Expression and Functional Analysis of Two Cytochrome P450 Monooxygenase Genes and a UDP-Glycosyltransferase Gene Linked with Thiamethoxam Resistance in the Colorado Potato Beetle

Simple Summary Several differentially expressed genes encoding cytochrome P450 monooxygenases (P450s) and UDP-glycosyltransferases (UGTs), namely, CYP9Z140, CYP9AY1, and UGT321AP1, were screened and verified between thiamethoxam-susceptible and resistant populations of Leptinotarsa decemlineata (Say). The expression of the three genes was significantly enhanced after exposure to thiamethoxam. RNA interference of three genes increased mortality of test adults following thiamethoxam treatment. The findings reveal that the three genes have roles in the thiamethoxam resistance of L. decemlineata. Abstract Cytochrome P450 monooxygenases (P450s) and UDP-glycosyltransferases (UGTs) are involved in the evolution of insecticide resistance. Leptinotarsa decemlineata (Say), the Colorado potato beetle (CPB), is a notorious insect that has developed resistance to various insecticides including neonicotinoids. This study investigated whether the differentially expressed P450 genes CYP9Z140 and CYP9AY1 and UGT gene UGT321AP1, found in our transcriptome results, conferred resistance to thiamethoxam in L. decemlineata. Resistance monitoring showed that the sampled field populations of L. decemlineata adults collected from Urumqi City and Qapqal, Jimsar, and Mulei Counties of Xinjiang in 2021–2023 developed low levels of resistance to thiamethoxam with resistance ratios ranging from 6.66- to 9.52-fold. Expression analyses indicated that CYP9Z140, CYP9AY1, and UGT321AP1 were significantly upregulated in thiamethoxam-resistant populations compared with susceptible populations. The expression of all three genes also increased significantly after thiamethoxam treatment compared with the control. Spatiotemporal expression patterns showed that the highest expression of CYP9Z140 and CYP9AY1 occurred in pupae and the midgut, whereas UGT321AP1 was highly expressed in adults and Malpighian tubules. Knocking down all three genes individually or simultaneously using RNA interference increased the sensitivity of adult L. decemlineata to thiamethoxam. These results suggest that overexpression of CYP9Z140, CYP9AY1, and UGT321AP1 contributes to the development of thiamethoxam resistance in L. decemlineata and provides a scientific basis for improving new resistance management of CPB.


Introduction
The Colorado potato beetle (CPB), Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), is a notorious insect pest of solanaceous crops causing considerable economic losses.In China, the beetle is mainly distributed in the potato-growing areas north of Tianshan in Xinjiang and has spread to northeast China in recent years, posing a serious threat to potato production [1].Currently, the application of various insecticides is still the most effective way to control CPB.The neonicotinoid agent thiamethoxam has been commonly applied for CPB in Xinjiang for nearly two decades; however, such excessive reliance has led inevitably to resistance developing in local CPB populations [2,3].
In addition to P450s, as key phase II enzymes in detoxification, insect uridine diphosphate glycosyltransferases (UGTs) have also received attention in insecticide resistance research.For example, the midgut-specific overexpression of UGT341A4, UGT344B49, and UGT344M2 significantly increased insensitivity to cyantraniliprole in A. gossypii [23], whereas the upregulated expression of FoUGT466B1, FoUGT468A3, and FoUGT468A4 contributed to spinosad resistance in Frankliniella occidentalis [24].UGT352A5 was also reported to be responsible for conferring thiamethoxam resistance in B. tabaci [25], while Kaplanoglu et al. [22] found that the overexpression of UGT2 was related to imidacloprid resistance in resistant L. decemlineata.
Recently, RNA interference (RNAi) has become a novel pest control technology with high specificity, selectivity, and safety.A major breakthrough has been made in biopesticides based on RNAi, with the registration of Ledprona, a dsRNA that targets the proteasome subunit beta type-5 (PSMB5) gene of CPB, by the United States in 2023 [26].Therefore, screening detoxification enzyme genes related to resistance will also help in the development of this new biopesticide.
Studies indicate that different insect species and even different populations of the same insect have different metabolic resistance mechanisms to the same insecticide.However, there is limited information about which genes are involved in the molecular metabolic mechanism of resistance to thiamethoxam in CPB.In this study, the main goal was to uncover the role of P450 and UGT genes in the thiamethoxam resistance of L. decemlineata.Thus, transcriptome analysis was performed to screen genes encoding detoxifying enzymes that were differentially expressed between thiamethoxam-resistant and sensitive CPB populations in Xinjiang.The expression of two upregulated P450 CYP9e2-like genes (CYP9Z140 and CYP9AY1) and one UGT gene (UGT321AP1) was further verified and analyzed in different field populations, stages, and tissues, and in response to thiamethoxam via quantitative real-time PCR (RT-qPCR).RNAi was then used to suppress the expression

Bioassay
The contact toxicity of thiamethoxam in CPB adults was assayed using a topical application method.Thiamethoxam (97% powder, Jiangsu Bangsheng Biotechnology Co., Ltd., Huaian, China) was diluted to at least five different concentrations with analytical-grade acetone to result in a 10-100% mortality range of test insects.Ten adults were treated individually with 1.1 µL of insecticide solution or acetone as control, which was applied to their ventral area using a microapplicator (Hamilton Company, Reno, NV, USA), and then added to Petri dishes (9 cm in diameter and 1.5 cm in height) containing fresh potato leaves and maintained under the conditions described above.Each treatment had three replicates.The standard reference for dead beetles was based on Liu et al. [2], and beetle mortality was recorded after 72 h.

