Profiles of Amino Acids and Acylcarnitines Related with Deltamethrin Resistance in Aedes Albopictus

Profiles of Amino Acids and Acylcarnitines Related with Deltamethrin Resistance in Aedes Albopictus | 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 Article Profiles of Amino Acids and Acylcarnitines Related with Deltamethrin Resistance in Aedes Albopictus Lianfen Huang, Jun Li, Xinghua Su, Ruili Xie, Lilan Peng, Chenfang Tang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-135058/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: Fast emerging and increasing insecticide resistance in Asian tiger mosquito, Aedes albopictus brings alarm to the dengue epidemic over the world. The genetic and transcriptional sequencing has greatly advanced our understanding of the molecular basis for this crucial adaptation, but less is known about the regulation of insecticide resistance physiology at the metabolomic level. Methods: We captured the relative abundance of 17 amino acids and 34 AcylCNs in the 3 rd to 4 th instar larvae of three geographically close strains of Ae. albopictus , including a laboratory insecticide susceptible strain (Foshan) and two field deltamethrin-resistant strains (Yuexiu/Baiyun), as well as a laboratory induced deltamethrin-resistant (R18) and its parental insecticide susceptible strains (R0) to characterize their amino acids and acylcarnitines (AcylCNs) profiles by LC-MS/MS spectrometry. Results: Heatmap and PCA analysis of amino acids and AcylCNs profile indicated a clear separation of Foshan from Yuexiu/Baiyun populations, as well as a partly overlapping between Yuexiu and Baiyun populations. All strains were abundant in five amino acids (Pro, Val, Arg, Glu, His) and two AcylCNs (C0, C2). Five amino acids (Tyr, Cit, Pro, Thr, Gly) were significantly higher, while four amino acids (His, Val, Glu, Ala) were obviously lower in Yuexiu strain than Baiyun population. R18 displayed a differential amino acid and AcylCNs profile from its parental R0 strain. Conclusion: This study displayed a distinction of amino acids and AcylCNs profiles between insecticide-resistant and geographically or genetically-close insecticide-susceptible strain of Ae. albopictus , suggesting a possible application of amino acids and AcylCNs profiles for rapid diagnosis of deltamethrin resistance in Ae. albopictus in the future. Infectious Diseases Amino acids and acylcarnitines profiles Deltamethrin resistance Aedes albopictus Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Asian tiger mosquito, Aedes albopictus ( Ae. albopictus ), has spread to 70 countries all over the world out of its native home in Southeast Asia [ 1 ], with strong capacity in the transmission of various arboviruses like Dengue and Zika viruses [ 2 ]. At present, vector population control strategies, especially usage of insecticides targeting the larval and adult stages, are the most popular way to limit Ae. albopictus mosquitoes and virus transmission [ 3 , 4 ]. Pyrethroids are the most frequently utilized adulticides to control the spread of Ae.albopictus to prevent dengue virus transmission as for their low mammalian toxicity and rapid knockdown effect [ 4 – 6 ]. However, extensive and prolonged use of pyrethroids like deltamethrin has imposed selection pressure on Ae. albopictus and eventually caused resistance [ 4 , 7 ]. Fast emerging and increasing resistance to insecticides like deltamethrin in Ae. albopictus brings alarm to the dengue epidemic over the world. Pyrethroids resistance in Ae. albopictus occurs mainly through the F1534S and F1534L mutation in the VGSC gene [ 7 , 8 ], also known as knockdown resistance (kdr) resistance, as well as up-regulation of specific detoxification enzymes like glutathione S-transferase (GST) (GSTE1, AGAP009195-PA) and cytochrome P450 monooxygenases (P450s) CYP6A8, CYP9Z4, CYP6D4 [ 9 ]. Pyrethroids resistance was also regarded to be associated with pathways involved in metabolic process and pathways [ 10 , 11 ], protein digestion and absorption [ 11 ]. Metabolic pathways and biosynthesis of secondary metabolites have been identified to be the top-two differential pathway categories between deltamethrin-resistant and susceptible individuals of Ae. albopictus [ 9 ]. Metabolomics could reflect systematic human metabolic variations in accordance with phenotypic changes, such as resistance to insulin [ 12 ] and trastuzumab [ 13 ]. Characteristic of amino acids and AcylCNs acylcarnitine profile has become is a powerful and popular tool for diagnostic of clinical metabolic alterations [ 14 – 18 ]. However, little is known of the characteristic of amino acids and AcylCNs profiles in mosquitoes. Altered profile of amino acids and AcylCNs has been reported in Culex quinquefasciatus L after exposure to chlorpyrifos, temephos and permethrin [ 19 ]. In this study, amino acids and AcylCNs profiles of deltamethrin-resistant and susceptible strains of Ae. albopictus were compared to observe their alterations in accordance with deltamethrin resistance. Materials And Methods Mosquito strains, analysis of insecticide resistance and kdr mutations Larvae of two field strains of Ae. albopictus were collected from public land like parks, schools and residential areas in 2017 in two districts named Yuexiu and Baiyun of Guangzhou city respectively, with dengue incidence of > 5 and 1–5 cases per 100,000 people respectively as previous report [ 7 ]. The larvae