RNA-seq screening of the CPGs in Culex pipiens pallens among cypermethrin-resistant populations

RNA-seq screening of the CPGs in Culex pipiens pallens among cypermethrin-resistant populations | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research RNA-seq screening of the CPGs in Culex pipiens pallens among cypermethrin-resistant populations Qiqi Shi, Peng Cheng, Chongxing Zhang, Lijuan Liu, Xiao Song, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.19260/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Long-lasting overdependence on insecticides has led to the rapid spread of pyrethroid resistance in mosquito vectors, which is of great concern to the general public. There are many studies on metabolic resistance and target resistance, but fewer studies have been conducted on cuticle resistance and behaviour resistance. The cuticle of mosquitoes has been hypothesized to play a role in insecticide resistance by reducing penetration or sequestering insecticides. Methods We used RNA sequencing (RNA-seq) to analyse the transcriptome of cypermethrin-resistant and cypermethrin-susceptible strains of Culex pipiens pallens . Sequenced 6 samples using an Illumina HiSeq platform, and generated approximately 6.66 Gb bases from each sample on average. Mapping the sequenced reads to a reference genome and reconstructing the transcripts, through gene expression analysis, we detected differentially expressed genes (DEGs) among the samples. Followed Gene Ontology (GO) classification and functional enrichment. Finally, we screened the genes of cuticle proteins associated with drug resistance throughout the genome, selected the significant DEGs with a log2 fold change>3.0 and Padj<0.05, and applied real-time fluorescence quantitative PCR to verify the DEGs. Results We obtained 13,517 novel transcripts, of which 8,653 were previously unknown splicing events for known genes, 665 were novel coding transcripts without any known features, and 4,199 were long noncoding RNA. A total of 1035, 944, and 657 genes were upregulated in comparisons between samples, and 2680, 1215, and 975 genes were downregulated in comparisons between samples. Finally, among all samples, 167 genes upregulated, and 145 genes downregulated. The GO classification and functional enrichment of DEGs as follows: molecular function, 224 genes; cellular component, 149 genes; and biological process, 272 genes. The expression of XM_001863852 and XM_001845881 in resistant strains of Culex pipiens pallens was lower than that in the laboratory sensitive strain, with fold changes in expression of 0.177 and 0.548, respectively; the expression of the XM_001845883.1 in the resistant strain was higher than that in the susceptible strain, and a 2.281-fold change in expression. Conclusions The results provide a reference for resistance mechanisms through the mosquito cuticle, furthermore, could provide a new perspective for disease vector control. Parasitology Culex pipiens pallens insecticide resistance cuticle protein gene RNA-seq Figures Figure 1 Figure 4 Figure 6 Figure 8 Figure 10 Background Culex pipiens pallens is the most common mosquito in northern urban areas and townships in China. In addition to stings and bites, it is also the main vector of several arboviruses, such as West Nile Virus (WNV), St. Louis encephalitis (SLE), Sindbis virus (SINV), Rift Valley fever (RVFV), Japanese encephalitis (JEV), and the main vector for lymphatic filariasis [ 1 , 2 ]. Because most mosquito-borne diseases (except JEV) currently do not have effective vaccines or therapeutic drugs, effective control of vector mosquitoes is the main measure to prevent mosquito-borne diseases. Among the measures, chemical control is the primary method of vector management[ 3 , 4 ].Pyrethroid insecticides are synthetic analogs of naturally occurring pyrethrins from Chrysanthemum spp. [ 5 ]. Due to their low mammalian toxicity, high insecticidal activity, fast action and ease of decomposition in the environment, pyrethroids are currently a dominant class of insecticides used globally against mosquitoes and other human disease vectors[ 6 ].However, with the long-term use of insecticides, mosquito resistance is becoming increasingly more serious, which not only greatly reduces the control effect of existing insecticides but also makes the development of new drugs more difficult. The emergence of insecticide resistance has become a major obstacle for controlling mosquito-borne diseases [ 7 ]. Insecticides, pyrethroids in particular, remain a mainstay for the control of these important vectors. In this paper, we review what is known about the levels, mechanisms and fitness costs of pyrethroid resistance in Cx. pipiens . Pyrethroid resistance in Cx. pipiens is a global problem, and resistance ratios of up to 7000-fold have been found in larvae of field collected mosquitoes[ 8 ]. High levels of resistance to pyrethroids in Culex mosquitoes have been widely reported [ 9 , 10 ]. Previous surveys have shown that Culex pipiens pallens/Cx. quinquefasciatus in southern China have different levels of resistance to pyrethroid insecticides. In Hainan and other provinces, the resistance level has reached several thousand fold[ 11 ]. The development of mosquito resistance has had an important impact on the control of Culex pipiens pallens . Mosquito resistance mechanisms include metabolic resistance, target resistance, cuticle resistance, and behavioural resistance [ 12 ]. Metabolic resistance refers to the degradation, isolation, or transportation/excretion of insecticides from cells prior to binding the target. Metabolic resistance results from increased detoxification caused by the overexpression of or conformational changes in the enzymes involved in chemical insecticide metabolism, sequestration, and excretion. P450-monooxygenases, glutathione S-transferases, and carboxy/cholinesterases are the main enzymes involved in this process [ 13 – 16 ].Target-site resistance, or mutations in target binding sites for insecticides, iscaused by a modification of the chemical insecticide site of action, reducing or preventing insecticide binding at that site. Mutations in the voltage sensitive sodium channel ( Vssc ) gene are the most common causes of target-site resistance [ 17 ]. Behavioural resistance results from selection pressure of mosquitoes under long-term exposure to pesticides, and mosquitoes thus show a series of behavioural changes to avoid pesticides. For example, long-term application of indoor residual spraying (IRS) and insecticide-treated nets (ITNS) caused mosquitoes to change from endophagy to exophagy and from endophily to exophily and caused peak bloodsucking to change from late night to dusk [ 18 ]. Cuticle thickening is implicated in insecticide resistance by reducing the uptake of the insecticide that reaches the target site in response to the modification of chemical composition of the cuticle[ 19 ]. However, the mechanism remains poorly understood, and its importance in Aedes species is yet to be confirmed[ 14 , 20 , 21 ].A study revealed that this mechanism may play a major role in the development of resistance where it normally happens simultaneously with other mechanism(s) [ 22 ], causing resistance to single or multiple insecticides [ 23 ]. It has been reviewed elsewhere that cuticle thickening is associated with metabolic detoxification whereby thicker cuticle causes gradual insecticide absorption rate that will increase the effectiveness of metabolic detoxification in Anopheles funestus [ 24 ]. Moreover, it is crucial to take note that insects with cuticular resistance will display resistance level of not more than 3-fold in comparison to susceptible insects, but the co-occurrence of other resistance mechanism will lead to a surge in insecticide resistance level markedly [ 25 ]. This is demonstrated by Anopheles gambiae in Benin [ 26 ] through which overexpression of cuticular genes and P450 genes gave rise to a relatively high resistance level. High-throughput sequencing, i.e., RNA-seq, has become the main choice for measuring expression levels [ 27 ]. RNA-seq can be performed without prior knowledge of the reference or sequence of interest and allows a wide variety of applications such as the ‘de novo’ reconstruction of a transcriptome (without a reference genome), the evaluation of nucleotide variations, and the evaluation of methylation patterns [ 28 ]. RNA-seq technology has some advantages over the cDNA microarrays, such as a high level of data reproducibility, which reduces the number of technical replicates for experiments. In addition, RNA-seq allows identification and quantification of the expression of isoforms and unknown transcripts [ 29 ].A transcriptome is a collection of complete transcripts within a cell at a specific developmental stage and physiological state. The key goals of transcriptomics are to list transcripts of all species, including mRNAs, noncoding RNAs (ncRNAs) and small RNAs (sRNAs); to determine the transcriptional structure of genes, i.e., the transcriptional starting position, 5' and 3' ends, splicing patterns and other post-transcriptional modifications; and to quantify changes in the expression levels of each transcript under different developmental and treatment conditions. We used RNA-seq to analyse the transcriptome of cypermethrin-resistant and cypermethrin-susceptible strains of Culex pipiens pallens and obtained several cuticle protein genes that were differentially expressed in laboratory sensitive and resistant lines. However, these genes were unverified in the genome. We determined the sensitivity and resistance of mosquitoes to cypermethrin by real-time PCR and Centers for Disease Control and Prevention (CDC)Bottle Bioassay. In addition to the identification of the cypermethrin cuticle resistance genes, the relationship between the identified cuticle protein genes and mosquito resistance was verified to establish the specific mechanism of cypermethrin resistance in Culex pipiens pallens and provide new ideas for mosquito control and treatment. Methods Materials and methods Mosquito sample collection We collected laboratory sensitive and resistant strains of Culex pipiens pallens in the following developmental stages: I, II, III, and IV instar larvae, pupa and female Culex pipiens pallens 3 days after hatching that had not fed on blood. A total of 200 mg of Culex pipiens pallens was collected at each developmental stage and placed into a 1.5 ml Eppendorf (EP) tube, to which 150 µl of TRIzol lysis buffer was added to soak the mosquitoes. The samples were quickly stored in a -80 °C freezer, and RNA was extracted and sent to the BGI group for transcriptome sequencing. The resistant strain was screened from sensitive strains in our laboratory in accordance with the larvae dipping method recommended by the WHO. RNA sequencing Three RNA-seq libraries per population (3 biological replicates) were prepared using TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, U.S.A.). The extracted RNA was treated with RNase-free DNase (Qiagen GmbH, Hilden, Germany) and purified using an RNeasy MinElute Cleanup Kit (Qiagen GmbH) to remove DNA. The amount of total RNA was measured in a NanoDrop 2000 (Thermo Fisher Scientific, Inc.). The quality of the extracted RNA was verified by agarose gel electrophoresis. Subsequently, the reaction systems to synthesize the first and second strands of cDNA were constructed, and after the second strand of cDNA was synthesized, the ends of the double-stranded cDNA were blunt ended with the EcoRI restriction sequence. After terminal phosphorylation and XhoI digestion, dscDNA was recovered using a recovery kit. An Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System were then used for quality tests, and the Illumina HiSeq platform was used for RNA-seq after dscDNA quality was confirmed. Sequenced reads were assigned to each sample (unplexing), and adaptors were removed. Read quality was assessed for each sample using FastQC. Reads were then filtered based on their length, pairing and quality using Trimmomatic [30] with the following parameters: Leading, 25; Trailing, 25; Minlen, 60; and Slidingwindow, 4-25. Only paired reads were kept. Reads were then mapped to the Culex pipiens quinquefasciatus genome using Tophat2 [31] with the following parameters: don’t report discordant pair alignments; final read mismatches = 3; intron length = 45-300000; and use coverage search. Only read pairs mapping at a unique location (mapQ > 50) were retained. Quantification of the transcription levels was performed using the Cuffdiff2 module of Cufflinks implemented in Galaxy pipeline (http://galaxyproject.org) based on fragment per kilobase exon model (FPKM) values obtained for each gene across all samples. Transcription ratios between each resistant and each susceptible strain were computed across all biological replicates using Cuff-diff. Genes showing an FC ≥ 3 (in either direction) and a q value ≤ 0.001 between a given resistant population and all 3 susceptible strains were considered DEGs. The DEseq2 and PossionDis algorithms were used to perform DEG detection. DEseq2 is differential analysis software based on the