant receptor 49b-like [Anoplophora glabripennis]  1e−115  52   OR4  317  No  XP_018577142.1 odorant receptor 4-like [Anoplophora glabripennis]  2e−90  45   OR5  419  Yes  XP_018577142.1 odorant receptor 4-like [Anoplophora glabripennis]  3e−122  43   OR6  384  Yes  AUF73037.1 odorant receptor [Anoplophora chinensis]  2e−75  39   OR7  361  Yes  XP_023309849.1 odorant receptor 49b-like [Anoplophora glabripennis]  4e−59  36   OR8  422  Yes  ALR72579.1 odorant receptor OR36 [Colaphellus bowringi]  2e−113  44   OR9  404  Yes  AUF73022.1 odorant receptor [Anoplophora chinensis]  7e−49  30   OR10  388  Yes  AUF73043.1 odorant receptor [Anoplophora chinensis]  1e−51  27   OR11  387  Yes  XP_018567969.1 odorant receptor Or2-like [Anoplophora glabripennis]  3e−118  43   OR12  397  Yes  ALR72575.1 odorant receptor OR32 [Colaphellus bowringi]  8e−50  31   OR13  175  No  XP_018564808.1 odorant receptor Or1-like [Anoplophora glabripennis]  2e−74  66   OR14  344  No  XP_023026692.1 odorant receptor 49b-like [Leptinotarsa decemlineata]  2e−159  63   OR15  151  No  XP_018567067.1 odorant receptor Or2-like [Anoplophora glabripennis]  2e−14  31   OR16  176  No  AUF73030.1 odorant receptor [Anoplophora chinensis]  2e−54  48   OR17  230  No  ALR72568.1 odorant receptor OR24 [Colaphellus bowringi]  5e−82  50   OR18  151  No  AIX97138.1 olfactory receptor 3, partial [Rhyzopertha dominica]  2e−19  30   OR19  186  No  ALR72565.1 odorant receptor OR20 [Colaphellus bowringi]  9e−30  35   OR20  242  No  XP_023310030.1 odorant receptor Or1-like [Anoplophora glabripennis]  7e−116  72   OR21  322  No  ALR72579.1 odorant receptor OR36 [Colaphellus bowringi]  1e−77  43   OR22  283  No  XP_022917715.1 odorant receptor 49b-like [Onthophagus taurus]  9e−17  29   OR23  180  No  XP_023311541.1 odorant receptor 22b-like [Anoplophora glabripennis]  4e−16  32   OR24  242  No  APC94230.1 odorant receptor 18 [Pyrrhalta maculicollis]  8e−82  49   OR25  152  No  XP_023023560.1 odorant receptor 47b-like [Leptinotarsa decemlineata]  4e−25  37   OR26  174  No  AUF73044.1 odorant receptor [Anoplophora chinensis]  7e−44  40   OR27  241  No  XP_023019277.1 odorant receptor 4-like [Leptinotarsa decemlineata]  9e−31  30   OR28  102  No  XP_023313103.1 odorant receptor 43a-like isoform X2 [Anoplophora glabripennis]  6e−41  65   OR29  52  No  AUF73036.1 odorant receptor [Anoplophora chinensis]  1e−18  76   OR30  103  No  AUF73020.1 odorant receptor [Anoplophora chinensis]  3e−39  63   OR31  102  No  XP_014363329.1 PREDICTED: odorant receptor Or1-like [Papilio machaon]  4e−17  44   OR32  96  No  XP_018575789.2 putative odorant receptor 71a [Anoplophora glabripennis]  7e−27  51  Ionotropic glutamate receptor (iGluR) and ionotropic receptor (IR)   iGluR1  930  Yes  XP_018563905.1 glutamate receptor ionotropic, kainate 2-like [Anoplophora glabripennis]  0.0  63   iGluR2  223  No  XP_018569924.1 glutamate receptor ionotropic, kainate 2-like [Anoplophora glabripennis]  5e−102  66   iGluR3  645  No  XP_023312109.1 glutamate receptor ionotropic, kainate 2-like isoform X4 [Anoplophora glabripennis]  0.0  92   iGluR4  670  No  XP_018570638.1 glutamate receptor ionotropic, kainate 2-like isoform X1 [Anoplophora glabripennis]  0.0  59   iGluR5  561  No  XP_023024797.1 glutamate receptor ionotropic, kainate 2-like [Leptinotarsa decemlineata]  9e−136  40   iGluR6  758  No  XP_018563904.1 glutamate receptor ionotropic, kainate 2-like isoform X2 [Anoplophora glabripennis]  0.0  59   iGluR7  611  No  XP_018570638.1 glutamate receptor ionotropic, kainate 2-like isoform X1 [Anoplophora glabripennis]  0.0  71   iGluR8  248  No  XP_018570639.1 glutamate receptor ionotropic, kainate 1-like isoform X2 [Anoplophora glabripennis]  2e−103  65   IR1  170  No  AUF73085.1 ionotropic receptor [Anoplophora chinensis]  