RNA-Sequencing Data Analysis
Twelve adults (three beetles for each repetition) of each population, including a thiamethoxam-susceptible population and two resistant populations with low-level resistance to thiamethoxam, were sent on dry ice to Biomarker Technologies Co., Ltd.(Beijing, China), for RNA extraction, cDNA library construction, and RNA sequencing.Sequencing was performed on an Illumina Novaseq 6000 platform (company, city, country) using a 150 bp paired-end sequencing strategy.The clean reads were aligned to the reference genome of L. decemlineata from the relevant genome website https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000500325.1/(accessed on 31 October 2021).Differential expression levels between susceptible and resistant populations were analyzed using the DESeq2 R package (1.20.0), based on fragments per kilobase per million (FPKM).The false discovery rate (FDR) was used to identify the threshold of the p-value in multiple tests to compute the significant difference.Genes with an absolute value of log 2 Fold Change > 1 and FDR core < 0.05 found by DESeq2 were considered to be differentially expressed.
The cloud blast feature in Blast2GO v.2 software was used to annotate the transcripts by comparing the sequences with the arthropod non-redundant protein database with a Blast expectation value (e-value) of 1.0 × 10 -5 as a cutoff.Gene Ontology (GO) enrichment analysis was performed using Perl script by plotting the GO information of the differentially expressed genes (DEGs) retrieved from Blast2GO against all GOs from the L. decemlineata genome data.The obtained annotation was enriched and refined using TopGo (R package).Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were assigned to the assembled sequences by Perl script.

Sequence and Phylogenetic Analysis
P450 and UGT genes identified from the transcriptome and genome data of L. decemlineata were cloned and verified by reverse transcription PCR (RT-PCR).Total RNA was extracted from a mixture of eggs, first-to fourth-instar larvae, pupae, and adults of L. decemlineata.Multiple alignments of sequences were performed using GeneDoc EXE, and the structural domains were detected based on comparison with other identified sequences.The theoretical isoelectric points (pIs) and molecular weights (Mws) were analyzed by ExPASy (https://web.expasy.org/protparam/(accessed on 15 October 2022)).MEGA 7 was utilized to construct the phylogenetic trees via the neighbor-joining method with 1000 bootstrap replications based on the amino acid sequences of CYP9e2 and UGT genes from other insects acquired through similarity searches of the NCBI database.The three verified genes were named by the P450 (David R. Nelson, Department of Molecular Sciences, University of Tennessee, Memphis, TN, USA) and UGT nomenclature committees (https://labs.wsu.edu/ugt/(accessed on 31 March 2022) as CYP9Z140, CYP9AY1, and UGT321AP1, respectively.

Preparation of Samples for Expression Analysis
Three adults were sampled from each population from the eight sample sites in Xinjiang (detailed in Table 1) to determine the expression difference in three candidate genes among different CPB field populations.In order to extract RNA of similar concentration, the number of the beetle samples at different stages of development collected was different to make their quality similar.We collected 30 eggs (E), 30 1st-instar larvae (L1), 20 2nd-instar larvae (L2), 10 3rd-instar larvae (L3), as well as 3 4th-instar larvae (L4), 3 pupae (P) and 3 adults (A), from the URMQA population to examine the stage-specific expression of three genes.To compare the tissue expression of candidate genes, the foreguts, midguts, hindguts, Malpighian tubules, fat bodies, head, thorax, and abdomen were dissected from five adults of URMQA, respectively.Three adults were sampled from the survivors of URMQA treated with either LD 50 of thiamethoxam, or acetone treatment (as control) for 72 h was used to determine the inducible expression profiles of three genes.The sample size above is taken as a biological replication, and each treatment (population) had three biological replicates.All samples were frozen quickly in liquid nitrogen and stored at -80 • C until use.

Total RNA Isolation and cDNA Synthesis
Total RNA from the above-mentioned samples was isolated using Yfx Total RNA Extraction Reagent (Yi Fei Xue Biotechnology Co., Ltd., Nanjing, China), following the manufacturer's protocol.The concentration of the RNA samples was analyzed on a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).The first-strand cDNA was then synthesized by using a PrimeScript RT reagent kit (TaKaRa Biotechnology Co., Ltd., Dalian, China).
Potato leaves of similar size were dipped in bacterial solutions containing dsCYP9Z140, dsCYP9AY1, dsUGT321AP1, and dsGFP (as control) for 30 min, and then placed in plastic feeding chambers with 17 cm in length, 11.7 cm in width, and 5 cm in height after air drying.Adult CPB collected from URMQA were carefully transferred into each chamber containing the treated leaves.Thirty beetles were used for each treatment, and all treatments were replicated 6 times.A fresh supply of treated potato leaves was provided daily.To silence the three genes simultaneously, the beetles were fed a mixture of dsRNA of the three genes at a 1:1:1 ratio.After 6 d of continuous feeding on treated leaves, four replicates from each treatment group (12 adults) were used to extract total RNA for measuring the expression levels of the target genes, as detailed above.The remaining beetles from each treatment group were used to determine the susceptibility to thiamethoxam.Adults were treated with a median lethal dose (LD 50 ) of thiamethoxam (0.2963 µg/adult), with the same amount of acetone used as a control.Each treatment was repeated four times with 15-20 adults each.The number of dead beetles in each group was then recorded, as described above.

