Identification of Chemosensory Genes Based on the Antennal Transcriptomic Analysis of Plagiodera versicolora

Simple Summary In this study, we conducted a transcriptome analysis of adult antennae in Plagiodera versicolora (Coleoptera: Chrysomelidae) and identified a total of 98 candidate chemosensory genes, encoding 40 odorant receptors (ORs), 7 ionotropic receptors (IRs), 13 gustatory receptors (GRs), 10 chemosensory proteins (CSPs), 24 odorant binding proteins (OBPs), and 4 sensory neuron membrane proteins (SNMPs). The tissue expression profiles showed that almost all PverOBPs and PverORs were highly expressed in the antennae. In addition, the results revealed that PverOBP10, PverOBP12, PverOBP18, PverOR24, and PverOR35 showed female-biased expression profiles, indicating that these receptors may be involved in some female-specific behaviors such as oviposition site seeking. This work greatly promotes the understanding of the olfactory system and will help provide insight for functional studies of the chemoreception mechanism in P. versicolora. Abstract Insects can sense surrounding chemical signals by their accurate chemosensory systems. This system plays a vital role in the life history of insects. Several gene families participate in chemosensory processes, including odorant receptors (ORs), ionotropic receptors (IRs), gustatory receptors (GRs), chemosensory proteins (CSPs), odorant binding proteins (OBPs), and sensory neuron membrane proteins (SNMPs). Plagiodera versicolora (Coleoptera: Chrysomelidae), is a leaf-eating forest pest found in salicaceous trees worldwide. In this study, a transcriptome analysis of male and female adult antennae in P. versicolora individuals was conducted, which identified a total of 98 candidate chemosensory genes including 40 ORs, 7 IRs, 13 GRs, 10 CSPs, 24 OBPs, and 4 SNMPs. Subsequently, the tissue expression profiles of 15 P. versicolora OBPs (PverOBPs) and 39 ORs (PverORs) were conducted by quantitative real-time PCR. The data showed that almost all PverOBPs and PverORs were highly expressed in the male and female antennae. In addition, several OBPs and ORs (PverOBP10, PverOBP12, PverOBP18, PverOR24, and PverOR35) had higher expression levels in female antennae than those in the male antennae, indicating that these genes may be taking part in some female-specific behaviors, such as find mates, oviposition site, etc. This study deeply promotes further understanding of the chemosensory system and functional studies of the chemoreception genes in P. versicolora.


Quantitative Real-Time PCR (RT-qPCR) Analysis
RT-qPCR was conducted to determine the expression profiles of male and female insects. The cDNAs were synthesized with HiScript ® III RT SuperMix for RT-qPCR (+gDNA wiper) (Vazyme, Nanjing, China), based on the manufacturer's instructions. The specific primers were designed and are listed in Table S1. RT-qPCR was conducted on the CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA) with a ChamQTM Universal SYBR ® RT-qPCR Master Mix (Vazyme, Nanjing, China), following the manufacturer's instructions. Reaction programs were set at 95 • C for 30 s, followed by 40 cycles of 95 • C for 5 s and 60 • C for 34 s. The RPS18 gene [53] was used as a reference to normalize the relative expression levels of OR and OBP genes. For each gene, three biological replicates were conducted. Gene expression levels were analyzed using the 2 −∆∆CT method [54]. The one-way analysis of variance (ANOVA) followed by the Tukey's HSD test was used to test gene expression using SPSS 26.0 software (SPSS Inc., Chicago, IL, USA).