were housed [ 7 ] and have been tested for their larval resistance to four insecticides: (1) organophosphate, temephos; (2) microbial bacterial toxin, Bti; (3) hormonal insect growth regulators, pyriproxyfen (PPF); (4) the chitin biosynthesis inhibitor, hexaflumuron; as well as their adult resistance to five adulticides including deltamethrin, permethrin, malathion, bendiocarb, DDT following WHO guidelines [ 7 ]. The larvae housed from both Yuexiu and Baiyun districts were also tested for their resistance to adulticides including deltamethrin, permethrin, DDT, bendiocarb, malathion following WHO guidelines as previous report [ 7 , 20 ]. For larval bioassays, the resistant status was evaluated by the resistant ratio (RR 50 ) (RR 50 < 5, susceptible; 5 < RR 50 10, highly resistant), for adult bioassays, resistant status was defined by mortality rate: resistant if mortality 98% [ 7 ]. The kdr mutation in resistant and susceptible individuals from Yuexiu and Baiyun districts in 2017 has been genotyped at the voltage-gated sodium channel (VGSC) gene after exposure to deltamethrin, permethrin, and DDT to detect mutations within domains II, III and IV [ 7 ], samples exposed to bendiocarb have been genotyped at the ace-1 gene to detect mutations within G119 [ 7 ]. The Foshan strain of Ae. albopictus , an insecticide-susceptible stain which was reared in laboratory without exposure to any insecticides since its collection in 1983 in Foshan (a smaller city adjacent to Guangzhou city), was used as the control strain for metabolomic comparison with these field strains of Ae. albopictus . R18 is a laboratory resistant strain (LC 50 = 0.039, RR 50 = 13) after deltamethrin selection of an insecticide susceptible Ae. albopictus populations from Shanghai CDC for 17 generations, which was primarily screened by 0.005 mg/L of deltamethrin (Sigma, USA) from the 1st to the 10th generation, followed by deltamethrin screening at 50% lethal concentration (LC 50 ) from the 11th to the 22nd generation as described previously [ 21 ]. The parental strain of Ae. albopictus without any exposure to insecticides (R0) was used as an insecticide-susceptible control strain for R18 strains of Ae. albopictus in this study, LC 50 (95%) = 0.003 (0.001, 0.006) mg/L. The larval and adult resistance of R18 to deltamethrin was also analyzed following WHO guidelines as previous report [ 7 , 20 ]. The resistant and susceptible adults against deltamethrin from R18 strains of Ae. albopictus were also genotyped for kdr mutation as previous report [ 7 ]. LC-MS/MS sample preparation and metabolites extraction Six replicates of ten numbers of 3rd to 4th instar larvae (approximately 2 mg/larvae) of Foshan, Yuexiu, Baiyun, R0, R18 strains were sampled respectively. Then, they were homogenized (TIANGEN OSE-Y10-plus) in 500 µl of ddH 2 O, followed by centrifugation at 4 ℃,13000 rpm for 10 min. The lysate was transferred to a sterile syringe which was attached to a 0.22 µm acrodisc for filtration. Finally, 30 µl of the filtered lysate was added to the filter paper (S&S903) and dried for storage. A paper circle with 3.2 mm diameter was punched from the dry larval lysate filter paper by a Panthera-Puncher TM 9 Puncher (PerkinElmer, Waltham, MA, USA)]. A NeoBase non-derivatized MS/MS kit (Perkin Elmer) was employed for extraction of amino acids and AcylCNs. Metabolomics analysis LC-MS/MS (API 2000, ABSciex, Framingham, MA, USA) platform, with stable isotopes labelled amino acid standards (Cambrige Isotope Labs) as an internal solution, was selected for quantification of 17 amino acids including alanine, arginine, aspartate, citrulline, glutamine, glutamate, glycine, histidine, leucine, methionine, ornithine, phenylalanine, proline, serine, threonine, tyrosine, valine) and 34 AcylCNs including C0, C2, C3, C3DC, C4, C4-OH, C4DC, C5, C5:1, C5-OH, C5DC, C6, C6:1, C6DC, C8, C8:1, C10, C10:1, C10:2, C12, C12:1, C14, C14-OH, C14:1, C14:2, C16, C16:1, C16-OH, C16:1-OH, C18, C18:1, C18-OH, C18:2, C18:1-OH (34AC). Analysis of 17 amino acids and 34 AcylCNs was interpreted in multiple reaction monitoring (MRM) mode with Analyst 1.6.2 software (ABSciex) and NeoBase database. Statistical analysis Finally, the contents of 17 amino acids and 34 AcylCNs were scaled according to the preparation procedure of dry larval lysate filter paper. The concentration of the detected metabolites was normalized and analyzed by orthogonal pattern recognition methods principal component analysis (PCA). PCA with performed using Metaboanalyst (v3.0) ( https://www.metaboanalyst.ca/faces/home.xhtml ) to display the characteristic of amino acids and AcylCNs profiles of Ae. albopictus with different levels of deltamethrin resistance. SPSS 17.0 was applied for the spearman correlation analysis between the deltamethrin LC 50 values and the contents of amino acids and AcylCNs in Ae. albopictus. LC 50 and KDT 50 were estimated using the log-probit models [ 7 ]. For larvae bioassays, the resistant status was measured by the resistant ratio (RR 50 ), i.e., the ratio of LC 50 (or IE 50 ) for the field population over LC 50 (or IE 50 ) for the laboratory-susceptible strain [ 7 ]. Larval resistance status was defined as susceptible if RR 50 < 5, moderately resistant if 5 < RR 50 10 [ 7 ]. For adult bioassays, resistant status was defined by mortality rate: Resistant if mortality 98% [ 7 , 9 ]. The relationship between nonsynonymous mutations and