Statistical Analysis
Bioassay data were corrected for control mortality by Abott's formula.Median lethal doses (LD 50 ) and 95% fiducial limits (FLs) were estimated via PoloPlus 2.00 software (Leora Software, Berkeley, CA, USA).The resistance ratio (RR) was calculated by dividing the LD 50 value of the field population by the LD 50 value of the susceptible population and was quantified according to Shi et al. [3].The quantitative data of three genes and mortality of test beetles exposed to thiamethoxam after RNAi were expressed as the mean ± standard error (SE) from at least three biological replicates.Data on transcriptome validation and inducible expression were analyzed to compare the difference between the two treatments using Student's t-test.The remaining data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests.Statistical analysis was carried out using GraphPad Prism 8.02 and SPSS statistics (IBM SPSS Statistics 27 software, Chicago, IL, USA).Statistical differences were considered significant at p < 0.05.

Resistance Levels of L. decemlineata Populations to Thiamethoxam
The sensitivity to thiamethoxam of L. decemlineata field populations collected from Xinjiang in 2021, 2022, and 2023 were assayed by topical application (Table 3).The URMQY population was considered to be relatively sensitive.In 2021, the ML population remained sensitive to thiamethoxam, with an RR of 2.18-fold, whereas the URMQA population showed decreased susceptibility, with an RR of 3.04-fold.The JMSL and QPQLZ populations developed 7.18-and 8.33-fold low levels of resistance to thiamethoxam, respectively.In 2022, the URMQA and JMST populations had low levels of resistance and decreased susceptibility, with RRs of 9.53-and 3.23-fold, respectively.In 2023, Both ML and JMSD1 developed low resistance, with RRs of 7.42-and 6.66-fold, respectively.The JMSQ, URMQA, and JMSD2 populations showed decreased susceptibility with RRs ranging from 3.04-fold to 4.63-fold.QPQLB remained sensitive to thiamethoxam throughout the study period.The results of resistance monitoring to thiamethoxam may provide the basis for effective control of L. decemlineata.

Transcriptome Analysis
Illumina short-read sequences from mRNAs isolated from the URMQY, QPQLZ, and JMSL populations were compiled into a transcriptome, generating 26,114,887, 21,000,522, and 24,582,542 usable reads, respectively.The percentage of Q30 bases was 93.39% and above, and the GC content of each population ranged from 40.52% to 41.23% (Table 4).Analysis of the log-fold change in expression of genes that were significantly upor downregulated in all samples revealed 813 DEGs between the URMQY and JMSL populations, of which 263 (32.35%) were upregulated and 550 (67.65%) were downregulated.In addition, there were 883 DEGs detected between the URMQY and QPQLZ populations, of which 254 (28.77%) were upregulated and 629 (71.23%) were downregulated.
GO analyses indicated that the annotated DEGs could be divided into three different categories as follows: biological process (BP); cellular component (CC); and molecular function (MF) (Figure 1).In each of these three main categories, the terms "metabolic process", "cellular process", "cell part", "binding", and "catalytic activity" were the most dominant.The top 20 enriched KEGG pathways were mainly linked with the metabolism of xenobiotics (Figure 2).
Several classes of detoxifying enzymes involved in enzymatic detoxification mechanisms were upregulated between susceptible and resistant populations (Table 5).Two P450 genes with a fold change > 2 and FDR score < 0.0001 (ID: 111518298 and 111508919) and two UGT genes were used for subsequent analysis.RT-qPCR was performed to confirm the transcript expression obtained from the RNA-sequencing data.The expression levels of CYP9Z140 (ID: 111518298) and CYP9AY1 (ID: 111508919) in the QPQLZ population and UGT321AP1 (ID: 111517685) in the JMSL population were significantly enhanced by 1.92, 2.04, and 36.4 times, respectively (p < 0.05), compared with the URMQY population, which was consistent with the transcriptome results (Figure 3).However, UGT324BR1 (ID: 111518183) was not upregulated significantly in the QPQLZ population compared with the URMQY population and, thus, was not considered as a candidate gene for follow-up studies.lar function (MF) (Figure 1).In each of these three main categories, the terms "metabolic process", "cellular process", "cell part", "binding", and "catalytic activity" were the most dominant.The top 20 enriched KEGG pathways were mainly linked with the metabolism of xenobiotics (Figure 2).Several classes of detoxifying enzymes involved in enzymatic detoxification mechanisms were upregulated between susceptible and resistant populations (Table 5).Two P450 genes with a fold change > 2 and FDR score < 0.0001 (ID: 111518298 and 111508919) and two UGT genes were used for subsequent analysis.

Gene Structure and Phylogenetic Analysis
The structural features of the two P450 genes (CYP9Z140 and CYP9AY1) are illu trated in Figure 4A.The full-length genes contained a 1578 bp open reading frame (OR encoding 525 amino acid residues.The theoretical isoelectric points (pIs) were 8.95 an 5.73, and the molecular weights (Mw) were 60.435 and 60.727 kDa, respectively.Co served domains in the genes are common to cytochrome P450s and include the C-he motif (WxxxR), I-helix motif (GxE/DTT/S), K-helix motif (ExLR), the conserved ami acid sequence PxxFxP motif, and the heme-binding motif (PFxxGxxxCxG).Structur features of the UGT gene UGT321AP1 are shown in Figure 5A.The full-length cDNA UGT321AP1 encoded 517 amino acids.Its pI and Mw were 6.88 and 59.20 kDa, respe tively.Similar to UGTs in other insects, the signal peptide of UGT321AP1 was found the N terminus, and the signature motif situ ed in the middle of the C-terminal domain; two sugar donor-binding site domai (DBR1 and DBR2) were also predicted for the amino acid sequences.A hydrophob transmembrane domain containing ~29 hydrophobic amino acid residues was found the C terminus.