principle of negative binomial distribution, and the analysis was conducted according to the method used in Michael I et al. [32]. The PossionDis difference analysis algorithm is based on the Poisson distribution model, and the analysis was conducted according to the method described in Audic S et al. [33]. Screening and verification of DEGs between sensitive and resistant strains Based on the results of the abovementioned transcriptome sequencing analysis, we analysed the genes related to cuticle proteins among the susceptible and resistant strain DEGs and identified 3 mRNAs with a fold change greater than 2 (-log2Ratio ≥ 1) and FDR ≤ 0.001 for subsequent real-time PCR verification. Referring to the Culex pipiens pallens genome data in the gene library, real-time quantitative PCR primers were designed using Primer Premier 5 software and the nucleotide sequences of the selected mRNAs, and β-actin was used as the quantitative internal mRNA reference. The primers were synthesized by Shenzhen BGI. The base sequences of the specific primers are provided in Table 1. RNA was extracted using TRIzol reagent, and cDNA was synthesized. cDNA was synthesized using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Inc.) using oligo(dT)18. qRT-PCR was performed using a CFX96 Touch (Bio-Rad Laboratories, Inc., Hercules, CA, U.S.A.). Twenty-five nanograms of cDNA and 500 nM of each forward and reverse primer were used in each reaction. The relative expression of each gene in resistant and susceptible mosquitoes was calculated by the ΔΔCt method [34] using actin (ADIR001186-RA) as a control. Real-time PCR data were analysed using reliability simulation tool (REST) software and hypothesis testing; other data were expressed as the mean ± standard deviation (XJ ± S) and analysed with STATA7.0 software. Student's T test was performed for comparisons between groups, and p<0.05 was used as a basis for determining statistical significance. Table 1 Nucleotide sequence of each gene primer and related information Gene ID Primer sequence (5'-3') Primer length Product size XM_001845883.1-F TGCCATCCGTTTCTTCCA 18 103 XM_001845883.1-R GGGCTCAACCAGGGAGTAAG 20 XM_001863852-F ATGCCATCGTGAAGGGTGT 19 93 XM_001863852 -R GACTCTTGATGTCTCCGGTGTG 22 actin-F AGGACTCGTACGTCGGTGAC 20 - actin-R TGGTGCCAGATCTTCTCTCCAT 22 XM_001845881-F CACATTCGGATTACAAAATG 20 196 XM_001845881-R GTGGTAGCTGTAGCTGTACTG 21 Results Overview of RNA-seq data We selected 3 samples from different physiological stages of the sensitive and resistant strains, for a total of 6 samples, and each sample produced an average of 6.66 Gb of data. The sequenced clean reads were compared with the reference genome of Culex pipiens pallens , and the transcripts were reintegrated. A total of 13,517 new transcripts were detected, of which 8,653 were new alternative splicing isoforms of existing known protein-coding genes, 665 were transcripts of unknown protein-coding genes, and 4,199 were lncRNAs (see Tables 2, 3). Table 2 Summary of differentially expressed genes VS Upregulated Downregulated Cx_S_strain-VS-Cx_R_strain.DEseq2 167 145 Cx_S_strain-VS-Cx_R_strain.DEseq3 1035 2680 Cx_S_strain-VS-Cx_R_strain.DEseq4 944 1215 Cx_S_strain-VS-Cx_R_strain.DEseq5 657 975 Table 3 Summary of whole genome expression Sample name Total gene number Number of known genes Number of novel genes Total transcript number Known transcript number Novel transcript number R_strain_1 14597 14036 561 19094 11413 7681 R_strain_2 14507 13921 586 19460 11415 8045 R_strain_3 14592 14016 576 19733 11533 8200 S_strain_1 14603 14040 563 19381 11440 7941 S_strain_2 14551 13980 571 19671 11562 8109 S_strain_3 14568 13999 569 19679 11501 8178 Prediction of new transcripts After comparing the clean reads to the Culex pipiens pallens genome, we used StringTie [35] software to perform transcript reintegration for each sample and then used cuffmerge and cuffcompare software (both are packages in Cufflinks [36]) to compare the reintegrated transcripts with the annotation information for the Culex pipiens genome. We selected transcripts with a class code type of u, i, o, and j as candidates for novel transcripts. A total of 13,517 new transcripts were detected. Detailed statistical information is provided in Table 4. [Due to technical limitations, Table 4 could not be displayed here. Please see the supplementary files section to access the table.] Detection of SNPs and INDELs After comparing clean reads to the Culex pipiens pallens genome, we used Genome Analysis Toolkit (GATK) [37] software to call each chromosome, identify single nucleotide polymorphisms (SNPs) and insertion and deletion (INDEL) sites for each sample, and store the final results in variant call format (VCF). The SNP statistical information for all samples is provided in Table 5. We then analysed the site information for each SNP and INDEL, as shown in Figure 1 and Figure 2. [See supplementary files for Table 5.] Numbers, functional categorization and pathway analysis of DEGs DEGs were obtained by comparing the expression levels of differential genes between the sample groups. The results are shown in Figure 3. DEGs were compared with the GO library for functional classification. GO included 3 major categories: molecular function, cellular composition and biological process. Subsequently, the 3 categories were separately classified. The results are provided in Figure 4. The obtained DEGs were compared with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and the related metabolic pathways were identified. The results are provided in Figure 5. Validation of the 3 target cuticle protein genes To ensure amplification of the target genes and the housekeeping gene, we performed primer verification. The results showed that the amplification curve for the primers was good and that the melting curve was monomodal, and the electrophoresis results revealed specific target fragments. The average Ct value for the actin gene in the sensitive group was 25.905; the average Ct value of the actin gene in the resistant group was 26.227; the average Ct value of the XM_001863852 gene in the sensitive group was 26.813; the average Ct value of the XM_001863852 gene in the resistant group was 29.633; the average Ct value of the XM_001845883.1 gene in the sensitive group was 36.797; the average Ct value of the XM_001845883.1 gene in the resistant group was 35.93; the average Ct value of the XM_001845881 gene in the sensitive group was 32.647; and the average Ct value of the XM_001845881 gene in the resistant group was 33.837 (see Tables 6 and 7 for details). Subsequently, we used the 2- △△ Ct method to analyse the expression of target gene mRNA in the extracted RNA and normalized the result based on the housekeeping gene. The specific calculation method is as follows: △△ Ct = (Ct target gene - Ct housekeeping gene) experimental group - (Ct target gene - Ct housekeeping gene) control group The relative expression level of the target gene = 2^- △△ Ct, which indicates the fold change in expression for the target gene in the experimental group relative to the control group. The calculation results are provided in Tables 6 and 7. Table 6 Quantitative PCR results Target ID Target Name Sample ID Sample Name Ct Avg (SDM) Rel. Qty (SDM) T001 Actin S001 S (sensitive) 1 25.475 1.00E+00 T001 Actin S002 S 2 26.205 1.00E+00 T001 Actin S003 S 3 26.035 1.00E+00 T001 Actin S004 R (resistance) 1 25.37 1.00E+00 T001 Actin S005 R 2 26.19 1.00E+00 T001 Actin S006 R 3 27.12 1.00E+00 T002 XM_001863852 S001 S 1 31.79 1.00E+00 T002 XM_001863852 S002 S 2 24.00 3.67E+02 T002 XM_001863852 S003 S 3 24.65 2.08E+02 T002 XM_001863852 S004 R 1 30.235 2.73E+00 T002 XM_001863852 S005 R 2 30.61 3.72E+00 T002 XM_001863852 S006 R 3 28.055 4.16E+01 T003 XM_001845883.1 S001 S 1 36.19 1.00E+00 T003 XM_001845883.1 S002 S 2 37.07 9.01E-01 T003 XM_001845883.1 S003 S 3 37.13 7.68E-01 T003 XM_001845883.1 S004 R 1 35.43 1.58E+00 T003 XM_001845883.1 S005 R 2 35.94 1.95E+00 T003 XM_001845883.1 S006 R 3 36.42 2.67E+00 T004 XM_001845881 S001 S 1 36.48 1.00E+00 T004 XM_001845881 S002 S 2 29.33 2.36E+02 T004 XM_001845881 S003 S 3 32.13 3.01E+01 T004 XM_001845881 S004 R 1 33.04 1.01E+01 T004 XM_001845881 S005 R 2 35.15 4.13E+00 T004 XM_001845881 S006 R3 33.32 2.80E+01 Table 7 Calculation of the relative quantitative Ct values for target genes and the internal reference gene β-actin XM_001863852 XM_001845883.1 XM_001845881 Average Ct value in the sensitive group 25.905 26.813 36.797 32.647 Average Ct value in the resistant group 26.227 29.633 35.93 33.837 △ Ct value in the sensitive group ----- 0.908 10.892 6.742 △ Ct value in the resistant group ----- 3.406 9.703 7.61 △△ Ct value ----- 2.498 -1.189 0.868 2^- △△ Ct value ----- 0.177 2.281 0.548 It can be seen from Table 7 that the expression levels of the target genes XM_001863852 and XM_001845881 were similar between the sensitive and resistant strains of Culex pipiens pallens ; the fold changes in expression were 0.177 and 0.548, respectively. The expression level of the target gene XM_001845883.1 in the resistant strain was higher than that in the sensitive strain, and the fold change in expression was 2.281. Discussion It has been reported that at least 504 insect species worldwide exhibit chemical insecticide resistance [ 38 ], that more than 109 (subspecies) vector mosquitoes are resistant to one or more insecticides [ 39 ], and that the number of species and the quantities of resistant insects have increased year to year. Culex pipiens pallens is an important vector for lymphatic filariasis and Japanese encephalitis virus. It is one of the main stinging and harassing mosquito species in cities and towns. Chemical pesticides have played an important role in the prevention and treatment of malaria and filariasis in Shandong Province. However, in recent years, due to the need to establish healthy and civilized cities, the large, continuous and irregular use of pesticides has led to the development of drug resistance and caused environmental pollution, while the degree of resistance by mosquitoes has also shown an increasing trend. The occurrence and development of resistance in mosquitos has limited the further application of chemical pesticides and has become a prominent problem in the prevention and control of insect-borne diseases in China and abroad. Based on the high chemical resistance of Culex pipiens pallens in Shandong Province[ 40 ], this study used high-throughput transcriptome sequencing technology to qualitatively and quantitatively study cypermethrin-resistant strains of Culex pipiens pallens and identified the cuticle protein genes responsible for the drug-resistant phenotype. Real-time quantitative PCR were used to verify the identified cuticle protein genes and to confirm whether there is a high correlation between cuticle protein genes and the drug resistance phenotype. Mosquito resistance generally includes 4 types: metabolic resistance, target resistance, cuticle resistance, and behavioural resistance. In 1963, a survey of houseflies ( Fannia canicularis ) identified the cause of insecticide resistance. It was found that the penetration of chemical pesticides in resistant lines was slower than in sensitive lines, suggesting that slower penetration could be the cause for dichlorodiphenyltrichloroethane (DDT) and pyrethroid resistance [ 41 ]. Studies have shown that pyrethroid-resistant female Anopheles sinensis have thicker cuticles than do sensitive female Anopheles sinensis and that female mosquitoes also have thicker cuticles than do male mosquitoes [ 42 ]. In addition, Lily et al. [ 43 ] confirmed that cuticle thickening is present in pyrethroid-resistant strains of Cimex lectularius . Cuticle analysis by electron microscopy and characterization of lipid extracts showed that resistant mosquitoes had a thicker outer skin layer and a higher hydrocarbon content (approximately 29%) [ 44 ]. Multiple studies have confirmed that the cuticle of resistant lines is much thicker than that of sensitive lines [ 43 , 44 ]. The cuticular protein (CP) family was first discovered in 2007 by tandem mass spectrometry analysis of epidermal exfoliation from Anopheles gambiae [ 45 ]. Most gene family members have the prefix CPLC (cuticular protein of low complexity), and such members often play an important role in protein-protein interaction networks [ 46 , 47 ]. The above evidence suggests that insect cuticle proteins play an indispensable role in mosquito resistance. The expression of the target genes XM_001863852 and XM_001845881 in the Culex pipiens pallens laboratory resistant strain was lower than that in the laboratory sensitive strain, and the fold changes in expression were 0.177 and 0.548, respectively, while the expression of XM_001845883.1 in the resistant strain was higher than that in the sensitive strain, and with a 2.281-fold change in expression. GO function analysis indicated that all 3 were genes responsible for cuticle structural components, and an NR database comparison also showed that these genes code cuticle proteins in Culex pipiens . In fact, when we screened the genes, we initially identified 3 different genes in the XM_001845 series: XM_001845880, XM_001845881 