2e−42  48   IR2  200  No  AST36363.1 putative ionotropic receptor IR75q.1 [Cydia fagiglandana]  8e−15  30   IR8a  664  No  ALR72538.1 ionotropic receptor 8a [Colaphellus bowringi]  2e−159  78   IR21a  158  No  XP_023313061.1 ionotropic receptor 21a-like [Anoplophora glabripennis]  4e−75  72   IR25a  935  Yes  XP_018574744.1 ionotropic receptor 25a [Anoplophora glabripennis]  0.0  85   IR40a  65  No  XP_023310509.1 ionotropic receptor 40a [Anoplophora glabripennis]  5e−33  91   IR41a.1  635  Yes  AKC58587.1 chemosensory ionotropic receptor 41a [Anomala corpulenta]  2e−132  37   IR41a.2  232  No  AKC58587.1 chemosensory ionotropic receptor 41a [Anomala corpulenta]  2e−32  34   IR41a.3  240  No  NP_001345663.1 ionotropic receptor 41a [Aedes aegypti]  3e−39  37   IR64a.1  392  No  XP_023311151.1 glutamate receptor 2-like [Anoplophora glabripennis]  0.0  66   IR64a.2  90  No  XP_018571126.1 glutamate receptor 2-like [Anoplophora glabripennis]  4e−34  62   IR68a  193  No  XP_018568048.1 glutamate receptor ionotropic, kainate 5 [Anoplophora glabripennis]  2e−88  59   IR75q  623  Yes  APC94348.1 ionotropic receptor 5, partial [Pyrrhalta aenescens]  4e−125  36   IR75q.1  601  Yes  APC94348.1 ionotropic receptor 5, partial [Pyrrhalta aenescens]  5e−137  38   IR75q.2  427  No  ANQ46495.1 ionotropic receptor 3 [Phyllotreta striolata]  0.0  62   IR75s  601  Yes  XP_018562688.2 probable glutamate receptor [Anoplophora glabripennis]  0.0  70   IR76b  561  Yes  XP_018568700.1 glutamate receptor ionotropic, delta-1 [Anoplophora glabripennis]  0.0  65   IR93a  705  No  XP_018576792.1 ionotropic receptor 93a isoform X1 [Anoplophora glabripennis]  0.0  67  Gustatory receptor (GR)   GR1  339  Yes  XP_018565150.1 G-protein coupled receptor moody-like isoform X1 [Anoplophora glabripennis]  0.0  85   GR2  335  Yes  AKC58582.1 gustatory receptor 5 [Anomala corpulenta]  4e−157  68   GR3  134  No  ALR72527.1 gustatory receptor 1, partial [Colaphellus bowringi]  2e−29  44   GR4  79  No  XP_023310039.1 putative gustatory receptor 2a [Anoplophora glabripennis]  5e−19  51   GR5  113  No  AUF73059.1 gustatory receptor, partial [Anoplophora chinensis]  7e−33  57  Sensory neuron membrane protein (SNMP)   SNMP1a  521  Yes  ALR72542.1 sensory neuron membrane protein SNMP1a [Colaphellus bowringi]  0.0  65   SNMP1b  531  Yes  ALR72543.1 sensory neuron membrane protein SNMP1b [Colaphellus bowringi]  0.0  59   SNMP2a  520  Yes  XP_018566911.1 sensory neuron membrane protein 2 [Anoplophora glabripennis]  0.0  63   SNMP2b  505  Yes  XP_018566912.1 sensory neuron membrane protein 2-like [Anoplophora glabripennis]  0.0  67  View Large Fig. 2. View largeDownload slide Candidate gustatory receptor gene family in X. quadripes. (A) Maximum-likelihood tree of GRs from Coleoptera and D. melanogaster. CO2, fructose, and sugar receptors are highlighted. Bootstrap values with <0.80 are shown in circles. Achi, A. chinensis; Agla, A. glabripennis; Dmel, D. melanogaster and Tcas, T. castaneum. (B) Expression profile of three X. quadripes GRs. Information on the quality and quantity of the reference gene and tissue abbreviations is shown in Fig. 1. Fig. 2. View largeDownload slide Candidate gustatory receptor gene family in X. quadripes. (A) Maximum-likelihood tree of GRs from Coleoptera and D. melanogaster. CO2, fructose, and sugar receptors are highlighted. Bootstrap values with <0.80 are shown in circles. Achi, A. chinensis; Agla, A. glabripennis; Dmel, D. melanogaster and Tcas, T. castaneum. (B) Expression profile of three X. quadripes GRs. Information on the quality and quantity of the reference gene and tissue abbreviations is shown in Fig. 1. Candidate Ionotropic Receptors We identified a total of 18 IRs and eight iGluRs from the transcriptome. The nomenclature of IRs were followed the