Overview of the Sequence Assembly
The next-generation sequencing of the cDNA library, using the Illumina Novaseq platform, was constructed from the male and female adult antennae of P. versicolora. In total, 59,893,741 clean reads were obtained with a Q20 percentage of 97.83%. About 24,862 unigenes, with a total length of 34,555,981 and an N50 length of 2675 bp, were identified. Statistics showed that 63.1% of the 15,687 unigenes were greater than 500 bp in length ( Figure 1). In total, 11,925 unigenes were matched to entries in the NCBI non-redundant (NR) protein database (http://www.ncbi.nlm.nih.gov/protein (accessed on 10 April 2021) by a BLASTX search.
Statistics showed that 63.1% of the 15,687 unigenes were greater than 500 bp in length ( Figure 1). In total, 11,925 unigenes were matched to entries in the NCBI non-redundant (NR) protein database (http://www.ncbi.nlm.nih.gov/protein (accessed on 10 April 2021) by a BLASTX search.

Overview of Gene Ontology (GO) Annotation
The transcripts were classified into different functional categories based on their GO annotation. Overall, these unigenes could be placed into three functional categories: cellular components (11,847), biological processes (16,293), and molecular function (7150) (Figure 2). In the class of molecular function, the genes expressed in the antennae were mostly related to binding (2966), catalytic (2887), and transporter activities (406), indicating that some unigenes in these sub-categories might have a connection with chemosensory behavior in insects.

Overview of Gene Ontology (GO) Annotation
The transcripts were classified into different functional categories based on their GO annotation. Overall, these unigenes could be placed into three functional categories: cellular components (11,847), biological processes (16,293), and molecular function (7150) (Figure 2). In the class of molecular function, the genes expressed in the antennae were mostly related to binding (2966), catalytic (2887), and transporter activities (406), indicating that some unigenes in these sub-categories might have a connection with chemosensory behavior in insects.
Statistics showed that 63.1% of the 15,687 unigenes were greater than 500 bp in length ( Figure 1). In total, 11,925 unigenes were matched to entries in the NCBI non-redundant (NR) protein database (http://www.ncbi.nlm.nih.gov/protein (accessed on 10 April 2021) by a BLASTX search.

Overview of Gene Ontology (GO) Annotation
The transcripts were classified into different functional categories based on their GO annotation. Overall, these unigenes could be placed into three functional categories: cellular components (11,847), biological processes (16,293), and molecular function (7150) ( Figure 2). In the class of molecular function, the genes expressed in the antennae were mostly related to binding (2966), catalytic (2887), and transporter activities (406), indicating that some unigenes in these sub-categories might have a connection with chemosensory behavior in insects.

Identification of the Candidate Chemosensory Genes
Based on similarity analyses of the sequences tested, a total of 98 candidate chemosensory genes from the male and female antennae transcriptomes of P. versicolora were identified. These included 40 ORs, 7 IRs, 13 GRs, 10 CSPs, 24 OBPs, and 4 SNMPs (Tables S2  and S3). When compared with insects of Coleoptera, where the chemosensory genes had been identified by transcriptome tests, the number of chemosensory genes identified in this study was similar to those found in C. bowringi (104 chemosensory genes), D. ponderosae (111 chemosensory genes), and was higher than that of M. alternatus (52 chemosensory genes).

OBPs
We obtained 24 unigenes encoding candidate OBPs in P. versicolora (PverOBPs), which is less than that observed in M. alternatus (29) and C. bowringi (26), but more than that observed in D. helophoroides (23). Sequence analysis showed that 23 OBPs have complete ORFs and encoded 125 to 226 amino acids, but only three OBPs have no signal peptide sequences (Table S2). The result of the phylogenetic tree showed that PverOBP4 and PverOBP12 were clustered with the functionally characterized MaltOBP13 and MaltOBP10, respectively. In addition, several PverOBPs (OBP18, 10, 14, 16, 19, 7, 2, and 4) were clustered with CbowOBPs (OBP25, 26, 12, 3, 6, 5, 7, and 20, respectively) ( Figure 3). The tissue expression profiles revealed that three PverOBPs (PverOBP10, 12 and 18) had a higher expression level in female antennae than male antennae. Among these PverOBPs, except for PverOBP15, the remaining 14 candidate genes were specifically expressed in the antennae with low or no expression level in the body ( Figure 4).