and XM_001845883.1. However, when designing the primers, the primers designed for these 3 were not ideal, and there were varying degrees of non-specific amplification. We compared the designed primer fragments to the BLAST database and found that the primers were highly consistent with an unknown conserved hypothetical protein. We questioned if the 3 different genes in the XM_001845 series were different splicing isoforms of the same gene, and if so, whether such frequent splicing could promote resistance in mosquitoes. After considering the experimental cost, experimental significance and feasibility of the experiment, we selected 2 of the genes to conduct further validation, and the experimental results for XM_001845881 and XM_001845883.1 were opposite to each other. Therefore, we questioned whether the 2 genes had antagonistic effects in the genetic pathways leading to the formation of cuticle resistance in mosquitoes; for example, XM_001845883.1 is responsible for promoting the formation of cuticle resistance, while XM_001845881 is responsible for regulating the expression of other upstream or downstream cuticle protein genes, preventing the overexpression of other related cuticle proteins. Because the 3 identified target genes in our study are novel, the original identification process was relatively complicated and innovative; however, the specific regulatory networks and the in vivo function of the respective genes need to be further explored. Chemical control has become the main means of controlling disease vectors and reducing vector infectious diseases, including the indoor and outdoor use of adult mosquito insecticides and the use of larval insecticides at mosquito breeding sites. However, the emergence and spread of pesticide resistance seriously affects disease vector control. Due to the lack of knowledge about the geographical distribution of potential pesticide resistance mechanisms, it is difficult to develop effective pesticide resistance management strategies. In addition, the spatiotemporal principles for the selection and use of pesticides also need further planning and implementation. Conclusions These data provide transcriptomic information related to the resistance of Culex pipiens pallens and preliminarily verify the relationship between the identified cuticle protein genes and mosquito drug resistance, partially explaining the specific mechanism of mosquito cuticle resistance, providing a scientific basis for the study of new target insecticides, and providing new ideas for the prevention and control of mosquitoes. Abbreviations RNA-seq RNA sequencing CPGs cuticular protein genes CP cuticular protein DEGs differentially expressed genes GO Gene Ontology PCR Polymerase Chain Reaction WNV West Nile Virus SLE St. Louis encephalitis SINV Sindbis virus RVFV Rift Valley fever JEV Japanese encephalitis Vssc voltage sensitive sodium channel IRS indoor residual spraying ITNS insecticide-treated nets ncRNAs noncoding RNAs sRNAs small RNAs CDC Centers for Disease Control and Prevention EP Eppendorf dscDNA double strands cDNA FPKM fragment per kilobase exon model REST reliability simulation tool lncRNAs long noncoding RNAs GATK Genome Analysis Toolkit SNPs single nucleotide polymorphisms INDEL insertion and deletion VCF variant call format KEGG Kyoto Encyclopedia of Genes and Genomes DDT dichlorodiphenyltrichloroethane CPLC cuticular protein of low complexity WHO World Health Organization Declarations Acknowledgements We are grateful to BGI-genomics, BGI-Shenzhen (Shenzhen, 518083, China) for their assistance with the sequencing and bioinformatics analysis. Funding This work was supported by grants from the National Natural Science Foundation of China [81871685 (MQG), 81672059 (MQG), 81471985(MQG)], the Innovation Project of Shandong Academy of Medical Sciences (MQG), the key research and development program of Shandong province (2018GSF118092, 2019GSF111006) Availability of data and materials All data generated or analyzed during this study are included in this published article and its additional files. Authors’ contributions QQS, PC, CXZ, IJL, XS, XXG, HFW, YW, HML provided the samples collection and wrote the manuscript. HWW and MQG reviewed and edited the manuscript. All authors read and approved the final manuscript. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References [1] Grigoraki L, Puggioli A, Mavridis K, Douris V, Montanari M, Bellini R, et al. 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Cuticle thickening associated with pyrethroid resistance in the major malaria vector Anopheles funestus . Parasit Vectors. 2010;3:67. [43]Lilly DG, Latham SL, Webb CE, Doggett SL. Cuticle thickening in apyrethroid-resistant strain of the common bed bug, Cimexlectularius L (Hemiptera: Cimicidae). PLoS One. 2016;11(4): e0153302.doi: 10.1371/journal.pone.0153302. [44] Jacobs CG, Braak N, Lamers GE, van der Zee M. Elucidation of the serosal cuticle machinery in the beetle Tribolium by RNA sequencing and functional analysis of Knickkopf1,Retroactive and Laccase2. Insect Mol Biol. 2015;60:7-12. [45]He N, Botelho JM, McNall RJ, Belozerov V, Dunn WA, Mize T, et al. Proteomic analysis of cast cuticles from Anopheles gambiae by tandem mass spectrometry. Insect Mol Biol. 2007;37(2):135-146. [46] Cornman RS, Willis JH. Annotation and analysis of low-complexity protein families of Anopheles gambiae that are associated with cuticle. Insect Mol Biol. 2009;18(5):607-622. [47] Mc Dougall C, Aguilera F, Degnan BM. Rapid evolution of pearl oyster shellmatrix proteins with repetitive, low-complexity domains. J R Soc Interface.2013;10(82):20130041. Supplementary Files Table5.docx Table5.docx Table4.docx GraphicalAbstract.docx GraphicalAbstract.docx Table4.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9873","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":260205,"identity":"074a96a8-c175-43de-ae5a-e190983dd549","order_by":1,"name":"Qiqi Shi","email":"","orcid":"https://orcid.org/0000-0002-0182-9887","institution":"shandong institute of parasitic diseases,shandong academy of medical sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qiqi","middleName":"","lastName":"Shi","suffix":""},{"id":260206,"identity":"c72d8dbd-44ac-4bda-bc92-e966bc56c7e3","order_by":2,"name":"Peng 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20:52:27","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":27082,"visible":true,"origin":"","legend":"","description":"","filename":"Table4.docx","url":"https://assets-eu.researchsquare.com/files/b18a97d7-6554-4bcb-b250-e8a69c54fa66/v1/Table 4 .docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eRNA-seq screening of the CPGs in \u003cem\u003eCulex pipiens pallens \u003c/em\u003eamong cypermethrin-resistant populations\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003e \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCulex pipiens pallens\u003c/span\u003e is the most common mosquito in northern urban areas and townships in China. In addition to stings and bites, it is also the main vector of several arboviruses, such as West Nile Virus (WNV), St. Louis encephalitis (SLE), Sindbis virus (SINV), Rift Valley fever (RVFV), Japanese encephalitis (JEV), and the main vector for lymphatic filariasis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Because most mosquito-borne diseases (except JEV) currently do not have effective vaccines or therapeutic drugs, effective control of vector mosquitoes is the main measure to prevent mosquito-borne diseases. Among the measures, chemical control is the primary method of vector management[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].Pyrethroid insecticides are synthetic analogs of naturally occurring pyrethrins from Chrysanthemum spp. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Due to their low mammalian toxicity, high insecticidal activity, fast action and ease of decomposition in the environment, pyrethroids are currently a dominant class of insecticides used globally against mosquitoes and other human disease vectors[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].However, with the long-term use of insecticides, mosquito resistance is becoming increasingly more serious, which not only greatly reduces the control effect of existing insecticides but also makes the development of new drugs more difficult. The emergence of insecticide resistance has become a major obstacle for controlling mosquito-borne diseases [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInsecticides, pyrethroids in particular, remain a mainstay for the control of these important vectors. In this paper, we review what is known about the levels, mechanisms and fitness costs of pyrethroid resistance in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCx. pipiens\u003c/span\u003e. Pyrethroid resistance in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCx. pipiens\u003c/span\u003e is a global problem, and resistance ratios of up to 7000-fold have been found in larvae of field collected mosquitoes[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHigh levels of resistance to pyrethroids in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCulex\u003c/span\u003e mosquitoes have been widely reported [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Previous surveys have shown that \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCulex pipiens pallens/Cx. quinquefasciatus\u003c/span\u003e in southern China have different levels of resistance to pyrethroid insecticides. In Hainan and other provinces, the resistance level has reached several thousand fold[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The development of mosquito resistance has had an important impact on the control of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCulex pipiens pallens\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eMosquito resistance mechanisms include metabolic resistance, target resistance, cuticle resistance, and behavioural resistance [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Metabolic resistance refers to the degradation, isolation, or transportation/excretion of insecticides from cells prior to binding the target. Metabolic resistance results from increased detoxification caused by the overexpression of or conformational changes in the enzymes involved in chemical insecticide metabolism, sequestration, and excretion. P450-monooxygenases, glutathione S-transferases, and carboxy/cholinesterases are the main enzymes involved in this process [\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].Target-site resistance, or mutations in target binding sites for insecticides, iscaused by a modification of the chemical insecticide site of action, reducing or preventing insecticide binding at that site. Mutations in the voltage sensitive sodium channel (\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eVssc\u003c/span\u003e) gene are the most common causes of target-site resistance [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Behavioural resistance results from selection pressure of mosquitoes under long-term exposure to pesticides, and mosquitoes thus show a series of behavioural changes to avoid pesticides. For example, long-term application of indoor residual spraying (IRS) and insecticide-treated nets (ITNS) caused mosquitoes to change from endophagy to exophagy and from endophily to exophily and caused peak bloodsucking to change from late night to dusk [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCuticle thickening is implicated in insecticide resistance by reducing the uptake of the insecticide that reaches the target site in response to the modification of chemical composition of the cuticle[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, the mechanism remains poorly understood, and its importance in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eAedes\u003c/span\u003e species is yet to be confirmed[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].A study revealed that this mechanism may play a major role in the development of resistance where it normally happens simultaneously with other mechanism(s) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], causing resistance to single or multiple insecticides [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It has been reviewed elsewhere that cuticle thickening is associated with metabolic detoxification whereby thicker cuticle causes gradual insecticide absorption rate that will increase the effectiveness of metabolic detoxification in Anopheles funestus [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Moreover, it is crucial to take note that insects with cuticular resistance will display resistance level of not more than 3-fold in comparison to susceptible insects, but the co-occurrence of other resistance mechanism will lead to a surge in insecticide resistance level markedly [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This is demonstrated by Anopheles gambiae in Benin [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] through which overexpression of cuticular genes and P450 genes gave rise to a relatively high resistance level.