resistance was verified by Fisher’s exact test or the χ2 -test (when all n > 5), and the odds ratio (OR) was calculated for each mutation [ 7 ]. Results Insecticide resistance and kdr mutation of Ae. albopictus larvae from Yuexiu and Baiyun districts in 2017 Ae. albopictus larvae from both Yuexiu and Baiyun districts had moderate to high resistance to adulticides like deltamethrin and permethrin, both of them remained susceptible to malathion (Table 1 ). Moreover, larvae from Yuexiu district had developed much higher level of resistance to deltamethrin than the larvae from Baiyun district, but larvae from Baiyun district was more resistant to both permethrin and DDT (Table 1 ). In 2017, the larvae and adult populations of Ae. albopictus from Baiyun/Yuexiu districts of Guangzhou city, China has developed high resistance to currently used insecticides (deltamethrin, permethrin, DDT and bendiocarb, mortality 98%) [ 7 ]. The mortality rate against deltamethrin, permethrin, DDT and bendiocarb was 5.6%, 55.2%, 52.1%, 42.4% in the adult populations from Yuexiu district, and 38.4%, 68.8%, 74.7% and 51.2 in the adult population from Baiyun district, respectively [ 7 ]. Ae. albopictus larvae from both districts were still sensitive to Bti and hexaflumuron (RR 50 10) [ 7 ]. Table 1 The larval resistance of Ae. albopictus from both Yuexiu and Baiyun districts to five adulticides (deltamethrin, permethrin, DDT, bendiocarb, malathion) in 2017 deltamethrin Permethrin DDT Bendiocarb Malathion LC 50 (95%)(mg/L) RR 50 LC 50 (95%)(mg/L) RR 50 LC 50 (95%)(mg/L) RR 50 LC 50 (95%)(mg/L) RR 50 LC 50 (95%)(mg/L) RR 50 Baiyun 0.027(0.020,0.049) 27 0.628(0.527,0.730) 15 5.281(5.073,5.538) 12.1 1.460(0.995,1.745) 0.77 0.073(0.068,0.080) 1.7 Yuexiu 0.066(0.061,0.094) 66 0.356(0.113,0.533) 8.47 1.912(1.560,2.227) 4.37 5.37(4.826,6.031) 5.54 0.092(0.081,0.109) 2.14 Foshan 0.001(0.001,0.006) 1 0.042(0.031,0.053) 1 0.438(0.389,0.484) 1 0.969(0.908,1.038) 1 0.043(0.039,0.046) 1 Kdr mutation associates with deltamethrin resistance in R18 strain The larvae and adult populations of Ae. albopictus from R18 has developed high resistance to deltamethrin (larval LC 50 = 0.039, RR 50 = 13, adult mortality = 85%). Sequences of domains II (480 bp), III (346 bp) and IV (280 bp) of the VGSC gene were also obtained from the resistant and susceptible mosquitoes after deltamethrin adult bioassays for R18 [ 7 ]. Kdr genotyping showed non-synonymous mutations of F1534S could be detected at codon 1534 in domain III of R18, where wild-type TTC (Phe) was changed to TCC (Ser) at a high frequency of 78.38% (29/37), indicating a significant correlation with deltamethrin resistance (Table 2 , p < 0.01). Table 2 Kdr mutations at position F1534 of the VGSC gene in adults of Ae. albopictus from R18 strain and its parental susceptible R0 strain . Insecticide N Genotype Odds Ratio(95%Cl) FF FS SS FL LL F1534S F1534L Susceptible strain (R0) 40 40 0 0 0 0 1 --- R18 37 9 23 6 0 0 123.00 (14.30,1013.39)** -- **: p < 0.01 Differential larval amino acids and AcylCNs profiles between insecticide-resistant Ae. albopictus from Baiyun/Yuexiu and susceptible Foshan strain Amino acids and AcylCNs profile analysis demonstrated that Pro, Val, Arg, Glu and His were the five most abundant amino acids (Fig. 1A) whereas C0, C2 were the richest AcylCNs in the larvae of Ae. albopictus (Table S2) & (Figs. 1B and 1C). The metabolites in Baiyun/Yuexiu larvae displaced fluctuation in comparison to Foshan larvae. T test analysis showed the concentration of five amino acids Tyr, Cit, Pro, Thr, Gly (Fig. 2A) and five AcylCNs C3DC, C6-1, C10, C10-1,C10-2 (Fig. 2B) were significantly higher, while the levels of C2 (Fig. 2B) and four amino acids His, Val, Glu, Ala (Fig. 2A) were obviously lower in Yuexiu strain in comparison to Foshan strain. Similarly, Cit and Arg were increased while His was decreased in Baiyun strain (Fig. 2C). Heatmap and PCA analysis of 17 amino acids and 34 AcylCNs contents indicated significant discrepancy of amino acids and AcylCNs profile between Foshan and Baiyun/Yuexiu larvae (Figs. 3A and 3B). However, profile of Yuexiu larvae was partly overlapped with that of Baiyun larvae (Fig. 3B). Differential larval amino acids and AcylCNs profiles between deltamethrin-resistant (R18) and its parental susceptible (R0) strains Amino acids and AcylCNs profile analysis demonstrated that Pro, Val, Arg, Glu and His were the five most abundant amino acids whereas C0, C2 were the richest AcylCNs in the deltamethrin-resistant (R18) and -susceptible (R0) larvae of Ae. albopictus (Table S2). Between deltamethrin-resistant R18 and susceptible (R0) strains, no significant difference of amino acids and AcylCNs were found, but the heatmap and PCA analysis of 17 amino acids and 34 AcylCNs contents indicated discrepancy of amino acids and AcylCNs profile (Figs. 4A and 4B). Table 3 Kdr mutations at position F1534 of the VGSC gene in resistant and susceptible adults of Ae. albopictus against deltamethrin, permethrin and DDT from both Yuexiu and Baiyun districts in 2017 [ 7 ] Insecticide Phenotype N Genotype Odds Ratio(95%Cl) FF FS SS FL LL F1534S F1534L Deltamethrin S 20 17 3 0 0 0 1 1 R 41 2 1 17 3 18 4.44(1.12,17.52)* NA permethrin S 19 11 5 0 1 1 1 1 R 42 1 4 22 5 11 4.55(1.38,15.05)* 27.20(4.60,160.69)** DDT S 18 15 2 1 0 0 1 1 R 44 5 7 15 11 6 5.00(1.27,19.74) NA** NA: Not Available. Significance level. *p < 0.05,**P < 0.01. Discussion