Antennae-enriched expression of candidate odorant degrading enzyme genes in the turnip aphid, Lipaphis erysimi

Aphids heavily rely on their olfactory system for foraging behavior. Odorant-degrading enzymes (ODEs) are essential in preserving the olfactory acuity of aphids by removing redundant odorants in the antennae. Certain enzymes within this group stand out as being enriched and/or biased expressed in the antennae, such as carboxylesterases (CXEs), cytochrome P450 (CYPs), glutathione S-transferases (GSTs), and UDP-glycosyltransferases (UGTs). Here, we performed a comparative transcriptome analysis of antennae and body tissue to isolate the antennal ODE genes of turnip aphid Lipaphis erysimi. A dataset of one CXE, seven CYPs, two GSTs, and five UGTs enriched in the antennae was identified and subjected to sequence analysis. Furthermore, qRT-PCR analyses showed that 13 ODE genes (LeCXE6, LeCYP4c1, LeCYP6a2, LeCYP6a13, LeCYP6a14.2, LeCYP6k1, LeCYP18a1, LeGST1, LeUGT1-7, LeUGT2B7, LeUGT2B13, LeUGT2C1.1, and LeUGT2C1.2) were specifically or significantly elevated in antennal tissues. Among these antennae-enriched ODEs, LeCYP4c1, LeCYP6a2, LeCYP6a13, LeCYP6a14.2, LeCYP18a1, LeUGT2B7, and LeUGT2B13 were found to exhibit significantly higher expression levels in alate aphids compared to apterous and nymph aphids, suggesting their putative role in detecting new host plant location. The results presented in this study highlight the identification and expression of ODE genes in L. erysimi, paving the path to investigate their functional role in odorant degradation during the olfactory processes.


Introduction
Insect antennae are intricate sensory organs essential in detecting a variety of lipophilic volatiles from the environment, helping insects secure food, find mates, lay eggs, and steer clear of potential predators (Leal, 2013;Cheema et al., 2021). During these biologic processes, the exogenous odor molecules are initially bound with insect odorant-binding proteins (OBPs) and chemosensory proteins (CSPs); they then move through the sensillum lymph and interact with olfactory receptors (ORs) situated on the membrane surface of olfactory sensory neurons. The ORs convert the chemical signals from the odor molecules into electrophysiological signals, which can be OPEN ACCESS EDITED BY deciphered by the brain (Leal, 2013;Pelosi et al., 2018;Cheema et al., 2021;Zhou and Jander, 2022). Subsequently, antennal enzymes called odorant-degrading enzymes (ODEs) present in the vicinity of ORs become critical for the rapid degradation of odorant molecules, allowing for the insect's olfactory system to recover and maintain its sensitivity (Younus et al., 2014;Blomquist et al., 2021;Chertemps and Maïbèche, 2021).
The turnip aphid, Lipaphis erysimi Kaltenbach, poses a significant threat to the cultivation of Brassica vegetables and oilseed crops due to its direct feeding and/or transmission of harmful plant viruses. RNA interference (RNAi) has emerged as a promising strategy for controlling aphids, and antennal ODEs hold great promise as the optimal target genes for disrupting foraging behaviors (Yu et al., 2014;Yu et al., 2016;Wei et al., 2021;Wu et al., 2022;Ma et al., 2023). In this study, we aimed to identify antennae-enriched ODE genes of this aphid species by conducting as follows: 1) performing comparative analysis on the antennal and body transcriptomes of L. erysimi; 2) isolating and in silico analysis of candidate ODE genes; 3) identifying the expression profile of the ODE genes among different tissues and developmental stages.

Insect rearing
The colony of L. erysimi utilized in this study was established in 2020, based on the field populations from Xinfeng in the Jiangxi province of China (Kuang et al., 2023). The population was continuously maintained on Chinese cabbage Shanghaiqing (Brassica rapa var. chinensis) without exposure to any insecticides under controlled conditions (27°C-28°C, 60%-65% RH, 14:10 L:D photoperiod).

Sample preparation, RNA extraction, and cDNA synthesis
Samples of L. erysimi were collected at five different stages, which include 1st instar nymph, 2nd instar nymph, 3rd instar nymph, 4th instar nymph, 1-day apterous and alate adults. The apterous aphids anesthetized on ice were dissected under a stereomicroscope to collect various tissues of L. erysimi, such as antenna, head, leg, and cuticle. Total RNA extraction was performed according to the manufacturer's protocol of Trizol reagent (Sigma, St. Louis, MO, United States). The purity and concentration of RNAs were determined using a NanoDrop One C spectrophotometer from Thermo Fisher Scientific (Waltham, MA, United States). The first-strand cDNA synthesis was synthesized with 500 ng of purified RNA, utilizing the highly effective EasyScript ® One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech, Beijing, China). The resulting cDNA was properly stored at −20°C until ready to be used.