\u003c/p\u003e \u003cp\u003eHigh-throughput sequencing, i.e., RNA-seq, has become the main choice for measuring expression levels [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. RNA-seq can be performed without prior knowledge of the reference or sequence of interest and allows a wide variety of applications such as the \u0026lsquo;de novo\u0026rsquo; reconstruction of a transcriptome (without a reference genome), the evaluation of nucleotide variations, and the evaluation of methylation patterns [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. RNA-seq technology has some advantages over the cDNA microarrays, such as a high level of data reproducibility, which reduces the number of technical replicates for experiments. In addition, RNA-seq allows identification and quantification of the expression of isoforms and unknown transcripts [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].A transcriptome is a collection of complete transcripts within a cell at a specific developmental stage and physiological state. The key goals of transcriptomics are to list transcripts of all species, including mRNAs, noncoding RNAs (ncRNAs) and small RNAs (sRNAs); to determine the transcriptional structure of genes, i.e., the transcriptional starting position, 5' and 3' ends, splicing patterns and other post-transcriptional modifications; and to quantify changes in the expression levels of each transcript under different developmental and treatment conditions.\u003c/p\u003e \u003cp\u003eWe used RNA-seq to analyse the transcriptome of cypermethrin-resistant and cypermethrin-susceptible strains of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCulex pipiens pallens\u003c/span\u003e and obtained several cuticle protein genes that were differentially expressed in laboratory sensitive and resistant lines. However, these genes were unverified in the genome. We determined the sensitivity and resistance of mosquitoes to cypermethrin by real-time PCR and Centers for Disease Control and Prevention (CDC)Bottle Bioassay. In addition to the identification of the cypermethrin cuticle resistance genes, the relationship between the identified cuticle protein genes and mosquito resistance was verified to establish the specific mechanism of cypermethrin resistance in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCulex pipiens pallens\u003c/span\u003e and provide new ideas for mosquito control and treatment.\u003c/p\u003e "},{"header":"Methods","content":" \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eMaterials and methods\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eMosquito sample collection\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:18.75pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eWe collected laboratory sensitive and resistant strains of \u003cem\u003eCulex pipiens pallens\u003c/em\u003e in the following developmental stages: I, II, III, and IV instar larvae, pupa and female \u003cem\u003eCulex pipiens pallens\u003c/em\u003e 3 days after hatching that had not fed on blood. A total of 200 mg of \u003cem\u003eCulex pipiens pallens\u003c/em\u003e was collected at each developmental stage and placed into a 1.5 ml Eppendorf (EP) tube, to which 150 µl of TRIzol lysis buffer was added to soak the mosquitoes. The samples were quickly stored in a -80 °C freezer, and RNA was extracted and sent to the BGI group for transcriptome sequencing. The resistant strain was screened from sensitive strains in our laboratory in accordance with the larvae dipping method recommended by the WHO.\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eRNA sequencing\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eThree RNA-seq libraries per population (3 biological replicates) were prepared using TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, U.S.A.). The extracted RNA was treated with RNase-free DNase (Qiagen GmbH, Hilden, Germany) and purified using an RNeasy MinElute Cleanup Kit (Qiagen GmbH) to remove DNA. The amount of total RNA was measured in a NanoDrop 2000 (Thermo Fisher Scientific, Inc.). The quality of the extracted RNA was verified by agarose gel electrophoresis. Subsequently, the reaction systems to synthesize the first and second strands of cDNA were constructed, and after the second strand of cDNA was synthesized, the ends of the double-stranded cDNA were blunt ended with the EcoRI restriction sequence. After terminal phosphorylation and XhoI digestion, dscDNA was recovered using a recovery kit. An Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System were then used for quality tests, and the Illumina HiSeq platform was used for RNA-seq after dscDNA quality was confirmed.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eSequenced reads were assigned to each sample (unplexing), and adaptors were removed. Read quality was assessed for each sample using FastQC. Reads were then filtered based on their length, pairing and quality using Trimmomatic [30] with the following parameters: Leading, 25; Trailing, 25; Minlen, 60; and Slidingwindow, 4-25. Only paired reads were kept. Reads were then mapped to the \u003cem\u003eCulex pipiens quinquefasciatus\u003c/em\u003e genome using Tophat2 [31] with the following parameters: don’t report discordant pair alignments; final read mismatches = 3; intron length = 45-300000; and use coverage search. Only read pairs mapping at a unique location (mapQ \u0026gt; 50) were retained. Quantification of the transcription levels was performed using the Cuffdiff2 module of Cufflinks implemented in Galaxy pipeline (http://galaxyproject.org) based on\u0026nbsp;fragment per kilobase exon model (FPKM) values obtained for each gene across all samples.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eTranscription ratios between each resistant and each susceptible strain were computed across all biological replicates using Cuff-diff. Genes showing an FC ≥ 3 (in either direction) and a q value ≤ 0.001 between a given resistant population and all 3 susceptible strains were considered DEGs.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eThe DEseq2 and PossionDis algorithms were used to perform DEG detection. DEseq2 is differential analysis software based on the principle of negative binomial distribution, and the analysis was conducted according to the method used in Michael I et al. [32]. The PossionDis difference analysis algorithm is based on the Poisson distribution model, and the analysis was conducted according to the method described in Audic S et al. [33].\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.05pt;line-height:150%;font-size:16px;font-family:SimSun;text-indent:-.05pt;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eScreening and verification of DEGs between sensitive and resistant strains\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eBased on the results of the abovementioned transcriptome sequencing analysis, we analysed the genes related to cuticle proteins among the susceptible and resistant strain DEGs and identified 3 mRNAs with a fold change greater than 2 (-log2Ratio ≥ 1) and FDR ≤ 0.001 for subsequent real-time PCR verification.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eReferring to the \u003cem\u003eCulex pipiens pallens\u003c/em\u003e genome data in the gene library, real-time quantitative PCR primers were designed using Primer Premier 5 software and the nucleotide sequences of the selected mRNAs, and β-actin was used as the quantitative internal mRNA reference. The primers were synthesized by Shenzhen BGI. The base sequences of the specific primers are provided in Table 1. RNA was extracted using TRIzol reagent, and cDNA was synthesized. cDNA was synthesized using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Inc.) using oligo(dT)18. qRT-PCR was performed using a CFX96 Touch (Bio-Rad Laboratories, Inc., Hercules, CA, U.S.A.). Twenty-five nanograms of cDNA and 500 nM of each forward and reverse primer were used in each reaction. The relative expression of each gene in resistant and susceptible mosquitoes was calculated by the ΔΔCt method [34] using actin (ADIR001186-RA) as a control. Real-time PCR data were analysed using reliability simulation tool (REST) software and hypothesis testing; other data were expressed as the mean ± standard deviation (XJ ± S) and analysed with STATA7.0 software. Student's T test was performed for comparisons between groups, and p\u0026lt;0.05 was used as a basis for determining statistical significance.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\n \u003cbr\u003e\n\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eTable 1 Nucleotide sequence of each gene primer and related information\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"margin-left:5.15pt;border-collapse:collapse;border:none;\" width=\"561\" cellspacing=\"0\" cellpadding=\"0\" border=\"1\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:120.75pt;border-top:solid windowtext 1.5pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\" width=\"28.698752228163993%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:15px;line-height: 107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eGene ID\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:191.35pt;border-top:solid windowtext 1.5pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\" width=\"45.45454545454545%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:15px;line-height: 107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003ePrimer sequence (5'-3')\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.55pt;border-top:solid windowtext 1.5pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\" width=\"10.16042780748663%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:15px;line-height: 107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003ePrimer length\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.3pt;border-top:solid windowtext 1.5pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\" width=\"15.686274509803921%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:15px;line-height: 107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eProduct size\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120.75pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"28.698752228163993%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eXM_001845883.1-F\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191.35pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"45.45454545454545%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eTGCCATCCGTTTCTTCCA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"10.16042780748663%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;text-indent:14.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e18\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 66.3pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"15.686274509803921%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e103\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120.75pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"34.038054968287526%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eXM_001845883.1-R\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191.35pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"53.911205073995774%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eGGGCTCAACCAGGGAGTAAG\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"12.050739957716702%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;text-indent:14.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e20\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120.75pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"28.698752228163993%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eXM_001863852-F\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191.35pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"45.45454545454545%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eATGCCATCGTGAAGGGTGT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"10.16042780748663%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;text-indent:14.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e19\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 66.3pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"15.686274509803921%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e93\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120.75pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"34.038054968287526%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eXM_001863852 -R\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191.35pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"53.911205073995774%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eGACTCTTGATGTCTCCGGTGTG\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"12.050739957716702%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;text-indent:14.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e22\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120.75pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"28.698752228163993%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eactin-F\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191.35pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"45.45454545454545%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eAGGACTCGTACGTCGGTGAC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"10.16042780748663%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;text-indent:14.