For insecticide resistance monitoring, the bioassays like WHO susceptibility test [22] and CDC bottle assay [23], molecular detection of knock-down resistant gene mutations, analysis of metabolic enzymes GSTs, p450s and CCEs activities have been frequently used, but limitations existed, such as time and labor-consuming, difficult for standardization with complicated procedures, requirement of special insectary laboratory to rear mosquitoes and subjective judgement of larval and adult death, etc. Amino acids and AcylCNs profile have been popular for diagnostic of clinical metabolic diseases. In this study, we first tried to explore the characteristic of amino acids and AcylCNs profile among geographically or genetically-close strains of Ae. albopictus with differential resistance to insecticides especially deltamethrin, and compared their differences. The insecticide resistance status and the corresponding possible genetic mechanism of the test strains was analyzed thoroughly before their metabolomic analysis. Deltamethrin was the most popular adulticide for vector control in urban areas of Guangzhou city [7]. Accordingly, the most significant difference of insecticide resistance between the larvae and adults from different districts were their differential resistance to deltamethrin, such as significantly higher larval and adult resistance to deltamethrin in Yuexiu strain (larval RR 50 = 66, adult mortality of 5.6%) than Baiyun strain (larval RR 50 = 27, adult mortality of 38.4%) (Table 1) [7]. In Ae. albopictus adults from both districts, the resistance to pyrethroids (deltamethrin, permethrin) and DDT has been proved to be significantly associated with non-synonymous 1534 codon mutations from homozygote wild-type TTC (Phe) (F1534F) to homozygote TCC (Ser) (F1534S) or CTC (Leu) (F1534L) to at domain III (346 bp) of the VGSC gene, with five detected kdr genotype (FF, FS, SS, FL, LL) in the resistant and susceptible individuals of Ae. albopictus adults for three insecticides (deltamethrin, permethrin and DDT) from both districts (Table 3) [7] as previous report [8]. Similarly, Kdr genotyping detected F1534S mutation at codon 1534 in domain III of R18 strain at a frequency of 78.38% (29/37), indicating a significant correlation of F1534S mutation with laboratory-induced deltamethrin resistance in Ae. albopictus (Table 2, p < 0.01). The PCA analysis of amino acids and AcylCNs profiles among different strains demonstrated that Ae. albopictus larvae with differential deltamethrin resistant level displaced differential amino acids and AcylCNs profiles, including clear distinction of amino acids and AcylCNs profile between insecticide-susceptible larvae (Foshan strain) and larvae collected from geographically-close and insecticide-resistant Baiyun/Yuexiu districts, or obvious difference between a laboratory-induced deltamethrin-resistant strain (R18) and its parental susceptible Shanghai strain (R0), whereas it was relatively similar between insecticide-resistant larvae from relative geographically-close Baiyun and Yuexiu districts. This may suggest a potential role of amino acids and AcylCNs profile in predicting larval insecticide resistance of Ae. albopictus . Moreover, five amino acids (Tyr, Cit, Pro, Thr, Gly) were significantly higher, whereas four amino acids (His, Val, Glu, Ala) were obviously lower in larvae from Yuexiu district than the susceptible Foshan strain. However, only Cit and Arg were increased while His was decreased in larvae from Baiyun district than the susceptible Foshan strain, this phenomenon was probably due to their differential resistance to insecticides especially deltamethrin. Amino acids are metabolites required for protein synthesis, and have roles in metabolism as energy sources and nucleic acid precursors, they could be transformed into carbon-hydrates and fatty acids. The significant difference of amino acids between insecticide-susceptible larvae (Foshan strain) and insecticide-resistant larvae collected from geographically-close Baiyun/Yuexiu districts, was probably associated with the amino acid changes in correspondence with the deltamethrin-resistance correlated genetic changes [7] and altered expression of P450s, cuticle proteins, glutathione S transferase, serine proteases, heat shock protein cytochrome s, esterase [9], as well as P450 gene SNPs such as Arg226Ser in CYP6A8, Pro175Gln in CYP9B2, His877Tyr in CYP9B2, Cys212Ser in CYP1A1 in Ae. albopictus [9]. The change of amino acids and proteins was also reported to be involved in insecticide resistance of other mosquitoes, such as arginine (ARG) changes for energy production and insecticide detoxification of Culex quinquefasciatus L [19], elevated P450s and esterases (EST) in the insecticide-resistant housefly M. domestica [24], more proteins involved in oxidoreductase, transferase, hydrolase, transporter and catalytic activity, cuticle formation in the pyrethroid resistant strain of culex pipiens pallens [25, 26]. Changes of amino acids and insect proteases also occurred under insecticide exposure for protein metabolism, such as for re-synthesis of up-regulated detoxification enzymes, suggesting a role of protein metabolism under insecticide pressure [24, 27]. Carnitine is derived from amino acids lysine and methionine, AcylCNs are metabolites of organic acids and beta oxidation of fats. In this study, C0 and C2 were the two most abundant AcylCNs in larvae