conventions of D. melanogaster and T. castaneum based on the phylogeny and orthology. Two candidate IRs were likely to be members of the D-IRs subfamily and were named as IR1 and IR2. iGluRs were named with the Arabic numerals from 1 to 8. Six IRs (IR25a, IR41a.1, IR75q, IR75q.1, IR75s, and IR76b) and one iGluR (iGluR1) exhibited full-length ORFs. Of them, IR25a shared the longest sequence encoding 935 AAs, while IR76b showed the shortest sequence encoding 561 AAs. Sequence alignment analysis indicated that both XquaIR75q and XquaIR75q.1 exhibited a high identity (58%). A conserved coreceptor XquaIR25a had the highest identity (85%) to AglaIR25a (Table 1 and Supplementary Additional File 1). Phylogenetic analysis clustered the XquaIRs into 10 orthologous groups, consisting of IR8a, IR25a, IR21a, IR40a, IR41a, IR64a, IR68a, IR75, IR76b, and IR93a. Two other IRs of IR1 and IR2 were grouped into the D-IRs subfamily of T. castaneum and D. melanogaster. As expected, two co-receptors of IR8a and IR25a were close to the iGluR family in phylogeny. Three A-IRs lineage-expansions in X. quadripes were observed with two copies in IR64a, three in IR41a, and four in IR75 (Fig. 3A). Fig. 3. View largeDownload slide View largeDownload slide Candidate ionotropic receptor gene family in X. quadripes. (A) Maximum-likelihood tree of IRs and iGluRs from Coleoptera and D. melanogaster. Based on the phylogeny and sequence orthology of IRs, various groups are divided with highlighted clades of co-receptors and lineage-expansions. Newly identified XquaiGluRs and IRs are labeled with red. Bootstrap values with <0.80 are shown in circles. Other iGluR subfamilies are used as the outgroup to root the tree. Blon, B. longissima; Dpon, D. ponderosae; Ityp, I. typographus; Tcas, T. castaneum; Xqua, X. quadripes and Dmel, D. melanogaster. (B) Expression profile of eight X. quadripes iGluRs. (C) Expression profile of 15 X. quadripes IRs. Information on the quality and quantity of the reference gene and tissue abbreviations is shown in Fig. 1. Fig. 3. View largeDownload slide View largeDownload slide Candidate ionotropic receptor gene family in X. quadripes. (A) Maximum-likelihood tree of IRs and iGluRs from Coleoptera and D. melanogaster. Based on the phylogeny and sequence orthology of IRs, various groups are divided with highlighted clades of co-receptors and lineage-expansions. Newly identified XquaiGluRs and IRs are labeled with red. Bootstrap values with <0.80 are shown in circles. Other iGluR subfamilies are used as the outgroup to root the tree. Blon, B. longissima; Dpon, D. ponderosae; Ityp, I. typographus; Tcas, T. castaneum; Xqua, X. quadripes and Dmel, D. melanogaster. (B) Expression profile of eight X. quadripes iGluRs. (C) Expression profile of 15 X. quadripes IRs. Information on the quality and quantity of the reference gene and tissue abbreviations is shown in Fig. 1. In the expression analysis of iGluRs, five genes (iGluR4-8) were present in all tissues and the remaining three iGluRs (iGluR1-3) were detected in at least four tissue types of male and female adults (Fig. 3B). Similarly, most of IR genes including three candidate co-receptors IR8a, IR25a and IR76b were also expressed widely in all tested tissues with antennal-predominant levels. Intriguingly, some genes displayed sex-differential expression. For example, both IR1 and IR2 had female-biased expression, while IR21a and IR25a expression was more abundant in male antennae relative to that of females. Except three co-receptors, some IRs (e.g., three copies of IR41a, IR75s, and IR93a) were also present in nonchemosensory tissues such as heads without antennae, thoraxes, or abdomens (Fig. 3C). Candidate SNMPs Based on the transcriptome, we found four SNMPs, namely SNMP1a, SNMP1b, SNMP2a, and