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e20\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 66.3pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"15.686274509803921%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120.75pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"34.038054968287526%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eactin-R\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191.35pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"53.911205073995774%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eTGGTGCCAGATCTTCTCTCCAT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"12.050739957716702%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;text-indent:14.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e22\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120.75pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"28.698752228163993%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eXM_001845881-F\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191.35pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"45.45454545454545%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eCACATTCGGATTACAAAATG\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"10.16042780748663%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;text-indent:14.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e20\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 66.3pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 13.5pt;vertical-align: bottom;\" width=\"15.686274509803921%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e196\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120.75pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 10.85pt;vertical-align: bottom;\" width=\"34.038054968287526%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eXM_001845881-R\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191.35pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 10.85pt;vertical-align: bottom;\" width=\"53.911205073995774%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:.25in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eGTGGTAGCTGTAGCTGTACTG\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 10.85pt;vertical-align: bottom;\" width=\"12.050739957716702%\" valign=\"bottom\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;text-indent:14.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e21\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Results","content":"\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eOverview of RNA-seq data\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eWe selected 3 samples from different physiological stages of the sensitive and resistant strains, for a total of 6 samples, and each sample produced an average of 6.66 Gb of data. The sequenced clean reads were compared with the reference genome of \u003cem\u003eCulex pipiens pallens\u003c/em\u003e, and the transcripts were reintegrated. A total of 13,517 new transcripts were detected, of which 8,653 were new alternative splicing isoforms of existing known protein-coding genes, 665 were transcripts of unknown protein-coding genes, and 4,199 were lncRNAs (see Tables 2, 3).\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\n \u003cbr\u003e\n\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:center;text-indent:28.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eTable 2 Summary of differentially expressed genes\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\" width=\"568\" cellspacing=\"0\" cellpadding=\"0\" border=\"1\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 180.9pt;border-color: rgb(79, 129, 189) currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0in 5.4pt;vertical-align: top;\" width=\"42.429577464788736%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eVS\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120.6pt;border-color: rgb(79, 129, 189) currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0in 5.4pt;vertical-align: top;\" width=\"28.345070422535212%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eUpregulated\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124.6pt;border-color: rgb(79, 129, 189) currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0in 5.4pt;vertical-align: top;\" width=\"29.225352112676056%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eDownregulated\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 180.9pt;border: medium none;background: rgb(211, 223, 238) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"42.429577464788736%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eCx_S_strain-VS-Cx_R_strain.DEseq2\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120.6pt;border: medium none;background: rgb(211, 223, 238) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"28.345070422535212%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e167\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124.6pt;border: medium none;background: rgb(211, 223, 238) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"29.225352112676056%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e145\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 180.9pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"42.429577464788736%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eCx_S_strain-VS-Cx_R_strain.DEseq3\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120.6pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"28.345070422535212%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e1035\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124.6pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"29.225352112676056%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e2680\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 180.9pt;border: medium none;background: rgb(211, 223, 238) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"42.429577464788736%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eCx_S_strain-VS-Cx_R_strain.DEseq4\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120.6pt;border: medium none;background: rgb(211, 223, 238) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"28.345070422535212%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e944\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124.6pt;border: medium none;background: rgb(211, 223, 238) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"29.225352112676056%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e1215\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 180.9pt;border-color: currentcolor currentcolor rgb(79, 129, 189);border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\" width=\"42.429577464788736%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eCx_S_strain-VS-Cx_R_strain.DEseq5\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120.6pt;border-color: currentcolor currentcolor rgb(79, 129, 189);border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\" width=\"28.345070422535212%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e657\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124.6pt;border-color: currentcolor currentcolor rgb(79, 129, 189);border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\" width=\"29.225352112676056%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e975\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;text-indent:18.7pt;\"\u003e\n \u003cbr\u003e\n\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;text-indent:18.7pt;\"\u003e\n \u003cbr\u003e\n\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;text-align:center;text-indent:18.7pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eTable 3 Summary of whole genome expression\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\" width=\"565\" cellspacing=\"0\" cellpadding=\"0\" border=\"1\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76.3pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0in 5.4pt;vertical-align: top;\" width=\"17.98941798941799%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eSample name\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eTotal gene number\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eNumber of known genes\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eNumber of novel genes\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eTotal transcript number\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0in 5.4pt;vertical-align: top;\" width=\"14.991181657848324%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eKnown transcript number\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eNovel transcript number\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76.3pt;border: medium none;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"17.98941798941799%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eR_strain_1\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e14597\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e14036\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e561\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e19094\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border: medium none;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"14.991181657848324%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e11413\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e7681\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76.3pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"17.98941798941799%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eR_strain_2\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e14507\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e13921\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e586\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e19460\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"14.991181657848324%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e11415\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e8045\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76.3pt;border: medium none;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"17.98941798941799%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eR_strain_3\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New 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style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e576\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e19733\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border: medium none;background: rgb(198, 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Roman\u0026quot;,serif;color:black;\"\u003e8200\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76.3pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"17.98941798941799%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eS_strain_1\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e14603\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e14040\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e563\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e19381\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"14.991181657848324%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e11440\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e7941\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76.3pt;border: medium none;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"17.98941798941799%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eS_strain_2\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e14551\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e13980\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e571\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e19671\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border: medium none;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"14.991181657848324%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e11562\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: medium none;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e8109\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76.3pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\" width=\"17.98941798941799%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eS_strain_3\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e14568\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e13999\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e569\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e19679\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\" width=\"14.991181657848324%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e11501\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\" width=\"13.403880070546737%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e8178\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;\"\u003e\n \u003cbr\u003e\n\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 19px; line-height: 150%; font-family: \u0026quot;Times New Roman\u0026quot;, serif;\"\u003ePrediction of new transcripts\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eAfter comparing the clean reads to the \u003cem\u003eCulex pipiens pallens\u0026nbsp;\u003c/em\u003egenome, we used StringTie [35] software to perform transcript reintegration for each sample and then used cuffmerge and cuffcompare software (both are packages in Cufflinks [36]) to compare the reintegrated transcripts with the annotation information for the \u003cem\u003eCulex pipiens\u003c/em\u003e genome. We selected transcripts with a class code type of u, i, o, and j as candidates for novel transcripts. A total of 13,517 new transcripts were detected. Detailed statistical information is provided in Table 4.