Comparative transcriptome analysis
To isolate the antennal-biased genes in L. erysimi, the highthroughput transcriptome data sets were retrieved from our former study (GenBank accession number PRJNA947784), which included conducting Illumina sequencing on the antennae and bodies (excluding antennae) of adult apterous aphids, transcriptome de novo assembly, as well as functional annotation of the unigenes (Kuang et al., 2023). The differential gene expression analysis was carried out using the antennal and body transcriptome data as described by Yu et al. (2023). Briefly, the transcript abundances were determined by RSEM (version 1.2.12); DESeq2 (version 1.4.5) was utilized to identify differentially expressed genes (DEGs) between samples, and a gene was considered differentially expressed if the corrected p-value was ≤ 0.05.

Identification of candidate ODE genes
The antennae-biased ODE genes with FPKM ≥10 were selected from the gene repertories obtained through comparative transcriptome analysis. Candidate ODEs were confirmed using the BLASTX algorithm, and their open reading frames (ORFs) were predicted using the ORF finder tool (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The amino acid components, theoretical isoelectric points (pIs), and molecular weights (MWs) of ODE genes were calculated using

Quantitative real-time PCR analysis
The quantitative real-time PCR (qRT-PCR) reactions were conducted in a 20 μL volume that comprised of 4 μL of diluted cDNA, 0.4 μM of each primer, and 10 μL of PerfectStart ® Green qPCR SuperMix (TransGen Biotech, Beijing, China). Reactions were performed on a Roche LightCycler 96 ® system (Roche Diagnostics, Mannheim, Germany) with the following thermal program: initial denaturation for 10 min at 95°C, followed by a 40-cycle two-step amplification profile of 95°C for 5 s and 60°C for 30 s. Two reference genes, actin (GenBank accession number OQ626608) and GAPDH (GenBank accession number OQ626609), were employed to standardize the quantity of cDNA added to the PCR reactions. The relative expression of each ODE gene was analyzed using the 2 −ΔΔCt method (Livak and Schmittgen, 2001). Reactions were performed in triplicate, and the gene-specific primers are listed in Table 1.

Statistical analysis
The statistical differences of ODE gene expression levels among different developmental stages and tissues were analyzed using analysis of variance (ANOVA), followed by Tukey multiple comparison test. The statistical analysis was conducted using GRAPHPAD PRISM software (version 6.0; GraphPad Software Inc., La Jolla, CA, United States) and a

FIGURE 1
The differentially expressed genes (DEGs) between Lipaphis erysimi antennal and body transcriptomes. Red dots indicated the unigenes upregulated in antennae; blue dots indicated the downregulated unigenes in antennae by comparing with body samples.

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frontiersin.org 04 p-value of ≤ 0.05 was set as the threshold for statistical significance.

Differentially expressed genes analysis
Comparative analyses of the antennal and body transcriptomes in this study provide useful information to identify the antennae-abundant and/or antennae-biased genes. A total of 8,932 differentially expressed genes (DEGs) with a Q value ≤ 0.05 were identified, and 4,797 DEGs were significantly upregulated in the antennae (Figure 1). Among the antennae-abundant DEGs, one CXE, seven CYPs, two GSTs, and five UGTs were identified by blasting against the Nr database. The candidate ODEs were designated according to gene names of the top blast hits in NCBI. Detailed information on these ODE enzymes is shown in Table 2.

Identification of putative CXE genes
One putative CXE gene, LeCXE6, showed a high level of expression in the antennae, with FPKM values over 10 times higher than in the rest of the body. LeCXE6 encodes a 564 amino-acid protein, with a signal sequence cleavage site predicted between Gly-18 and Phe-19 at the N-terminus. The predicted protein has a theoretical molecular mass of 62.73 kDa and an isoelectric point of 5.57, as determined using ProtParam tool in Expasy server (Table 2). Conserved domain and sequence alignment analysis revealed that LeCXE6 contained the typical motif of carboxylesterase family, including a conserved pentapeptide (Gly-X-Ser-X-Gly) and an oxyanion hole (Gly-Gly-Ala; Supplementary Figure S1). Phylogenetic analysis showed that LeCXE6 fell into the beta-esterase clade and was closely clustered with the well-studied odorant-degrading enzymes, PjapPDE and ApolPDE (Figure 2).