of all five strains. The changes of free carnitine (C0) and acetyl-carnitine (C2) were regarded to be involved in energy production and insecticide detoxification of Culex quinquefasciatus L [19]. In this study, five AcylCNs (C3DC, C6-1, C10, C10-1, C10-2) were found to be significantly higher in larvae from Yuexiu strain than the susceptible Foshan strain, while the levels of C2 were obviously lower, suggesting a probable variation of these AcylCNs in association with the development of insecticide resistance in Ae. albopictus . Conclusions This study first captured the characteristic of amino acids and AcylCNs profile of Ae. albopictus larvae with differential resistance to insecticides especially deltamethrin, indicating the possibility of LC-MS/MS based amino acids and AcylCNs profile as a tool for fast discrimination of deltamethrin-resistant strain from -susceptible strain of Ae. albopictus , as well as a potential way for seeking potential biomarkers for early diagnosis of insecticide resistance like deltamethrin in Ae. albopictus . Abbreviations AA, amino acids. AC, acylcarnitine. AcylCNs, acylcarnitine. Ae. albopictus, Aedes albopictus . PCA, principal component analysis. Declarations Acknowledgements The authors would like to thank Hongxia Liu from Shanghai CDC for assistance in induction of laboratory deltamethrin-resistant strains of Ae. albopictus . Authors' contributions Conceptualization: Lianfen Huang, Ruili Xie, Xiao-Guang Chen, Hua Li. Data curation: Lianfen Huang, Xinhua Su, Chenfang Tang. Formal analysis: Lianfen Huang, Ruili Xie, Lilan Peng, Chenfang Tang, Jun Li, Xinhua Su. Funding acquisition: Xiao-Guang Chen, Hua Li. Investigation: Lianfen Huang, Ruili Xie, Lilan Peng, Xinhua Su. Methodology: Lianfen Huang, Ruili Xie, Lilan Peng, Chenfang Tang, Yonglan Huang, Xinhua Su. Project administration: Lianfen Huang, Ruili Xie, Jun Li. Supervision: Xiao-Guang Chen, Hua Li, Yonglan Huang. Final approval: all authors have read and approved the manuscript. Funding This study was supported by the National Institutes of Health, USA(R01AI083202), Guangzhou Science and technology project, China (202002030491) and the Medical Scientific Research Foundation of Guangdong Province, China (A2019512). Ethical approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of supporting data The insecticide resistance and Kdr mutation analysis data of the adults of Ae. albopictus from Yuexiu/Baiyun districts of Guangzhou city in 2017 is available from the paper (Su X, Guo Y, Deng J, Xu J, Zhou G, Zhou T, et al. (2019) Fast emerging insecticide resistance in Aedes albopictus in Guangzhou, China: Alarm to the dengue epidemic. PLoS Negl Trop Dis 13(9): e0007665.) at the website (https://doi.org/10.1371/journal. pntd.0007665). Competing interests The authors declare no conflicts of interest in this work. Authors' information 1 Department of Pathogen Biology and Experimental Teaching Center of Preventive Medicine, Guangdong Provincial Key Laboratory of Tropical Disease, School of Public Health, Southern Medical University, Guangzhou, China. 2 Clinical Laboratory, Guangzhou Women and Children’s Medical center, Guangzhou Medical University, Guangzhou, China. 3 Newborn Screening Center, Guangzhou Women and Children’s Medical center, Guangzhou Medical University, Guangzhou, China. References Manni M, Guglielmino CR, Scolari F, Vega-Rua A, Failloux AB, Somboon P, Lisa A, Savini G, Bonizzoni M, Gomulski LM, et al. Genetic evidence for a worldwide chaotic dispersion pattern of the arbovirus vector, Aedes albopictus . PLoS Negl Trop Dis. 2017;11(1):e5332. Bohers C, Mousson L, Madec Y, Vazeille M, Rhim A, M'Ghirbi Y, Bouattour A, Failloux AB. The recently introduced Aedes albopictus in Tunisia has the potential to transmit chikungunya, dengue and Zika viruses. 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Faseb J. 2019;33(1):796–807. Xu ZR, Zhu XY, Lu W, Sun WH, Cheng RQ, Ni JW, Xi L, Hussain K, Luo FH, Zhang MY. Altered serum amino acid and acylcarnitine profiles in hyperinsulinemic hypoglycemia and ketotic hypoglycemia. Front Endocrinol (Lausanne). 2020;11:577373. Gunther SH, Khoo CM, Tai ES, Sim X, Kovalik JP, Ching J, Lee JJ, van Dam RM. Serum acylcarnitines and amino acids and risk of type 2 diabetes in a multiethnic Asian population. BMJ Open Diabetes Res Care 2020, 8(1). Kasakin MF, Rogachev AD, Predtechenskaya EV, Zaigraev VJ, Koval VV, Pokrovsky AG: Changes in amino acid and acylcarnitine plasma profiles for distinguish in patients with multiple sclerosis from healthy controls. Mult Scler Int 2020, 2020:9010937. Martin-Park A, Gomez-Govea MA, Lopez-Monroy B, Trevino-Alvarado VM, Torres-Sepulveda MD, Lopez-Uriarte GA, Villanueva-Segura OK, Ruiz-Herrera MD, Martinez-Fierro ML, Delgado-Enciso I, et al. Profiles of amino acids and acylcarnitines related with insecticide exposure in Culex quinquefasciatus (Say) . Plos One. 2017;12(1):e169514. World Health Organization. Guidelines for laboratory and field testing of mosquito larvicides. Geneva: World Health Organization; 2005. Huang LF, Li J, Peng L. The differential metabolic profiles between deltamethrin resistant and -susceptible strains of Aedes albopictus . J Med Entomol 2020. World Health Organization. Test procedures for insecticide resistance monitoring in malaria vectors, bio-efficacy and persistence of insecticides on treated surfaces: report of the WHO informal consultation, Geneva, 28–30 September 1998. Geneva World Health Organization 1998. Brogdon WG, McAllister JC. Simplification of adult mosquito bioassays through use of time-mortality determinations in glass bottles. J Am Mosq Control Assoc. 