Identification of the Candidate Chemosensory Genes
Based on similarity analyses of the sequences tested, a total of 98 cand chemosensory genes from the male and female antennae transcriptomes of P. versic were identified. These included 40 ORs, 7 IRs, 13 GRs, 10 CSPs, 24 OBPs, and 4 SN (Tables S2 and S3). When compared with insects of Coleoptera, where the chemosen genes had been identified by transcriptome tests, the number of chemosensory g identified in this study was similar to those found in C. bowringi (104 chemosen genes), D. ponderosae (111 chemosensory genes), and was higher than that of M. alter (52 chemosensory genes).

OBPs
We obtained 24 unigenes encoding candidate OBPs in P. versicolora (PverO which is less than that observed in M. alternatus (29) and C. bowringi (26), but more that observed in D. helophoroides (23). Sequence analysis showed that 23 OBPs have plete ORFs and encoded 125 to 226 amino acids, but only three OBPs have no signal tide sequences (Table S2). The result of the phylogenetic tree showed that PverOBP4 PverOBP12 were clustered with the functionally characterized MaltOBP13 MaltOBP10, respectively. In addition, several PverOBPs (OBP18, 10, 14, 16, 19, 7, 2, a were clustered with CbowOBPs (OBP25, 26, 12, 3, 6, 5, 7, and 20, respectively) (Figu The tissue expression profiles revealed that three PverOBPs (PverOBP10, 12 and 18 a higher expression level in female antennae than male antennae. Among these PverO except for PverOBP15, the remaining 14 candidate genes were specifically express the antennae with low or no expression level in the body ( Figure 4).

ORs
Forty different unigenes for candidate ORs were identified in P. versicolora (PverOR), among which 35 ORs contained a complete ORFs that encoded 372 to 479 amino acids (Table S3). The phylogenetic analysis showed that a PverOR gene displayed a high homology with the conserved Orco gene family in other three insects (C. bowringi, M. alternatus, and A. chinensis), which was designated as PverOrco. The results show that ORs were separated into five subfamilies, those being 1-3, 7a and 7b. We found that three PverORs (PverOR6, 10 and 32) and an McarOR20 that have been functionally characterized were clustered within a subgroup. Additionally, PverOR24 was clustered with CbowOR17 and AchiOR32 in the tree ( Figure 6). Among these PverORs, except for PverOR27 (which had a similar expression level between the antennae and bodies), the remaining candidate genes were specifically expressed at higher levels in the antennae than in the bodies. The results of RT-qPCR showed that PverOR24 and PverOR35 were highly expressed in female antennae (Figure 7). constructed using MEGA6 with the Neighbor-joining method.

ORs
Forty different unigenes for candidate ORs were identified in P. versicolora (PverOR), among which 35 ORs contained a complete ORFs that encoded 372 to 479 amino acids (Table S3). The phylogenetic analysis showed that a PverOR gene displayed a high homology with the conserved Orco gene family in other three insects (C. bowringi, M. alternatus, and A. chinensis), which was designated as PverOrco. The results show that ORs were separated into five subfamilies, those being 1-3, 7a and 7b. We found that three PverORs (PverOR6, 10 and 32) and an McarOR20 that have been functionally characterized were clustered within a subgroup. Additionally, PverOR24 was clustered with CbowOR17 and AchiOR32 in the tree ( Figure 6). Among these PverORs, except for PverOR27 (which had a similar expression level between the antennae and bodies), the remaining candidate genes were specifically expressed at higher levels in the antennae than in the bodies. The results of RT-qPCR showed that PverOR24 and PverOR35 were highly expressed in female antennae (Figure 7).

GRs
Bioinformatic analysis identified 13 candidate GRs in the P. versicolora (PverGRs) antennal transcriptome, seven of which have full-length ORFs (Table S3). GR sequences in P. versicolora and other insects were used for the phylogenetic analysis. The tree showed that PverGR1 was clustered in the CO2 receptors subfamily, two PverGRs (GR3 and GR10) were clustered together with the sugar receptor (including trehalose, glucose, sucrose, etc., expect for fructose) subfamily, and PverGR9 and PverGR12 were clustered together with the fructose receptor subgroup (Figure 8).