Identification of putative CYP genes
Seven DEGs abundant in L. erysimi antennae were identified to be CYPs by blasting against the Nr database. All candidate LeCYPs were found to contain full-length ORFs without any predicted signal peptide sequences. Their antennal RPKM values ranged from 29.83 to 1,220.17, representing more than a ten-fold increase in comparison to the body group. Among them, LeCYP6a13 was the most abundant CYP gene in antennae with a value exceeding 1,200, followed by LeCYP18a1 and LeCYP6a2 (Table 2). Phylogenetic analysis showed that the selected CYPs were well categorized into four subclasses, namely, CYP2, CYP3, CYP4, and mitochondrial CYP. The CYP3 class comprised of five antennal LeCYPs, including LeCYP6a13, LeCYP6a2, LeCYP6k1, LeCYP6a14.1, and LeCYP6a14.2. LeCYP18a1 was classified as a member of the CYP2 clan, while LeCYP4c1 was categorized into the CYP4 clade ( Figure 3).

Identification of putative GST genes
Two DEGs abundant in antennae were identified to be GSTs. Both LeGST1 and LeGST transcripts had full-length ORFs, encoding proteins that are 157 and 198 amino acids, respectively. It is noteworthy that LeGST1 showed an expression pattern that was particularly abundant in antennae, with an FPKM value of 93.81 that exceeded 18-fold higher than in the body (Table 2). Conserved domain analysis showed LeGST1 had a MAPEG (membraneassociated proteins in eicosanoid and glutathione metabolism) domain and was similar to the microsomal GST1, while LeGST had one GSH binding site (G-site) in the N-terminus and one hydrophobic substrate binding pocket (H-site) in the C-terminal region (Supplementary Figure S2). The phylogenetic analysis revealed that eight subclasses, namely, Microsomal-, Delta-, Epsilon-, Omega-, Sigma-, Theta-, Zeta-, and the unclassified-GST, were well clustered in their respective phylogenetic
Frontiers in Physiology frontiersin.org branches. LeGST1 was classified under the Microsomal-GST subclass, and LeGST was classified as a member of Sigma-GST (Figure 4).

Identification of putative UGT genes
A total of five antennal LeUGT genes were identified in the utilized transcript set. All LeUGT transcripts had full-length ORFs, encoding proteins ranging from 513 to 542 amino acids. Signal peptides were predicted in all candidate LeUGTs, with the exception of LeUGT2B13. FPKM analysis showed that LeUGT2B7 was the most antennaeabundant UGT with a value of 404.61, which was >20-fold higher than in the body (Table 1) Figure S3). Phylogenetic analysis showed that the candidate LeUGTs were grouped into three distinct subclades, with each subclade including several homologs from other aphid species. Specifically, LeUGT2B7, LeUGT2B13, and LeUGT2C1.2 were categorized into the UGT344 clade; LeUGT2C1.1 was clustered in the UGT343 subclade, and LeUGT1-7 was found to be a member of the UGT351 subclade ( Figure 5).

Developmental and tissue expression analysis for candidate ODE genes
The developmental and tissue expression profiles of LeCXE, LeCYP, LeGST, and LeUGT genes were analyzed using qRT-PCR. Developmental expression data showed that the candidate ODE genes were consistently detected throughout the various developmental stages of L. erysimi, spanning from the first instar nymph to the adult stage. Notably, LeCYP4c1, LeCYP6a2, LeCYP6a13, LeCYP6a14.2, LeCYP18a1, LeUGT2B7, and LeUGT2B13

FIGURE 3
Phylogenetic tree of 48 CYPs from the aphid species L. erysimi (Le, 7), A. pisum (Ap, 32), and the hemipteran insect D. citri (Dc, 7); as well as two wellstudied antennal CYPs, DponCYP345E2 and DponCYP6DE1, from Dendroctonus ponderosae. The neighbor-joining (NJ) tree was constructed using MEGA 11 with 1,000 bootstrap replicates. Seven L. erysimi CYPs are highlighted in blue. The CYP sequences used in this phylogenetic tree are provided in Supplementary Table S2.
Frontiers in Physiology frontiersin.org 07 exhibited significantly higher expression levels in alate aphids compared to apterous and nymph aphids ( Figure 6). Tissue expression analysis revealed that LeCYP6a14.1 and LeGST were highly expressed in both antenna and gut tissue, while the remaining 13 ODE genes displayed antennae-enriched expression profiles. In particular, the antennal expression levels of LeCYP6a13, LeCYP6k1, LeCYP6a14.2, LeGST1, LeUGT2B13, and LeUGT2C1.2 were >10-fold higher than in other tissues; LeCXE6, LeCYP4c1, LeCYP6a2, LeCYP18a1, LeUGT2B7, and LeUGT2C1.1 exhibited more than four times higher expression in antennae compared to non-olfactory tissues such as the head, leg, gut, and cuticle (Figure 7).