1998;14(2):159–64. Li M, Reid WR, Zhang L, Scott JG, Gao X, Kristensen M, Liu N. A whole transcriptomal linkage analysis of gene co-regulation in insecticide resistant house flies, Musca domestica . BMC GENOMICS. 2013;14:803. Lv Y, Wang W, Hong S, Lei Z, Fang F, Guo Q, Hu S, Tian M, Liu B, Zhang D, et al. Comparative transcriptome analyses of deltamethrin-susceptible and -resistant Culex pipiens pallens by RNA-sEq. Mol Genet Genomics. 2016;291(1):309–21. Wang W, Lv Y, Fang F, Hong S, Guo Q, Hu S, Zou F, Shi L, Lei Z, Ma K, et al. Identification of proteins associated with pyrethroid resistance by iTRAQ-based quantitative proteomic analysis in Culex pipiens pallens. Parasit Vectors. 2015;8:95. Wilkins RM. Insecticide resistance and intracellular proteases. Pest Manag Sci. 2017;73(12):2403–12. Supplementary Files TableS1.xlsx Additional file 1: Table S1. The concentration of 17AA and 34AC in deltamethrin-resistant (YX and BY) and -susceptible larvae (FS) of Ae. albopictus by LC-MS/MS spectrometry (XLSX) TableS2.xls Additional file 2: Table S2. The concentration of 17AA and 34AC in deltamethrin-resistant R18 and -susceptible larvae (R0) of Ae. albopictus by LC-MS/MS spectrometry (XLSX) 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-135058","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":7022559,"identity":"146c0780-4e79-4d1c-bf0a-e45542408041","order_by":0,"name":"Lianfen Huang","email":"","orcid":"","institution":"Guangzhou women and children's medical center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lianfen","middleName":"","lastName":"Huang","suffix":""},{"id":7022560,"identity":"8f1c6ab7-c8e1-49fd-b1bc-96aeab8030d5","order_by":1,"name":"Jun Li","email":"","orcid":"","institution":"Southern medical university, school of public health","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Li","suffix":""},{"id":7022561,"identity":"a4567b65-c776-440b-81b6-4dc4df96f786","order_by":2,"name":"Xinghua Su","email":"","orcid":"","institution":"Southern Medical University,school of public health","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xinghua","middleName":"","lastName":"Su","suffix":""},{"id":7022562,"identity":"0cc6765b-3fd2-4048-a730-4793bb37ef13","order_by":3,"name":"Ruili Xie","email":"","orcid":"","institution":"Southern Medical University School of Public Health","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ruili","middleName":"","lastName":"Xie","suffix":""},{"id":7022563,"identity":"d9b8607c-5b83-4546-840e-b76f22a62bd4","order_by":4,"name":"Lilan Peng","email":"","orcid":"","institution":"Southern medical university, school of public health","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lilan","middleName":"","lastName":"Peng","suffix":""},{"id":7022564,"identity":"53bc988a-12f8-49fa-88ef-f93bb2928f6a","order_by":5,"name":"Chenfang Tang","email":"","orcid":"","institution":"Guangzhou women and children's medical center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chenfang","middleName":"","lastName":"Tang","suffix":""},{"id":7022565,"identity":"b7fce99b-66aa-4140-83ab-96365fac0c6b","order_by":6,"name":"Jiabao Xu","email":"","orcid":"","institution":"Southern Medical University School of Public Health","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiabao","middleName":"","lastName":"Xu","suffix":""},{"id":7022566,"identity":"109582d4-7459-40db-bc61-3e752a00f388","order_by":7,"name":"Yonglan Huang","email":"","orcid":"","institution":"Guangzhou Women and Children's Medical 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Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqElEQVRIiWNgGAWjYLACxgYbHn7+BtK0pMlIzjhAmpbDNgYNCUSqlp92xnRz4Y7zPAYMBxg/fMwhQovB7Ryz2zPP3OYxZ25glpy5jRgt0kAtvG23eSwbDrAx8xKjRX42WMs5HoMDCURqYbgN1nKABC0Gt9PKbs9sS+aRnHGwmTi/yM9O3na7sM3Onp+/+eCHj0Q5DAiYIRRjA5HqEVpGwSgYBaNgFOAAAJhoNnkO7gkPAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-2466-0084","institution":"Guangzhou women and children's medical center","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2020-12-23 17:10:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-135058/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-135058/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":4560237,"identity":"f4ab823a-6f4d-41a6-b053-4871db7d4cf0","added_by":"auto","created_at":"2020-12-28 22:19:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":200857,"visible":true,"origin":"","legend":"The concentrations of amino acids and acylcarnitines in Foshan (FS), Baiyun (BY) and Yuexiu (YX) strains ","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-135058/v1/3d6a8603ac08d8ccc48a1777.png"},{"id":4560030,"identity":"bb0e89ce-cebd-4eb1-94c0-55797d141c3f","added_by":"auto","created_at":"2020-12-28 22:13:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":464849,"visible":true,"origin":"","legend":"Differential amino acids and AcylCNss between DS-FS and DR-BY/YX larvae. (A) \u0026 (B)significant differential amino acids and AcylCNs between susceptible Foshan (FS) and resistant Yuexiu (YX) larvae; (C) significant differential amino acids between susceptible DS-FS and Foshan (FS) and resistant Baiyun (BY) larvae.","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-135058/v1/efa11529c25a2b59119d6337.png"},{"id":4560034,"identity":"854da142-5197-41a0-8df7-ef992ab0894c","added_by":"auto","created_at":"2020-12-28 22:13:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":28869,"visible":true,"origin":"","legend":"Heatmap (A) and PCA analysis (B) showed Differential amino acids and AcylCNs profiles between Foshan (FS)and Baiyun (BY)/Yuexiu (YX) larvae.","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-135058/v1/cd1551ffa5e4197348f2943d.png"},{"id":4559966,"identity":"f4f95a45-f95a-4104-bd15-fc4ea1af92d5","added_by":"auto","created_at":"2020-12-28 22:10:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":422674,"visible":true,"origin":"","legend":"Heatmap (A) and PCA analysis (B) showed differential amino acids and AcylCNs profiles between larvae from deltamethrin-resistant R18 and its parental susceptible strains (R0)","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-135058/v1/9a4054857ddf0a8325b66a16.png"},{"id":13641448,"identity":"cf8c52ca-558a-47f8-a6f7-a563ae3d717c","added_by":"auto","created_at":"2021-09-17 09:03:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":965647,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-135058/v1/48e886f6-965b-466b-8c06-26c170ce3dbc.pdf"},{"id":4560145,"identity":"caeb708a-c73c-49cf-85ac-c39b42c9a838","added_by":"auto","created_at":"2020-12-28 22:16:08","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16102,"visible":true,"origin":"","legend":"Additional file 1: Table S1. The concentration of 17AA and 34AC in deltamethrin-resistant (YX and BY) and -susceptible larvae (FS) of Ae. albopictus by LC-MS/MS spectrometry (XLSX) ","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-135058/v1/73394c280d86d542afd3b511.xlsx"},{"id":4560328,"identity":"5bd06e3b-4ecf-41ef-b002-4bcd6f61c124","added_by":"auto","created_at":"2020-12-28 22:22:08","extension":"xls","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":32256,"visible":true,"origin":"","legend":"Additional file 2: Table S2. The concentration of 17AA and 34AC in deltamethrin-resistant R18 and -susceptible larvae (R0) of Ae. albopictus by LC-MS/MS spectrometry (XLSX)","description":"","filename":"TableS2.xls","url":"https://assets-eu.researchsquare.com/files/rs-135058/v1/be066ac281c076de4c19a8c4.xls"}],"financialInterests":"","formattedTitle":"\u003cp\u003eProfiles of Amino Acids and Acylcarnitines Related with Deltamethrin Resistance in Aedes Albopictus\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eAsian tiger mosquito, \u003cem\u003eAedes albopictus\u003c/em\u003e (\u003cem\u003eAe. albopictus\u003c/em\u003e), has spread to 70 countries all over the world out of its native home in Southeast Asia [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], with strong capacity in the transmission of various arboviruses like Dengue and Zika viruses [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. At present, vector population control strategies, especially usage of insecticides targeting the larval and adult stages, are the most popular way to limit \u003cem\u003eAe. albopictus\u003c/em\u003e mosquitoes and virus transmission [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Pyrethroids are the most frequently utilized adulticides to control the spread of \u003cem\u003eAe.albopictus\u003c/em\u003e to prevent dengue virus transmission as for their low mammalian toxicity and rapid knockdown effect [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, extensive and prolonged use of pyrethroids like deltamethrin has imposed selection pressure on \u003cem\u003eAe. albopictus\u003c/em\u003e and eventually caused resistance [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Fast emerging and increasing resistance to insecticides like deltamethrin in \u003cem\u003eAe. albopictus\u003c/em\u003e brings alarm to the dengue epidemic over the world.\u003c/p\u003e \u003cp\u003ePyrethroids resistance in \u003cem\u003eAe. albopictus\u003c/em\u003e occurs mainly through the F1534S and F1534L mutation in the VGSC gene [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], also known as knockdown resistance (kdr) resistance, as well as up-regulation of specific detoxification enzymes like glutathione S-transferase (GST) (GSTE1, AGAP009195-PA) and cytochrome P450 monooxygenases (P450s) CYP6A8, CYP9Z4, CYP6D4 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Pyrethroids resistance was also regarded to be associated with pathways involved in metabolic process and pathways [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], protein digestion and absorption [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Metabolic pathways and biosynthesis of secondary metabolites have been identified to be the top-two differential pathway categories between deltamethrin-resistant and susceptible individuals of \u003cem\u003eAe. albopictus\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Metabolomics could reflect systematic human metabolic variations in accordance with phenotypic changes, such as resistance to insulin [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and trastuzumab [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Characteristic of amino acids and AcylCNs acylcarnitine profile has become is a powerful and popular tool for diagnostic of clinical metabolic alterations [\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, little is known of the characteristic of amino acids and AcylCNs profiles in mosquitoes. Altered profile of amino acids and AcylCNs has been reported in \u003cem\u003eCulex quinquefasciatus L\u003c/em\u003e after exposure to chlorpyrifos, temephos and permethrin [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In this study, amino acids and AcylCNs profiles of deltamethrin-resistant and susceptible strains of \u003cem\u003eAe. albopictus\u003c/em\u003e were compared to observe their alterations in accordance with deltamethrin resistance.