SNMP2b, based on the phylogeny and NCBI Blast results. These genes encoded similar ORF lengths varying from 505 to 531 AAs. They were full-length cDNAs and shared moderate protein identities to SNMPs of other beetles (65% and 59% identities between XquaSNMP1a/CbowSNMP1a and XquaSNMP1b/CbowSNMP1b, respectively; 63% and 67% between XquaSNMP2a/AglaSNMP2 and XquaSNMP2b/AglaSNMP2-like). However, the identities among four XquaSNMPs were relatively low, varying from 22% to 40% (mean value = 29%) (Table 1 and Supplementary Additional File 1). Based on primarily the protein sequences of coleopteran SNMPs, we constructed the maximum-likelihood tree. Four XquaSNMPs were classified into two large groups of SNMP1 and SNMP2, which were further divided into four relatively independent clades of SNMP1-I, SNMP1-II, SNMP2-I, and SNMP2-II (Fig. 4A). Expression profile analysis revealed that SNMP1a was broadly expressed in tested tissues; SNMP1b expression was significantly enriched in antennae; SNMP2a also showed a relatively broad distribution with an obvious predominance in female wings and SNMP2b transcripts were detected in all tested tissues except heads without antennae (Fig. 4B). Fig. 4. View largeDownload slide Candidate sensory neuron membrane protein gene family in X. quadripes. (A) Maximum-likelihood tree of SNMPs from Coleoptera and D. melanogaster. Newly identified XquaSNMPs are labeled with red. Bootstrap values with <0.80 are shown in circles. In Coleoptera, four SNMP groups are distinguished according to the phylogenetic analysis. In the CD36 group, EMP-B, Epithelial Membrane Protein, isoform B; Crq-A, Croquemort isoform A and Pes, Peste. Achi, A. chinensis; Agla, A. glabripennis; Acor, Anomala corpulenta; Aqua, Ambrostoma quadriimpressum; Atum, Aethina tumida; Blon, Brontispa longissima; Cbow, Colaphellus bowringi; Dhel, Dastarcus helophoroides; Dmel, D. melanogaster; Dpon, D. ponderosae; Dval, D. valens; Ityp, I. typographus; Malt, Monochamus alternatus; Paen, Pyrrhalta aenescens; Pmac, Pyrrhalta maculicollis; Pstr, Phyllotreta striolata; Tcas, T. castaneum; Tmol, Tenebrio molitor, Tyun, T. yunnanensis and Xqua, X. quadripes. (B) Expression profile of four X. quadripes SNMPs. Information on the quality and quantity of the reference gene and tissue abbreviations are shown in Fig. 1. Fig. 4. View largeDownload slide Candidate sensory neuron membrane protein gene family in X. quadripes. (A) Maximum-likelihood tree of SNMPs from Coleoptera and D. melanogaster. Newly identified XquaSNMPs are labeled with red. Bootstrap values with <0.80 are shown in circles. In Coleoptera, four SNMP groups are distinguished according to the phylogenetic analysis. In the CD36 group, EMP-B, Epithelial Membrane Protein, isoform B; Crq-A, Croquemort isoform A and Pes, Peste. Achi, A. chinensis; Agla, A. glabripennis; Acor, Anomala corpulenta; Aqua, Ambrostoma quadriimpressum; Atum, Aethina tumida; Blon, Brontispa longissima; Cbow, Colaphellus bowringi; Dhel, Dastarcus helophoroides; Dmel, D. melanogaster; Dpon, D. ponderosae; Dval, D. valens; Ityp, I. typographus; Malt, Monochamus alternatus; Paen, Pyrrhalta aenescens; Pmac, Pyrrhalta maculicollis; Pstr, Phyllotreta striolata; Tcas, T. castaneum; Tmol, Tenebrio molitor, Tyun, T. yunnanensis and Xqua, X. quadripes. (B) Expression profile of four X. quadripes SNMPs. Information on the quality and quantity of the reference gene and tissue abbreviations are shown in Fig. 1. Discussion Most of insects, including the coffee white stemborer, use external environmental chemical cues produced by plants and animals to locate and recognize hosts and partners. Our current work has identified and characterized four crucial chemosensory transmembrane protein families of ORs, GRs, IRs, and SNMPs, serving as the perception and