\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\n \u003cbr\u003e\n\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e[Due to technical limitations, Table 4 could not be displayed here. Please see the supplementary files section to access the table.]\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\n \u003cbr\u003e\n\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eDetection of SNPs and INDELs\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:18.75pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eAfter comparing clean reads to the \u003cem\u003eCulex pipiens pallens\u0026nbsp;\u003c/em\u003egenome, we used Genome Analysis Toolkit (GATK) [37] software to call each chromosome, identify single nucleotide polymorphisms (SNPs) and insertion and deletion (INDEL) sites for each sample, and store the final results in variant call format (VCF). The SNP statistical information for all samples is provided in Table 5. We then analysed the site information for each SNP and INDEL, as shown in Figure 1 and Figure 2.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:18.75pt;\"\u003e\n \u003cbr\u003e\n\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:18.75pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e[See supplementary files for Table 5.]\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:18.75pt;\"\u003e\n \u003cbr\u003e\n\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eNumbers, functional categorization and pathway analysis of DEGs\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eDEGs were obtained by comparing the expression levels of differential genes between the sample groups. The results are shown in Figure 3.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eDEGs were compared with the GO library for functional classification. GO included 3 major categories: molecular function, cellular composition and biological process. Subsequently, the 3 categories were separately classified. The results are provided in Figure 4.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eThe obtained DEGs were compared with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and the related metabolic pathways were identified. The results are provided in Figure 5.\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eValidation of the 3 target cuticle protein genes\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eTo ensure amplification of the target genes and the housekeeping gene, we performed primer verification. The results showed that the amplification curve for the primers was good and that the melting curve was monomodal, and the electrophoresis results revealed specific target fragments. The average Ct value for the actin gene in the sensitive group was 25.905; the average Ct value of the actin gene in the resistant group was 26.227; the average Ct value of the XM_001863852 gene in the sensitive group was 26.813; the average Ct value of the XM_001863852 gene in the resistant group was 29.633; the average Ct value of the XM_001845883.1 gene in the sensitive group was 36.797; the average Ct value of the XM_001845883.1 gene in the resistant group was 35.93; the average Ct value of the XM_001845881 gene in the sensitive group was 32.647; and the average Ct value of the XM_001845881 gene in the resistant group was 33.837 (see Tables 6 and 7 for details).\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eSubsequently, we used the 2-\u003c/span\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;MS Mincho\u0026quot;;\"\u003e△△\u003c/span\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eCt method to analyse the expression of target gene mRNA in the extracted RNA and normalized the result based on the housekeeping gene.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eThe specific calculation method is as follows:\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;MS Mincho\u0026quot;;\"\u003e△△\u003c/span\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eCt = (Ct target gene - Ct housekeeping gene) experimental group - (Ct target gene - Ct housekeeping gene) control group\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eThe relative expression level of the target gene = 2^-\u003c/span\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;MS Mincho\u0026quot;;\"\u003e△△\u003c/span\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eCt, which indicates the fold change in expression for the target gene in the experimental group relative to the control group.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eThe calculation results are provided in Tables 6 and 7.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;text-indent:28.0pt;\"\u003e\n \u003cbr\u003e\n\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:center;text-indent:28.0pt;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eTable 6 Quantitative PCR results\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"margin-left:4.65pt;border-collapse:collapse;border:none;\" width=\"543\" cellspacing=\"0\" cellpadding=\"0\" border=\"1\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:.75in;border-top:solid windowtext 1.5pt;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:none;background:#C6D9F1;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"13.235294117647058%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eTarget ID\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:89.0pt;border-top:solid windowtext 1.5pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;background:#C6D9F1;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"21.875%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eTarget Name\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:.75in;border-top:solid windowtext 1.5pt;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:none;background:#C6D9F1;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"13.235294117647058%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eSample ID\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:69.0pt;border-top:solid windowtext 1.5pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;background:#C6D9F1;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"16.91176470588235%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eSample Name\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:71.0pt;border-top:solid windowtext 1.5pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;background:#C6D9F1;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"17.46323529411765%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eCt Avg (SDM)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:70.55pt;border-top:solid windowtext 1.5pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;background:#C6D9F1;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"17.279411764705884%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eRel. Qty (SDM)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:.75in;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"13.235294117647058%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eT001\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:89.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"21.875%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eActin\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:.75in;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"13.235294117647058%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eS001\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:69.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"16.91176470588235%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eS (sensitive) 1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:71.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"17.46323529411765%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e25.475\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:70.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"17.279411764705884%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e1.00E+00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:.75in;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"13.235294117647058%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eT001\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:89.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"21.875%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eActin\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:.75in;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"13.235294117647058%\"\u003e\n \u003cp 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style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e26.205\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:70.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"17.279411764705884%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e1.00E+00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:.75in;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"13.235294117647058%\"\u003e\n \u003cp 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style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eT004\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:89.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"21.875%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eXM_001845881\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:.75in;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"13.235294117647058%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eS003\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:69.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"16.91176470588235%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eS 3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:71.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"17.46323529411765%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e32.13\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:70.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"17.279411764705884%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e3.01E+01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:.75in;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"13.235294117647058%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eT004\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:89.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"21.875%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eXM_001845881\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:.75in;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"13.235294117647058%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eS004\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:69.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"16.91176470588235%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eR 1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:71.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"17.46323529411765%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e33.04\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:70.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"17.279411764705884%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e1.01E+01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:.75in;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"13.235294117647058%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eT004\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:89.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"21.875%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eXM_001845881\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:.75in;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"13.235294117647058%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eS005\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:69.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"16.91176470588235%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eR 2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:71.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"17.46323529411765%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e35.15\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:70.