Gene Structure and Phylogenetic Analysis
The structural features of the two P450 genes (CYP9Z140 and CYP9AY1) are illustrated in Figure 4A.The full-length genes contained a 1578 bp open reading frame (ORF) encoding 525 amino acid residues.The theoretical isoelectric points (pIs) were 8.95 and 5.73, and the molecular weights (Mw) were 60.435 and 60.727 kDa, respectively.Conserved domains in the genes are common to cytochrome P450s and include the C-helix motif (WxxxR), I-helix motif (GxE/DTT/S), K-helix motif (ExLR), the conserved amino acid sequence PxxFxP motif, and the heme-binding motif (PFxxGxxxCxG).Structural features of the UGT gene UGT321AP1 are shown in Figure 5A.The full-length cDNA of UGT321AP1 encoded 517 amino acids.Its pI and Mw were 6.88 and 59.20 kDa, respectively.Similar to UGTs in other insects, the signal peptide of UGT321AP1 was found at the N terminus, and the signature motif situated in the middle of the C-terminal domain; two sugar donor-binding site domains (DBR1 and DBR2) were also predicted for the amino acid sequences.A hydrophobic transmembrane domain containing ~29 hydrophobic amino acid residues was found at the C terminus.
The phylogenetic relationships among the three genes from L. decemlineata and related P450s and UGTs from other insects are shown in Figures 4B and 5B
family of Tenebrio molitor and Diabrotica virgifera virgifera from Coleoptera.UGT321AP1 was clustered in the branch of Tribolium castaneum (Coleoptera).
To study the effect of neonicotinoids on the expression of CYP9Z140, CYP9AY1, and UGT321AP1, the mRNA levels in URMQY adults treated with thiamethoxam LD50 for 72 h were determined and analyzed by RT-qPCR (Figure 7).Thiamethoxam treatment significantly increased the expression of all three genes by 2.31-(t = 3.217, p = 0.0324), 3.03-(t = 5.446, p = 0.0055), and 5.01-fold (t = 5.796, p = 0.0044), respectively, compared with the control.The expression levels of the three genes were normalized and calculated using EF-1α and RPL4 as internal reference genes.Different lowercase letters above the bars represent significant expression differences in each gene in different populations compared to susceptible populations using one-way ANOVA followed by Tukey's multiple comparisons (p < 0.05).
To study the effect of neonicotinoids on the expression of CYP9Z140, CYP9AY1, and UGT321AP1, the mRNA levels in URMQY adults treated with thiamethoxam LD 50 for 72 h were determined and analyzed by RT-qPCR (Figure 7).Thiamethoxam treatment significantly increased the expression of all three genes by 2.31-(t = 3.217, p = 0.0324), 3.03-(t = 5.446, p = 0.0055), and 5.01-fold (t = 5.796, p = 0.0044), respectively, compared with the control.
To identify the tissue-specific detoxification gene(s) that accounts for thiamethoxam resistance, the expression patterns of CYP9Z140, CYP9AY1, and UGT321AP1 were analyzed by RT-qPCR in the seven developmental stages (eggs, first to fourth instar larvae, pupae, and adults), different tissues (foregut, midgut, hindgut, fat body, and Malpighian tubules), and different body parts (head, thorax, and abdomen) of L. decemlineata.CYP9Z140, CYP9AY1, and UGT321AP1 were transcribed throughout all developmental stages of L. decemlineata.In the developmental stages, a similar pattern was detected between CYP9Z140 and CYP9AY1, whereas there were clear differences in the developmental expression levels of UGT321AP1 (Figure 8A).The expression of the two CYP450 genes was highest in pupae, followed by adult and fourth-instar larvae, and was lowest in the egg stage.The expression levels of CYP9Z140 and CYP9AY1 in the pupae, fourth-instar larvae, and adults were 31.10,14.81, and 14.45 (F = 331.277,df = 6,14; p < 0.05) and 20.79, 13.35, and 15.84 times (F = 67.698,df = 6,14; p < 0.05) higher than those in the egg stage, respectively.UGT321AP1 expression was highest in the second-instar larvae and adults, and lowest in the first-, second-, and fourth-instar larvae and pupae (F = 41.726,df = 6,14; p < 0.05).Tissue expression patterns showed that expression of the two P450 genes was highest in the midgut, whereas that of UGT321AP1 was highest in Malpighian tubules (Figure 8B).The expression of UGT321AP1 in the head and thorax was higher than that in the abdomen, with no significant difference in the expression of the two P450s in the different body parts of CPB adults (Figure 8C).Expression levels of P450 and UGT genes in L. decemlineata adults treated with LD50 of thiamethoxam (TMX).The mRNA expression levels of three genes in the URMQA population exposed to acetone for 72 h were used as controls.The expression of the test genes was normalized and calculated using EF-1α and RPL4 as internal reference genes.Different lowercase letters above the bars represent significant differences in mRNA levels between treatment and control for each gene by Student's t-test (n = 3, mean relative quantity ± SE, p < 0.05).
To identify the tissue-specific detoxification gene(s) that accounts for thiamethoxam resistance, the expression patterns of CYP9Z140, CYP9AY1, and UGT321AP1 were analyzed by RT-qPCR in the seven developmental stages (eggs, first to fourth instar larvae, pupae, and adults), different tissues (foregut, midgut, hindgut, fat body, and Malpighian tubules), and different body parts (head, thorax, and abdomen) of L. decemlineata.CYP9Z140, CYP9AY1, and UGT321AP1 were transcribed throughout all developmental stages of L. decemlineata.In the developmental stages, a similar pattern was detected between CYP9Z140 and CYP9AY1, whereas there were clear differences in the developmental expression levels of UGT321AP1 (Figure 8A).The expression of the two CYP450 genes was highest in pupae, followed by adult and fourth-instar larvae, and was lowest in the egg stage.The expression levels of CYP9Z140 and CYP9AY1 in the pupae, fourthinstar larvae, and adults were 31.10,14.81, and 14.45 (F = 331.277,df = 6,14; p < 0.05) and 20.79, 13.35, and 15.84 times (F = 67.698,df = 6,14; p < 0.05) higher than those in the egg stage, respectively.UGT321AP1 expression was highest in the second-instar larvae and adults, and lowest in the first-, second-, and fourth-instar larvae and pupae (F = 41.726,df = 6,14; p < 0.05).Tissue expression patterns showed that expression of the two P450 genes was highest in the midgut, whereas that of UGT321AP1 was highest in Malpighian tubules (Figure 8B).The expression of UGT321AP1 in the head and thorax was higher than that in the abdomen, with no significant difference in the expression of the two P450s in the different body parts of CPB adults (Figure 8C).Expression levels of P450 and UGT genes in L. decemlineata adults treated with LD 50 of thiamethoxam (TMX).The mRNA expression levels of three genes in the URMQA population exposed to acetone for 72 h were used as controls.The expression of the test genes was normalized and calculated using EF-1α and RPL4 as internal reference genes.Different lowercase letters above the bars represent significant differences in mRNA levels between treatment and control for each gene by Student's t-test (n = 3, mean relative quantity ± SE, p < 0.05).Relative expression levels of the three genes in different tissues of L. decemlineata adults.FG: foregut; MG: midgut; HG: hindgut; MT: Malpighian tubule; FB: fat body.(C) Relative expression levels of the three genes in different parts of L. decemlineata adults.H: head; T: thorax; Ab: abdomen.Data are expressed as mean relative quantity ± SEM.Different lowercase letters above the bars represent significant differences in mRNA levels for each gene in different stages or tissues using oneway ANOVA followed by Tukey's multiple comparisons (n = 3, p < 0.05).