recognition of chemical cues. As preliminary work of down-stream functional performance, this study on gene identification, phylogeny and expression profile is of particular significance for further functional studies and will allow for target experiments to identify potential molecular targets for the control of this pest. ORs primarily detect plant odorants and sex pheromones. They have been widely functioned in the orders of Diptera and Lepidoptera (Hallem and Carlson 2006, Zhang et al. 2014, de Fouchier et al. 2017), but very little is known in the cerambycid beetles. Our study identified a total of 33 OR candidates including a conserved Orco across insects (Vosshall and Hansson 2011). In comparison to three other cerambycid beetles of M. caryae (57 ORs) (Mitchell et al. 2012), A. glabripennis (131 ORs) (McKenna et al. 2016) and A. chinensis (44 ORs) (Wang et al. 2017), this coffee white stemborer has a relatively small sets of ORs, but more than nine in Monochamus alternatus (Wang et al. 2014). Meanwhile, this number is less than 341 in T. castaneum (Engsontia et al. 2008), 43 in I. typographus and 49 in D. ponderosae (Andersson et al. 2013), nearly equal to 34 in Ambrostoma quadriimpressum (Wang et al. 2016), and more than 22 in D. valens (Gu et al. 2015), 26 in Pyrrhalta aenescens, and 22 in Pyrrhalta maculicollis (Zhang et al. 2016a). The difference in gene numbers indicates that some IRs remain to be identified in X. quadripes, largely supported by results of our phylogenetic analysis as well as the fact that the identification is based solely on the transcriptome analysis rather than genomic data. For example, we did not detect the members of groups 4 and 5 in this species, but A. glabripennis ORs were present in the two groups (McKenna et al. 2016). To date, limited functional information on coleopteran ORs is available, including two conserved co-receptors Orco from D. armandi (Zhang et al. 2016b) and T. castaneum (Engsontia et al. 2008). As stimulus-specific ORs in the beetles, only three candidate pheromone receptors in M. caryae (McarOR3, OR5, and OR20) have been functionally characterized. They are respectively sensitive to (S)-2-methyl-1-butanol, 2-phenylethanol and (2S,3R)-2,3-hexanediol, sex pheromones of male beetles (Mitchell et al. 2012). Our study shows that XquaOR13, OR21, and OR17 are sisters to McarOR3, OR5, and OR20, respectively. Notably, 2-phenylethanol was also detected in male X. quadripes, and hence may be one of odorant candidates for XquaOR21. Additionally, some ketone analogs like 2S-hydroxy-3-decanone, 3-hydroxy-2-decanone and 2-hydroxy-3-octanone have been found from male X. quadripes (Hall et al. 2006). Coupled with antennal-enriched expression of three XquaORs, we suggest that they may respond to male-released compounds including sex pheromones. Expression profiling analysis reveals that most of ORs are specifically or highly expressed in antennae of both sexes, supporting their presence in the antennae and olfactory roles. Similar results have been reported in several other coleopteran species like Colaphellus bowringi (Li et al. 2015b), Anomala corpulenta (Li et al. 2015a), Holotrichia oblita (Li et al. 2017), and Phyllotreta striolata (Wu et al. 2016). In other tissues, OR expression indicates their functional diversities associated with gustatory and nonchemosensory functions (Kang and Koo 2012). Based on the analyses of sequence alignments and phylogenetic tree, 16 conserved XquaIRs show orthology to IRs of other coleopteran species and D. melanogaster, and thus are classified into the A-IRs subfamily (Croset et al. 2010). Notably, three orthologous groups of IR41a, IR64a, and IR75 generally possess multiple copies in X. quadripes, other Coleoptera and Diptera (Rytz et al. 2013). T. castaneum has