GRs
Bioinformatic analysis identified 13 candidate GRs in the P. versicolora (PverGRs) antennal transcriptome, seven of which have full-length ORFs (Table S3). GR sequences in P. versicolora and other insects were used for the phylogenetic analysis. The tree showed that PverGR1 was clustered in the CO 2 receptors subfamily, two PverGRs (GR3 and GR10) were clustered together with the sugar receptor (including trehalose, glucose, sucrose, etc., expect for fructose) subfamily, and PverGR9 and PverGR12 were clustered together with the fructose receptor subgroup (Figure 8). Insects 2022, 12, x FOR PEER REVIEW 9 of 15

IRs
Seven IR genes were identified in P. versicolora from the male and female antennal transcriptomes. Only four of these IRs had a full-length ORF (PverIR2, PverIR4, PverIR5, and PverIR6) that encoded 639 to 877 amino acids (Table S3). The phylogenetic analysis of IRs from six species of Coleopterans showed that ( Figure 9) these IRs can be divided into several different subfamilies. PverIR1 (named PverIR75q) clustered with CbowIR75q, DponIR75q, and TcasIR75q, suggesting it is part of the IR75q group. The results show that PverIR4 (named PverIR8a.1) and PverIR7 (named PverIR8a.2) were classified into IR8a coreceptor subgroup (Figure 9).

IRs
Seven IR genes were identified in P. versicolora from the male and female antennal transcriptomes. Only four of these IRs had a full-length ORF (PverIR2, PverIR4, PverIR5, and PverIR6) that encoded 639 to 877 amino acids (Table S3). The phylogenetic analysis of IRs from six species of Coleopterans showed that ( Figure 9) these IRs can be divided into several different subfamilies. PverIR1 (named PverIR75q) clustered with CbowIR75q, DponIR75q, and TcasIR75q, suggesting it is part of the IR75q group. The results show that PverIR4 (named PverIR8a.1) and PverIR7 (named PverIR8a.2) were classified into IR8a coreceptor subgroup (Figure 9).

SNMPs
Four SNMP genes with complete ORFs were obtained from the male and female antennal transcriptomes of P. versicolora (Table S3). This number is similar to that observed in C. bowringi but is higher than in other insects used in the phylogenetic tree. The results also show that four PverSNMP genes were clustered into the Coleoptera SNMP1 group (SNMP1a and SNMP1b subgroup) and SNMP2 group (SNMP2a and SNMP2b subgroup) ( Figure 10). Insects generally have two representatives of SNMPs (SNMP1 and SNMP2), although the copy numbers of each lineal orthologue seems to differ between species [55].

SNMPs
Four SNMP genes with complete ORFs were obtained from the male and female antennal transcriptomes of P. versicolora (Table S3). This number is similar to that observed in C. bowringi but is higher than in other insects used in the phylogenetic tree. The results also show that four PverSNMP genes were clustered into the Coleoptera SNMP1 group (SNMP1a and SNMP1b subgroup) and SNMP2 group (SNMP2a and SNMP2b subgroup) ( Figure 10). Insects generally have two representatives of SNMPs (SNMP1 and SNMP2), although the copy numbers of each lineal orthologue seems to differ between species [55].

Discussion
Although Coleoptera is the largest insect order when compared to Dipterans and Lepidopterans, there has been little research into the molecular mechanism of chemoreception. In the present study, the antennal transcriptome of a Coleoptera beetle, P. versi- Figure 10. Phylogenetic tree of insect SNMP. The P. versicolora genes are shown in blue. The tree was constructed using MEGA6 with the Neighbor-joining method.