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"17.279411764705884%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e4.13E+00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:.75in;border:none;border-bottom:solid windowtext 1.5pt;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"13.235294117647058%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eT004\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:89.0pt;border:none;border-bottom:solid windowtext 1.5pt;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"21.875%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eXM_001845881\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:.75in;border:none;border-bottom:solid windowtext 1.5pt;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"13.235294117647058%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eS006\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:69.0pt;border:none;border-bottom:solid windowtext 1.5pt;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"16.91176470588235%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eR3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:71.0pt;border:none;border-bottom:solid windowtext 1.5pt;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"17.46323529411765%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e33.32\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:70.55pt;border:none;border-bottom:solid windowtext 1.5pt;padding:0in 5.4pt 0in 5.4pt;height:12.75pt;\" width=\"17.279411764705884%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e2.80E+01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;text-align:justify;\"\u003e\n \u003cbr\u003e\n\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;text-align:justify;\"\u003e\n \u003cbr\u003e\n\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:SimSun;text-align:justify;\"\u003e\u003cspan style=\"font-size:19px;line-height:107%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eTable 7 Calculation of the relative quantitative Ct values for target genes and the internal reference gene\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\" width=\"568\" cellspacing=\"0\" cellpadding=\"0\" border=\"1\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 90.45pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"21.26537785588752%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.25pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"18.101933216168717%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eβ-actin\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.15pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"20.035149384885763%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eXM_001863852\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 88.1pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"20.56239015817223%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eXM_001845883.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.15pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;background: rgb(198, 217, 241) none repeat scroll 0% 0%;padding: 0in 5.4pt;vertical-align: top;\" width=\"20.035149384885763%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eXM_001845881\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 90.45pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"21.26537785588752%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eAverage Ct value in the sensitive group\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.25pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"18.101933216168717%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e25.905\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.15pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"20.035149384885763%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e26.813\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 88.1pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"20.56239015817223%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e36.797\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.15pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"20.035149384885763%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e32.647\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 90.45pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"21.26537785588752%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eAverage Ct value in the resistant group\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.25pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"18.101933216168717%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e26.227\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.15pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"20.035149384885763%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e29.633\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 88.1pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"20.56239015817223%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e35.93\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.15pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"20.035149384885763%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e33.837\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 90.45pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"21.26537785588752%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Cambria Math\u0026quot;,serif;\"\u003e△\u003c/span\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;Ct value in the sensitive group\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.25pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"18.101933216168717%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e-----\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.15pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"20.035149384885763%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0.908\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 88.1pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"20.56239015817223%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e10.892\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.15pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"20.035149384885763%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e6.742\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 90.45pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"21.26537785588752%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Cambria Math\u0026quot;,serif;\"\u003e△\u003c/span\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;Ct value in the resistant group\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.25pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"18.101933216168717%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e-----\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.15pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"20.035149384885763%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e3.406\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 88.1pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"20.56239015817223%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e9.703\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.15pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"20.035149384885763%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e7.61\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 90.45pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"21.26537785588752%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Cambria Math\u0026quot;,serif;\"\u003e△△\u003c/span\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;Ct value\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.25pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"18.101933216168717%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e-----\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.15pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"20.035149384885763%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e2.498\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 88.1pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"20.56239015817223%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e-1.189\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.15pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\" width=\"20.035149384885763%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0.868\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 90.45pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\" width=\"21.26537785588752%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e2^-\u003c/span\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Cambria Math\u0026quot;,serif;\"\u003e△△\u003c/span\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;Ct value\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.25pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\" width=\"18.101933216168717%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e-----\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.15pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\" width=\"20.035149384885763%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0.177\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 88.1pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\" width=\"20.56239015817223%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e2.281\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.15pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\" width=\"20.035149384885763%\" valign=\"top\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:16.0pt;font-size:16px;font-family:SimSun;\"\u003e\u003cspan style=\"font-size:19px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0.548\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:16px;font-family:SimSun;text-align:justify;\"\u003e\u003cspan style=\"font-size:19px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;It can be seen from Table 7 that the expression levels of the target genes XM_001863852 and XM_001845881 were similar between the sensitive and resistant strains of \u003cem\u003eCulex pipiens pallens\u003c/em\u003e; the fold changes in expression were 0.177 and 0.548, respectively. The expression level of the target gene XM_001845883.1 in the resistant strain was higher than that in the sensitive strain, and the fold change in expression was 2.281.\u003c/span\u003e\u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003eIt has been reported that at least 504 insect species worldwide exhibit chemical insecticide resistance [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], that more than 109 (subspecies) vector mosquitoes are resistant to one or more insecticides [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and that the number of species and the quantities of resistant insects have increased year to year. \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCulex pipiens pallens\u003c/span\u003e is an important vector for lymphatic filariasis and Japanese encephalitis virus. It is one of the main stinging and harassing mosquito species in cities and towns. Chemical pesticides have played an important role in the prevention and treatment of malaria and filariasis in Shandong Province. However, in recent years, due to the need to establish healthy and civilized cities, the large, continuous and irregular use of pesticides has led to the development of drug resistance and caused environmental pollution, while the degree of resistance by mosquitoes has also shown an increasing trend. The occurrence and development of resistance in mosquitos has limited the further application of chemical pesticides and has become a prominent problem in the prevention and control of insect-borne diseases in China and abroad. Based on the high chemical resistance of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCulex pipiens pallens\u003c/span\u003e in Shandong Province[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], this study used high-throughput transcriptome sequencing technology to qualitatively and quantitatively study cypermethrin-resistant strains of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCulex pipiens pallens\u003c/span\u003e and identified the cuticle protein genes responsible for the drug-resistant phenotype. Real-time quantitative PCR were used to verify the identified cuticle protein genes and to confirm whether there is a high correlation between cuticle protein genes and the drug resistance phenotype.