two copies of IR41a, three of IR64a, and three of IR75 (Croset et al. 2010). One of IR41a and eight of IR75 are identified in A. glabripennis, but no IR64a ortholog is found (McKenna et al. 2016). One, one, and four copies are present in IR41a, IR64a, and IR75 of D. melanogaster, respectively (Benton et al. 2009). Fifteen, one, and eleven of IR41a, IR64a, and IR75 are detected in A. aegypti, respectively (Croset et al. 2010, Rytz et al. 2013). Of the 18 XquaIRs identified, two IRs (IR1 and IR2) show no clear orthology to any receptors in coleopteran species or D. melanogaster, possibly as candidates for the species-specific D-IRs subfamily. In the analysis of expression profile, three broadly expressed co-receptors (IR8a, IR25a, and IR76b) provide evidence that at least a coreceptor IR is required for stimulus-specific IR functions (Abuin et al. 2011, Silbering et al. 2011). Of notice, XquaIR25a expression appears to be male-biased in antennae, consistent with EaffIR25a expression in the copepod Eurytemora affinis (Eyun et al. 2017) but in contrast to female-biased expression of H. oblita HoblIR25a (Li et al. 2017). The two other co-receptors (IR8a and IR76b) in X. quadripes display no differential expression between the sexes, similar to Drosophila species (Shiao et al. 2015), T. castaneum (Dippel et al. 2016), and H. melpomene (van Schooten et al. 2016). The remaining 12 IRs exhibit additional expression except the antennae, indicating their functional diversities in this beetle associated with olfactory, gustatory, and nonchemosensory functions. In comparison to other coleopteran species (220 GRs in T. castaneum and 234 in A. glabripennis) (Tribolium Genome Sequencing Consortium 2008, McKenna et al. 2016), our current study identified only five GRs from the antennal transcriptome of X. quadripes. This is possibly related to low or no expression of GRs in antennae. As indicated by previous studies of antennal transcriptome analyses from coleopteran and noncoleopteran species, GRs are typically expressed at low levels in the antennae (Liu et al. 2014, 2015; Wang et al. 2014; Li et al. 2015b; Hu et al. 2016). In T. castaneum, six copies of SNMPs have been identified with four SNMP1s, one SNMP2, and one SNMP3 (Dippel et al. 2016). Apart from that, other coleopteran species possess one to four SNMPs including two in A. glabripennis (Hu et al. 2016), three in A. chinensis (Wang et al. 2017), and one in M. alternatus (Wang et al. 2014). Four SNMPs in X. quadripes were identified and clustered in four groups, together with previous studies (Nichols and Vogt 2008, Vogt et al. 2009), possibly representing a complete set in this species. Of these four XquaSNMPs, SNMP1b has more specific expression in antennae possibly involved in olfaction. As expected, its orthologous SNMP1 has been demonstrated to be essential for the sensing of sex pheromones in Diptera (D. melanogaster) and Lepidoptera (H. virescens) (Benton et al. 2007, Jin et al. 2008, Pregitzer et al. 2014). Considering the fact that XquaSNMP1b has a close relationship with DmelSNMP1 in phylogeny, it is inferred that this gene may have a similar function linked to the detection of sex pheromones. Adults of the cerambycid beetles have important life activities involved in chemosensory behaviors such as mate recognition and host searching. The information on the identification and characterization of chemosensory-associated genes is of particular significance for the control of this pest. In this study, we have identified 60 novel candidates for the sensing of semiochemicals through the antennal transcriptome. 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Environmental EntomologyOxford University Press

Published: May 30, 2018

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