Discussion
CPB is a species characterized by the rapid development of resistance to a variety of insecticides [28].With the extensive application of neonicotinoids for the control of CPB in Xinjiang, it is necessary to continuously monitor such resistance.In the current study, the resistance levels to thiamethoxam of CPB from different areas of Xinjiang were investigated across three sample years (2021-2023).The LD50 value of the QPQLZ population to thiamethoxam (0.2592 μg⸳ adult −1 ) in our study was >0.0196 μg⸳ adult −1 of individuals from the same population collected in 2010 [2], while the resistance level of QPQLZ, with a RR of 8.33-fold (Table 3), was higher than the RR of 4.3-fold in the same population reported by Shi et al. [3].The resistance level to thiamethoxam in the ML population

Discussion
CPB is a species characterized by the rapid development of resistance to a variety of insecticides [28].With the extensive application of neonicotinoids for the control of CPB in Xinjiang, it is necessary to continuously monitor such resistance.In the current study, the resistance levels to thiamethoxam of CPB from different areas of Xinjiang were investigated across three sample years (2021-2023).The LD 50 value of the QPQLZ population to thiamethoxam (0.2592 µg adult −1 ) in our study was >0.0196 µg adult −1 of individuals from the same population collected in 2010 [2], while the resistance level of QPQLZ, with a RR of 8.33-fold (Table 3), was higher than the RR of 4.3-fold in the same population reported by Shi et al. [3].The resistance level to thiamethoxam in the ML population increased from 2.18-fold in 2021 to 7.42-fold in 2023, whereas the JMST, JMSQ, and JMSD2 populations exhibited decreased susceptibility to thiamethoxam.By contrast, the URMQA population in 2022, and the JMSD1 and JMSL populations, showed low resistance.We investigated and found that Arika suspension (thiamethoxam being the main ingredient) has been applied long-term in the Jimusar (JMS) region to control CBP.This could explain why all tested populations from Jimusar showed increased levels of tolerance to thiamethoxam.In addition, it can be found that the resistance levels to thiamethoxam of CPB populations tested are low.The possible reasons are as follows: firstly, the integrated control for the beetle in the local area is rather effective, including potato field rotation, alternative use of different types of insecticides, etc., which may delay the resistance development to thiamethoxam of CPB to a certain extent.Secondly, the highly resistant populations could not be obtained because of limited sampling sites.In subsequent investigations, we will add more sampling sites to monitor the resistance dynamics of local populations.
Developments in molecular biology and genomics have led to the mechanism of insecticide resistance mediated by genes encoding detoxification enzymes becoming a hot research topic.Many studies have reported that overexpression of P450 and UGT genes can lead to resistance of pests to neonicotinoid insecticides.For example, Zhu et al. [18] revealed 41 highly expressed P450 genes in imidacloprid-resistant populations of CPB from Long Island, New York, USA.Using transcriptional analysis, Clements et al. [19,20] found that expression levels of CYP9Z26 and CYP96K1 in imidacloprid-resistant adults from Wisconsin, USA, were significantly increased after imidacloprid treatment.In addition, qPCR analysis showed that CYP6K1 was also overexpressed in field populations under long-term use of neonicotinoid insecticides [29].Kaplanoglu et al. [22] revealed that overexpression of two genes encoding detoxifying enzymes (CYP4Q3 and UGT2) contributed to imidacloprid resistance in medium-level imidacloprid-resistant CPB populations.Based on these studies, it appears that overexpressed detoxification enzyme genes related to neonicotinoid resistance in CPB differ across populations with diverse resistance backgrounds.Thus, in the present study, transcriptome analysis was used to compare susceptible and resistant populations of L. decemlineata, revealing the DEGs CYP9Z140, CYP9AY, and UGT321AP1, as verified by qPCR.However, there was no evidence to suggest that these genes were directly involved in neonicotinoid resistance.
Further qPCR analysis showed that CYP9Z140, CYP9AY1, and UGT321AP1 were overexpressed significantly in thiamethoxam-resistant adults from the QPQLZ and JMSL populations in 2021, the URMQA population in 2022, and the JMSD1 and ML populations in 2023, comparable to CYP9e2-like genes reported to be overexpressed in resistant compared with susceptible adults [3].Therefore, we speculated that these genes were related to the resistance of L. decemlineata to thiamethoxam, based on the results of constitutive expression analyses of different resistant populations in Xinjiang.