\u003c/p\u003e \u003cp\u003eMosquito resistance generally includes 4 types: metabolic resistance, target resistance, cuticle resistance, and behavioural resistance. In 1963, a survey of houseflies (\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eFannia canicularis\u003c/span\u003e) identified the cause of insecticide resistance. It was found that the penetration of chemical pesticides in resistant lines was slower than in sensitive lines, suggesting that slower penetration could be the cause for dichlorodiphenyltrichloroethane (DDT) and pyrethroid resistance [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Studies have shown that pyrethroid-resistant female \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eAnopheles sinensis\u003c/span\u003e have thicker cuticles than do sensitive female \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eAnopheles sinensis\u003c/span\u003e and that female mosquitoes also have thicker cuticles than do male mosquitoes [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In addition, Lily et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] confirmed that cuticle thickening is present in pyrethroid-resistant strains of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCimex lectularius\u003c/span\u003e. Cuticle analysis by electron microscopy and characterization of lipid extracts showed that resistant mosquitoes had a thicker outer skin layer and a higher hydrocarbon content (approximately 29%) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Multiple studies have confirmed that the cuticle of resistant lines is much thicker than that of sensitive lines [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe cuticular protein (CP) family was first discovered in 2007 by tandem mass spectrometry analysis of epidermal exfoliation from \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eAnopheles gambiae\u003c/span\u003e [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Most gene family members have the prefix CPLC (cuticular protein of low complexity), and such members often play an important role in protein-protein interaction networks [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe above evidence suggests that insect cuticle proteins play an indispensable role in mosquito resistance. The expression of the target genes XM_001863852 and XM_001845881 in the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCulex pipiens pallens\u003c/span\u003e laboratory resistant strain was lower than that in the laboratory sensitive strain, and the fold changes in expression were 0.177 and 0.548, respectively, while the expression of XM_001845883.1 in the resistant strain was higher than that in the sensitive strain, and with a 2.281-fold change in expression. GO function analysis indicated that all 3 were genes responsible for cuticle structural components, and an NR database comparison also showed that these genes code cuticle proteins in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCulex pipiens\u003c/span\u003e. In fact, when we screened the genes, we initially identified 3 different genes in the XM_001845 series: XM_001845880, XM_001845881 and XM_001845883.1. However, when designing the primers, the primers designed for these 3 were not ideal, and there were varying degrees of non-specific amplification. We compared the designed primer fragments to the BLAST database and found that the primers were highly consistent with an unknown conserved hypothetical protein. We questioned if the 3 different genes in the XM_001845 series were different splicing isoforms of the same gene, and if so, whether such frequent splicing could promote resistance in mosquitoes. After considering the experimental cost, experimental significance and feasibility of the experiment, we selected 2 of the genes to conduct further validation, and the experimental results for XM_001845881 and XM_001845883.1 were opposite to each other. Therefore, we questioned whether the 2 genes had antagonistic effects in the genetic pathways leading to the formation of cuticle resistance in mosquitoes; for example, XM_001845883.1 is responsible for promoting the formation of cuticle resistance, while XM_001845881 is responsible for regulating the expression of other upstream or downstream cuticle protein genes, preventing the overexpression of other related cuticle proteins. Because the 3 identified target genes in our study are novel, the original identification process was relatively complicated and innovative; however, the specific regulatory networks and the in vivo function of the respective genes need to be further explored.\u003c/p\u003e \u003cp\u003eChemical control has become the main means of controlling disease vectors and reducing vector infectious diseases, including the indoor and outdoor use of adult mosquito insecticides and the use of larval insecticides at mosquito breeding sites. However, the emergence and spread of pesticide resistance seriously affects disease vector control. Due to the lack of knowledge about the geographical distribution of potential pesticide resistance mechanisms, it is difficult to develop effective pesticide resistance management strategies. In addition, the spatiotemporal principles for the selection and use of pesticides also need further planning and implementation.\u003c/p\u003e "},{"header":"Conclusions","content":"\u003cp\u003eThese data provide transcriptomic information related to the resistance of \u003cem\u003eCulex pipiens pallens\u003c/em\u003e and preliminarily verify the relationship between the identified cuticle protein genes and mosquito drug resistance, partially explaining the specific mechanism of mosquito cuticle resistance, providing a scientific basis for the study of new target insecticides, and providing new ideas for the prevention and control of mosquitoes.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eRNA-seq\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; RNA sequencing\u003c/p\u003e\n\u003cp\u003eCPGs\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; cuticular protein genes\u003c/p\u003e\n\u003cp\u003eCP\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; cuticular protein\u003c/p\u003e\n\u003cp\u003eDEGs\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; differentially expressed genes\u003c/p\u003e\n\u003cp\u003eGO\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Gene Ontology\u003c/p\u003e\n\u003cp\u003ePCR\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Polymerase Chain Reaction\u003c/p\u003e\n\u003cp\u003eWNV\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; West Nile Virus\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSLE\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; St. Louis encephalitis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSINV\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Sindbis virus\u003c/p\u003e\n\u003cp\u003eRVFV\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Rift Valley fever\u003c/p\u003e\n\u003cp\u003eJEV\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Japanese encephalitis\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eVssc \u003c/em\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;voltage sensitive sodium channel\u003c/p\u003e\n\u003cp\u003eIRS\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; indoor residual spraying\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eITNS\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;insecticide-treated nets\u003c/p\u003e\n\u003cp\u003encRNAs\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; noncoding RNAs\u0026nbsp;\u003c/p\u003e\n\u003cp\u003esRNAs\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; small RNAs\u003c/p\u003e\n\u003cp\u003eCDC\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Centers for Disease Control and Prevention\u003c/p\u003e\n\u003cp\u003eEP\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Eppendorf\u003c/p\u003e\n\u003cp\u003edscDNA\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; double strands cDNA\u003c/p\u003e\n\u003cp\u003eFPKM\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; fragment per kilobase exon model\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eREST\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; reliability simulation tool\u003c/p\u003e\n\u003cp\u003elncRNAs\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; long noncoding RNAs\u003c/p\u003e\n\u003cp\u003eGATK\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Genome Analysis Toolkit\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSNPs\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; single nucleotide polymorphisms\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eINDEL\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; insertion and deletion\u003c/p\u003e\n\u003cp\u003eVCF\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; variant call format\u003c/p\u003e\n\u003cp\u003eKEGG\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Kyoto Encyclopedia of Genes and Genomes\u003c/p\u003e\n\u003cp\u003eDDT\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; dichlorodiphenyltrichloroethane\u003c/p\u003e\n\u003cp\u003eCPLC\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; cuticular protein of low complexity\u003c/p\u003e\n\u003cp\u003eWHO\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; World Health Organization\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to BGI-genomics, BGI-Shenzhen (Shenzhen, 518083, China) for their assistance with the sequencing and\u0026nbsp;bioinformatics analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China [81871685 (MQG), 81672059 (MQG), 81471985(MQG)], the Innovation Project of Shandong Academy of Medical Sciences (MQG), the key research and development program of Shandong province (2018GSF118092, 2019GSF111006)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its additional files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQQS, PC, CXZ, IJL, XS, XXG, HFW, YW, HML provided the samples collection and wrote the manuscript. HWW and MQG reviewed and edited the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e[1] Grigoraki L, Puggioli A, Mavridis K,\u0026nbsp;Douris V, Montanari M, Bellini R, et al. Striking diflubenzuron resistance in \u003cem\u003eCulex pipiens\u003c/em\u003e, the prime vector of West Nile Virus. Sci Rep. 2017; 7(1):11699.\u003c/p\u003e\n\u003cp\u003e[2] Michael J. Turell. Members of the \u003cem\u003eCulex pipiens complex\u003c/em\u003e as vectors of viruses.J. 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J R Soc Interface.2013;10(82):20130041.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Culex pipiens pallens, insecticide resistance, cuticle protein gene, RNA-seq","lastPublishedDoi":"10.21203/rs.2.19260/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.19260/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground Long-lasting overdependence on insecticides has led to the rapid spread of pyrethroid resistance in mosquito vectors, which is of great concern to the general public. There are many studies on metabolic resistance and target resistance, but fewer studies have been conducted on cuticle resistance and behaviour resistance. The cuticle of mosquitoes has been hypothesized to play a role in insecticide resistance by reducing penetration or sequestering insecticides. \u003c/p\u003e\u003cp\u003eMethods We used RNA sequencing (RNA-seq) to analyse the transcriptome of cypermethrin-resistant and cypermethrin-susceptible strains of Culex pipiens pallens . Sequenced 6 samples using an Illumina HiSeq platform, and generated approximately 6.66 Gb bases from each sample on average. Mapping the sequenced reads to a reference genome and reconstructing the transcripts, through gene expression analysis, we detected differentially expressed genes (DEGs) among the samples. Followed Gene Ontology (GO) classification and functional enrichment. Finally, we screened the genes of cuticle proteins associated with drug resistance throughout the genome, selected the significant DEGs with a log2 fold change\u0026gt;3.0 and Padj\u0026lt;0.05, and applied real-time fluorescence quantitative PCR to verify the DEGs. \u003c/p\u003e\u003cp\u003eResults We obtained 13,517 novel transcripts, of which 8,653 were previously unknown splicing events for known genes, 665 were novel coding transcripts without any known features, and 4,199 were long noncoding RNA. A total of 1035, 944, and 657 genes were upregulated in comparisons between samples, and 2680, 1215, and 975 genes were downregulated in comparisons between samples. Finally, among all samples, 167 genes upregulated, and 145 genes downregulated. The GO classification and functional enrichment of DEGs as follows: molecular function, 224 genes; cellular component, 149 genes; and biological process, 272 genes. The expression of XM_001863852 and XM_001845881 in resistant strains of Culex pipiens pallens was lower than that in the laboratory sensitive strain, with fold changes in expression of 0.177 and 0.548, respectively; the expression of the XM_001845883.1 in the resistant strain was higher than that in the susceptible strain, and a 2.281-fold change in expression. \u003c/p\u003e\u003cp\u003eConclusions The results provide a reference for resistance mechanisms through the mosquito cuticle, furthermore, could provide a new perspective for disease vector control.\u003c/p\u003e","manuscriptTitle":"RNA-seq screening of the CPGs in Culex pipiens pallens among cypermethrin-resistant populations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2019-12-19 20:52:27","doi":"10.21203/rs.2.19260/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fb971da0-7514-435c-b98e-280d1a3c8c6d","owner":[],"postedDate":"December 19th, 2019","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":44252,"name":"Parasitology"}],"tags":[],"updatedAt":"","versionOfRecord":[],"versionCreatedAt":"2019-12-19 20:52:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9873","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"identity":"rs-9873","version":["v1"]},"buildId":"ApUGefWb6u5IBVtyqm6d5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}