Discussion
Although Coleoptera is the largest insect order when compared to Dipterans and Lepidopterans, there has been little research into the molecular mechanism of chemoreception. In the present study, the antennal transcriptome of a Coleoptera beetle, P. versicolora, was sequenced and analyzed. A total of 24,862 unigenes were identified and 76.5% of them were over 300 bp in length, suggesting the high depth and quality of the transcriptome. Busco analysis was conducted to evaluate the completeness of the transcriptome (File S2). Based on the transcriptome analysis, we identified 98 chemosensory genes in P. versicolora (Tables S2 and S3), and the phylogenetic trees were constructed with other insect chemosensory sequences (Figures 3, 5, 6 and 8-10). In addition, the spatial expression patterns of OBPs and ORs have been assessed through RT-qPCR analysis (Figures 4 and 7).
OBP-binding odorant molecules consist of the first step in the olfactory process. The number of PverOBPs (24) observed is similar to those found in C. bowringi (26) and D. helophoroides (23), and less than that of M. alternatus (29). The phylogenetic analysis showed that PverOBP4, 6 and 12 were clustered together with functionally characterized Dh-elOBP13, 21 and 10, respectively. The results indicated that the three PverOBPs may have same function as in D. helophoroides. In the present study, the majority of PverOBPs (14 out of 15 OBPs) were highly expressed in antennae, as shown by the RT-qPCR test, which is consistent with the expression profile of genes in other insects, such as Dioryctria abietella [56] and Galleria mellonella [57]. In addition, PverOBP10, 12 and 18 exhibited highly abundant expression levels in female antennae (Figure 4), suggesting that these OBPs may play an important role in antennal recognition processes, though further verification is needed. PverOBP15 was highly expressed in the bodies when compared to other OBPs (Figure 4), which may be involved in delivering and detecting some specific semiochemicals. In line with our results, several other studies showed that OBPs are differentially expressed in the body; for example, AtumOBP5, AtumOBP17, and AtumOBP21 have a higher expression in the forelegs [58].
PverGR1 was clustered in the CO 2 receptors subfamily, PverGR9 and PverGR12 in the fructose receptors subgroup, and PverGR3 and PverGR10 in the sugar receptors subfamily (Figure 8), indicating that these GRs might be taking part in the detection of CO 2 , sugar, and fructose [45,47,59]. Other GRs that do not belong to these three categories might be involved in other taste perception processes. Previous studies showed that an insect usually has three CO 2 receptors (these are allocated into three different groups: one, two, and three), e.g., AaegGr1-3 were reported in A. aegypti [51], AgamGr22-24 in A. gambiae [52], and HarmGr1-3 in H. armigera [42]. However, only one CO 2 receptor gene (PverGR1) was identified in P. versicolora, which belongs to the GR1 subfamily. Considering that gustatory sensilla are mainly distributed in the mouthparts (proboscises, labial palps), antennae, wings, legs, and ovipositor [60,61], we may identify more PverGR genes from the transcriptome of other tissues in the future.
The number of PverORs (40) is greater than that of harmonia axyridis (26) [62] and less than that of Rhynchophorus palmarum (63) [63]. In the OR phylogenetic tree, PverOR6, PverOR10, and PverOR32 were clustered into the same subgroup, with a functionally characterized pheromone receptor, McarOR20, the receptor of (2S,3R)-2,3-hexanediol and 3-hydroxyhexan-2-one in M. caryae [40]. The results indicate that these PverORs may be associated with the detection of the above pheromones or other active compounds. The discovery of new attractive substances would be helpful for the identification of sex pheromone compounds in P. versicolora. PverOR24 and PverOR35 genes were identified with significantly higher expression levels in female antennae than male antennae. Considering previous studies of the insect OR functions [64,65], female-biased PverORs may be involved in the detection of odors that play a critical role in female behavior, such as mating or oviposition. In addition, PverOR24 clusters with CbowOR17 and AchiOR32, as this might be relevant in the light of discovering pheromone receptors. The specific functions of these PverORs need to be explored in the future.