Many studies have shown that insect P450 and UGT genes can be induced by insecticides.For example, CYP6AX1 and CYP6AY1 of N. lugens and CYP6AY3v2 of Laodelphax striatellus were upregulated in the presence of imidacloprid [30,31].The expression of UGT352A4 and UGT352A5 in the thiamethoxam-resistant B. tabaci strain significantly increased after thiamethoxam treatment [24].Our study showed that thiamethoxam exposure significantly increased the expression of the three genes in URMQY adults.Similarly, the transcript level of CYP9e2 increased 4.2-fold in L. decemlineata exposed to clothianidin [32].In addition, CYP9e2-like genes are involved in insect resistance to a variety of insecticides.Oppert et al. [33] found that the expression of CYP9e2 in susceptible populations of T. castaneum increased when exposed to sublethal doses of phosphine.Jiang et al. [34] reported that the relative expression level of AcCYP9e2 in the midgut of Apis cerana workers was significantly higher than that of the control group after exposure to flumethrin.Using transcriptome analysis, Gao et al. [35] revealed that CYP9e2 of Plutella xylostella was upregulated after treatment with chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb, and spinosad.Therefore, we further speculate that CYP9Z140, CYP9AY1, and UGT321AP1 are associated with the detoxification of thiamethoxam in L. decemlineata.
The specific spatiotemporal expression patterns of genes encoding detoxifying enzymes are usually linked to their protein function.The current analysis showed that CYP9Z140, CYP9AY1, and UGT321AP1 were detected in all developmental stages and tissues of CPB, albeit with significantly different expression levels.The expression of CYP9Z140 and CYP9AY1 was highest in pupae and the midgut, while that of UGT321AP1 was highest in adults and Malpighian tubules.In addition, UGT321AP1 showed higher expression in the head and thorax than in the abdomen of CPB, whereas the expression of CYP9Z140 and CYP9AY1 showed no difference among the body parts.Similarly, CYP6FV12 of B. odoriphaga was highly expressed in the midgut but expressed at low levels in eggs [15], and CYP303a1 of Drosophila melanogaster was markedly overexpressed during the pupal stage [36].UGT353G2 in B. tabaci adults had the highest expression across different development stages [37].However, the highest stage-specific expression of CYP6FV12 was observed in fourth-instar nymphs of B. odoriphaga, and Cyp303a1 had the highest expression in the ring gland of D. melanogaster [15,36].The insect midgut and Malpighian tubules are important organs for detoxifying exogenic compounds, such as insecticides.Thus, our stage-and tissue-specific expression profiles suggested that these three genes were involved in CPB resistance to thiamethoxam and that the major detoxification action stages might occur in adults and pupae, followed by fourth-instar larvae.
Many studies have indirectly verified the roles of P450 and UGT genes in pest resistance through RNAi.For example, results from RNAi showed that CYP6ER1 not only had a role in the resistance of N. lugens to imidacloprid but was also closely related to the generation of thiamethoxam and dinotefuran resistance [13,38].In addition, the overexpressed gene CYP6CY14 was confirmed as having an important role in the thiamethoxam resistance of A. gossypii [14].The ingestion of dsRNAs for L. decemlineata successfully reduced the expression of CYP9Z26 and CYP9Z29 and increased the imidacloprid susceptibility of test beetles [21,22,28,39].In our study, after L. decemlineata adults were continuously fed with bacterial solutions containing individual or mixed dsRNA of three genes for 6 d, the expression levels of the target genes and the tolerance of test beetles to thiamethoxam were significantly decreased compared with dsGFP treatment.The roles of CYP9e2-like genes in the insecticide resistance of insects have also been reported.Bouafoura et al. [32] found that CYP9e2 knockdown increased the susceptibility of L. decemlineata to clothianidin.A cytochrome P450, CYP9E2, and a long non-coding RNA gene lncRNA-2 were found to be upregulated in a Spinosad-resistant population of CPB, and knockdown of these two genes using RNAi resulted in a significant increase in spinosad sensitivity, which implies CYP9E2 and lncRNA-2 jointly contribute to spinosad resistance [40].In addition, the suppression of UGT353G2 expression by RNAi substantially increased sensitivity to multiple neonicotinoids in resistant strains of B. tabaci, indicating the involvement of UGT353G2 in the neonicotinoid resistance of whitefly [36].The current study not only confirmed overexpression of the three target genes as an important resistance mechanism to neonicotinoids, but also indicated that different populations of L. decemlineata had different metabolic molecular mechanisms based on the RNAi effects of UGT321AP1, CYP9Z140, and CYP9AY1 on sensitivity to thiamethoxam.Our findings suggested that RNAi-triggered knockdown of CYP9Z140, CYP9AY1, and UGT321AP1 resulted in an increased susceptibility to thiamethoxam in the adults of the field populations, which may provide a scientific basis for improving new management of L. decemlineata.
Our study results showed that two P450 genes and one UGT gene conferred resistance to thiamethoxam, indicating that thiamethoxam resistance in L. decemlineata develop by a complex mechanism.Thus, other detoxification genes related to the thiamethoxam resistance of CPB need to be screened and identified.Furthermore, the regulatory mechanism of CYP9Z140, CYP9AY1, and UGT321AP1 expression remains to be elucidated.