Discussion
Insects depend on their antennae to detect and process hydrophobic odorant molecules (Krieger and Breer, 1999;Leal, 2013). Discovering the ODEs within the antennae would provide crucial insights into the odorant recognition mechanism of L. erysimi, which may help us control this destructive agricultural pest more effectively. In this study, comparing the transcriptome data of the antennal and body tissues identified one CXE, seven CYPs, two GSTs, and five UGTs. The developmental and tissue expression profiles of these ODE genes were determined to reveal their implications in odorant degradation during the process of olfactory perception. To our knowledge, this is the first report documenting the identification of ODE genes in this aphid species.
The widespread occurrence of CXEs enables a tremendous decrease in the concentration of ester compounds in insects. This leads to improved sensitivity of the olfactory system and minimizes the possible toxic impact of these compounds. Several antennae abundant CXEs have been functionally studied and confirmed as ODEs, and employed for the purpose of eliminating odorants in the antennae (Ishida and Leal, 2005;Durand et al., 2010;Durand et al., 2011;He et al., 2015;Wei et al., 2021). For example, two CXEs, SlCXE7 and SlCXE10, are predominantly expressed in
Frontiers in Physiology frontiersin.org the antennal sensilla, and play a key role in the degradation of pheromones and plant volatile components in the cotton leafworm, S. littoralis (Durand et al., 2010;Durand et al., 2011). A similar study in G. molesta has uncovered four antenna-enriched CXEs play a crucial role in regulating the insect's foraging and mating behaviors. Specifically, GmolCXE1 and GmolCXE5 are responsible for hydrolyzing the acetate sex pheromone (Z/E)-8dodecenyl, while GmolCXE14 and GmolCXE21 are involved in metabolizing the ester host plant volatiles ethyl butanoate and ethyl hexanoate (Wei et al., 2021). In our study, combined transcriptome and qRT-PCR analysis revealed that LeCXE6 was highly enriched in the antennae. Further phylogenetic analysis indicated that LeCXE6 was grouped into the "beta esterases" clade along with two well-characterized pheromone-degrading enzymes, ApolPDE of A. polyphemus and PjapPDE of P. japonica (Ishida and Leal, 2005;. These findings suggest that LeCXE6 may play a significant role in clearing redundant odorants during chemosensory processing. CYPs represent an essential family of detoxification enzymes that widely occur in both vertebrates and invertebrates. Accumulating studies have shown that insect CYPs, especially those found abundantly in antennae, play a significant role as ODEs in the metabolism of host plant volatiles and sex pheromones (Chiu et al., 2019a;Chiu et al., 2019b;Chiu et al., 2019c;Wu et al., 2022). In this study, a total of seven antennae enriched LeCYP genes were identified. Our number of antennal CYP genes in L. erysimi is comparable to those found in other insect species, such as seven antennae-abundant CYPs were documented in D. citri (Kuang et al., 2022), as well as four CYPs (CYP4L4, CYP4S4, CYP9A13, and CYP4G20) of Mamestra brassicae and four CYPs (CYP6DE1, CYP6DJ1, CYP6BW1, and CYP6BW3) of D. ponderosae have been found to be highly expressed in the antennae (Maïbèche-Coisne et al., 2002;Maïbèche-Coisne et al., 2005;Chiu et al., 2019a;Chiu et al., 2019b;Chiu et al., 2019c). Insect P450 genes are commonly divided into four clades, which include CYP2, CYP3, CYP4, and the mitochondrial CYP. Herein, we found five LeCYPs (i.e., LeCYP6a13, LeCYP6a2, LeCYP6k1, LeCYP6a14.1, and LeCYP6a14.2) were grouped into the CYP3 clan.

FIGURE 5
Phylogenetic relationship of 61 UGTs from the aphid species L. erysimi (Le, 5), M. persicae (Mp,19), Aphis gossypii (Ag, 17), and the hemipteran insect D. citri (Dc, 17); as well as the reported antennal SlUGT40R3 and SlUGT46A6 from Spodoptera littoralis, and DmeUgt35b from Drosophila melanogaster. The neighbor-joining (NJ) tree was constructed using MEGA 11 with 1,000 bootstrap replicates. Five L. erysimi UGTs identified in this study are highlighted in blue. The sequences used in this tree are provided in Supplementary Table S4.

FIGURE 6
The relative expression levels of candidate odorant degrading enzyme (ODE) genes among different developmental stages of L. erysimi. The expression level of the first instar nymph was arbitrarily assigned a value of 1. Different lowercase letters above the error bar indicate statistically significant differences among aphid developmental stages (p < 0.05; one-way ANOVA, Tukey's multiple comparisons test).

FIGURE 7
The relative expression levels of candidate ODE genes in different tissues of L. erysimi. The expression level in cuticle was arbitrarily given a value of 1, and the expression levels in other tissues were presented relative to the average cuticle. Significant differences of the relative abundance among aphid tissues were indicated by different letters above the error bar (p < 0.05; one-way ANOVA, Tukey's multiple comparisons test).