Conclusions
In summary, using resistance monitoring, this study showed that most test populations in Xinjiang developed low resistance to thiamethoxam.The results of RT-qPCR analysis determined not only that CYP9Z140, CYP9AY1, and UGT321AP1 were overexpressed in resistant populations but also that their expression was induced by thiamethoxam and that they were highly expressed in the midgut and Malpighian tubules.RNAi further confirmed the roles of the genes in the development of resistance to thiamethoxam against L. decemlineata.These results will facilitate the development of CPB resistance management strategies.

Figure 1 .
Figure 1.GO functional annotation of differentially expressed genes.

Figure 2 .
Figure 2. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment histogram of differentially expressed genes.
. The translated proteins of P450s shared the highest amino acid sequence identity with the CYP9 subfamily of Tenebrio molitor and Diabrotica virgifera virgifera from Coleoptera.UGT321AP1 was clustered in the branch of Tribolium castaneum (Coleoptera).

Figure 4 .
Figure 4. Bioinformatic analysis of two P450 genes CYP9Z140 and CYP9AY1 from L. decemlin (A) Alignment of amino acid sequences of CYP9Z140, CYP9AY1, and the related P450 g CYP9Z1 from Tribolium castaneum.Conserved motifs were highlighted in the sequences, includ the helix-C motif (WxxxR), the oxygen-binding motif (helix I) ([A/G] GX [E/D] T[T/S]), the hel motif (EXXRXXP), the conserved Meander motif (PXXFXP), and the heme-binding m (PFXXGXXXCXG).(B) Phylogenetic tree of CYP9Z140, CYP9AY1, and related P450s from othe sects.Bootstrap values (1000 replicates) are indicated next to the branches, and GenBank acces numbers are shown in parentheses.The black dot indicates CYP9Z140 and CYP9AY1 in L. dec lineata.

Figure 4 .
Figure 4. Bioinformatic analysis of two P450 genes CYP9Z140 and CYP9AY1 from L. decemlineata.(A) Alignment of amino acid sequences of CYP9Z140, CYP9AY1, and the related P450 gene CYP9Z1 from Tribolium castaneum.Conserved motifs were highlighted in the sequences, including the helix-C motif (WxxxR), the oxygen-binding motif (helix I) ([A/G] GX [E/D] T[T/S]), the helix K motif (EXXRXXP), the conserved Meander motif (PXXFXP), and the heme-binding motif (PFXXGXXXCXG).(B) Phylogenetic tree of CYP9Z140, CYP9AY1, and related P450s from other insects.Bootstrap values (1000 replicates) are indicated next to the branches, and GenBank accession numbers are shown in parentheses.The black dot indicates CYP9Z140 and CYP9AY1 in L. decemlineata.

Figure 5 .Figure 5 .
Figure 5. Bioinformatic analysis of UGT gene UGT321AP1 from L. decemlineata.(A) Alignment o amino acid sequences of UGT321AP1 and related UGT gene from T. castaneum, Sitophilos oryza and Anoplophora glabripennis.The signal peptides in the N terminus are shown with a blue under line.The UGT signature motif is shown with a yellow box.The transmembrane domains in the C terminal half and cytoplasmic tail are shown in green and purple underlines.The red bars unde the sequences indicate the two donor-binding regions (DBR1 and DBR2).(B) Phylogenetic tree o UGT321AP1 and related UGTs from other insects.Bootstrap values (1000 replicates) are indicate next to the branches, and GenBank accession numbers are shown in parentheses.The black do indicates UGT321AP1 in L. decemlineata.The phylogenetic relationships among the three genes from L. decemlineata and re lated P450s and UGTs from other insects are shown in Figures4B and 5B.The translate proteins of P450s shared the highest amino acid sequence identity with the CYP9 sub

Figure 6 .
Figure 6.Relative expression levels of CYP9Z140, CYP9AY1, and UGT321AP1 in different field populations of L. decemlineata in 2021, 2022, and 2023.Data are expressed as mean relative quantity ± SE.The expression levels of the three genes were normalized and calculated using EF-1α and RPL4 as internal reference genes.Different lowercase letters above the bars represent significant expression differences in each gene in different populations compared to susceptible populations using one-way ANOVA followed by Tukey's multiple comparisons (p < 0.05).

Figure 6 .
Figure 6.Relative expression levels of CYP9Z140, CYP9AY1, and UGT321AP1 in different field populations of L. decemlineata in 2021, 2022, and 2023.Data are expressed as mean relative quantity ± SE.The expression levels of the three genes were normalized and calculated using EF-1α and RPL4 as internal reference genes.Different lowercase letters above the bars represent significant expression differences in each gene in different populations compared to susceptible populations using one-way ANOVA followed by Tukey's multiple comparisons (p < 0.05).

Insects 2024 , 21 Figure 7 .
Figure 7. Expression levels of P450 and UGT genes in L. decemlineata adults treated with LD50 of thiamethoxam (TMX).The mRNA expression levels of three genes in the URMQA population exposed to acetone for 72 h were used as controls.The expression of the test genes was normalized and calculated using EF-1α and RPL4 as internal reference genes.Different lowercase letters above the bars represent significant differences in mRNA levels between treatment and control for each gene by Student's t-test (n = 3, mean relative quantity ± SE, p < 0.05).

Figure 7 .
Figure 7. Expression levels of P450 and UGT genes in L. decemlineata adults treated with LD 50 of thiamethoxam (TMX).The mRNA expression levels of three genes in the URMQA population exposed to acetone for 72 h were used as controls.The expression of the test genes was normalized and calculated using EF-1α and RPL4 as internal reference genes.Different lowercase letters above the bars represent significant differences in mRNA levels between treatment and control for each gene by Student's t-test (n = 3, mean relative quantity ± SE, p < 0.05).

Figure 8 .
Figure 8. Spatiotemporal expression patterns of CYP9Z140, CYP9AY1, and UGT321AP1 in L. decemlineata.(A)Relative expression levels of the three genes in developmental stages.L1: first-instar larva; L2: second-instar larva; L3: third-instar larva; L4: fourth-instar larva; P: pupa; A: adult.(B) Relative expression levels of the three genes in different tissues of L. decemlineata adults.FG: foregut; MG: midgut; HG: hindgut; MT: Malpighian tubule; FB: fat body.(C) Relative expression levels of the three genes in different parts of L. decemlineata adults.H: head; T: thorax; Ab: abdomen.Data are expressed as mean relative quantity ± SEM.Different lowercase letters above the bars represent significant differences in mRNA levels for each gene in different stages or tissues using oneway ANOVA followed by Tukey's multiple comparisons (n = 3, p < 0.05).

Figure 8 .
Figure 8. Spatiotemporal expression patterns of CYP9Z140, CYP9AY1, and UGT321AP1 in L. decemlineata.(A)Relative expression levels of the three genes in developmental stages.L1: first-instar larva; L2: second-instar larva; L3: third-instar larva; L4: fourth-instar larva; P: pupa; A: adult.(B) Relative expression levels of the three genes in different tissues of L. decemlineata adults.FG: foregut; MG: midgut; HG: hindgut; MT: Malpighian tubule; FB: fat body.(C) Relative expression levels of the three genes in different parts of L. decemlineata adults.H: head; T: thorax; Ab: abdomen.Data are expressed as mean relative quantity ± SEM.Different lowercase letters above the bars represent significant differences in mRNA levels for each gene in different stages or tissues using one-way ANOVA followed by Tukey's multiple comparisons (n = 3, p < 0.05).

Figure 9 .
Figure 9. Effects of RNA interference on mRNA expression of the three genes (A) and on sensitivity to thiamethoxam of adult L. decemlineata (B).(A) Quantitative PCR analysis was used to determine the expression of CYP9Z140, CYP9AY1, and UGT321AP1 in URMQA adults after feeding on a diet containing individual dsRNA or a mixture of dsRNA (dsCYP9Z140, dsCYP9AY, and dsUGT321AP1) for 6 days.The expression level obtained with dsRNA of target genes is shown relative to that obtained with dsGFP, which was assigned a value of 1. (B) Mortality was recorded for adult L. decemlineata exposed to thiamethoxam (0.2963 μg/adult) for 72 h after individual and simultaneous RNAi for 6 d.Adults were fed with dsGFP as control.All values are means + SEs of three biological replicates.Different lowercase letters above the bars represent significant differences for each treatment using one-way ANOVA followed by Tukey's multiple comparisons (n = 3, p < 0.05).

Figure 9 .
Figure 9. Effects of RNA interference on mRNA expression of the three genes (A) and on sensitivity to thiamethoxam of adult L. decemlineata (B).(A) Quantitative PCR analysis was used to determine the expression of CYP9Z140, CYP9AY1, and UGT321AP1 in URMQA adults after feeding on a diet containing individual dsRNA or a mixture of dsRNA (dsCYP9Z140, dsCYP9AY, and dsUGT321AP1) for 6 days.The expression level obtained with dsRNA of target genes is shown relative to that obtained with dsGFP, which was assigned a value of 1. (B) Mortality was recorded for adult L. decemlineata exposed to thiamethoxam (0.2963 µg/adult) for 72 h after individual and simultaneous RNAi for 6 d.Adults were fed with dsGFP as control.All values are means + SEs of three biological replicates.Different lowercase letters above the bars represent significant differences for each treatment using one-way ANOVA followed by Tukey's multiple comparisons (n = 3, p < 0.05).

Table 2 .
Primers and their application in this study.

Table 4 .
Sequencing results of different populations of L. decemlineata a .Clean reads: total number of pair-end reads of clean data.Clean bases: total number of bases.GC content: percentage of G and C bases in the DNA or RNA molecule.Q30%: percentage of bases with a mass value ≥ 30. a

Table 5 .
P450 and UGT genes upregulated significantly in transcriptomes of different groups of L. decemlineata.