\u003c/p\u003e "},{"header":"Materials And Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMosquito strains, analysis of insecticide resistance and kdr mutations\u003c/h2\u003e \u003cp\u003eLarvae of two field strains of \u003cem\u003eAe. albopictus\u003c/em\u003e were collected from public land like parks, schools and residential areas in 2017 in two districts named Yuexiu and Baiyun of Guangzhou city respectively, with dengue incidence of \u0026gt;\u0026thinsp;5 and 1\u0026ndash;5 cases per 100,000 people respectively as previous report [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The larvae were housed [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and have been tested for their larval resistance to four insecticides: (1) organophosphate, temephos; (2) microbial bacterial toxin, Bti; (3) hormonal insect growth regulators, pyriproxyfen (PPF); (4) the chitin biosynthesis inhibitor, hexaflumuron; as well as their adult resistance to five adulticides including deltamethrin, permethrin, malathion, bendiocarb, DDT following WHO guidelines [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The larvae housed from both Yuexiu and Baiyun districts were also tested for their resistance to adulticides including deltamethrin, permethrin, DDT, bendiocarb, malathion following WHO guidelines as previous report [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. For larval bioassays, the resistant status was evaluated by the resistant ratio (RR\u003csub\u003e50\u003c/sub\u003e) (RR\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;5, susceptible; 5\u0026thinsp;\u0026lt;\u0026thinsp;RR\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;10, moderately resistant; RR\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;10, highly resistant), for adult bioassays, resistant status was defined by mortality rate: resistant if mortality\u0026thinsp;\u0026lt;\u0026thinsp;90%, probably resistant if mortality was between 90 and 98%, and susceptible if mortality\u0026thinsp;\u0026gt;\u0026thinsp;98% [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The kdr mutation in resistant and susceptible individuals from Yuexiu and Baiyun districts in 2017 has been genotyped at the voltage-gated sodium channel (VGSC) gene after exposure to deltamethrin, permethrin, and DDT to detect mutations within domains II, III and IV [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], samples exposed to bendiocarb have been genotyped at the ace-1 gene to detect mutations within G119 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The Foshan strain of \u003cem\u003eAe. albopictus\u003c/em\u003e, an insecticide-susceptible stain which was reared in laboratory without exposure to any insecticides since its collection in 1983 in Foshan (a smaller city adjacent to Guangzhou city), was used as the control strain for metabolomic comparison with these field strains of \u003cem\u003eAe. albopictus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eR18 is a laboratory resistant strain (LC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.039, RR\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;13) after deltamethrin selection of an insecticide susceptible \u003cem\u003eAe. albopictus\u003c/em\u003e populations from Shanghai CDC for 17 generations, which was primarily screened by 0.005\u0026nbsp;mg/L of deltamethrin (Sigma, USA) from the 1st to the 10th generation, followed by deltamethrin screening at 50% lethal concentration (LC\u003csub\u003e50\u003c/sub\u003e) from the 11th to the 22nd generation as described previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The parental strain of \u003cem\u003eAe. albopictus\u003c/em\u003e without any exposure to insecticides (R0) was used as an insecticide-susceptible control strain for R18 strains of \u003cem\u003eAe. albopictus\u003c/em\u003e in this study, LC\u003csub\u003e50\u003c/sub\u003e (95%)\u0026thinsp;=\u0026thinsp;0.003 (0.001, 0.006) mg/L. The larval and adult resistance of R18 to deltamethrin was also analyzed following WHO guidelines as previous report [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The resistant and susceptible adults against deltamethrin from R18 strains of \u003cem\u003eAe. albopictus\u003c/em\u003e were also genotyped for kdr mutation as previous report [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eLC-MS/MS sample preparation and metabolites extraction\u003c/h2\u003e \u003cp\u003eSix replicates of ten numbers of 3rd to 4th instar larvae (approximately 2\u0026nbsp;mg/larvae) of Foshan, Yuexiu, Baiyun, R0, R18 strains were sampled respectively. Then, they were homogenized (TIANGEN OSE-Y10-plus) in 500\u0026nbsp;\u0026micro;l of ddH\u003csub\u003e2\u003c/sub\u003eO, followed by centrifugation at 4 ℃,13000\u0026nbsp;rpm for 10\u0026nbsp;min. The lysate was transferred to a sterile syringe which was attached to a 0.22\u0026nbsp;\u0026micro;m acrodisc for filtration. Finally, 30\u0026nbsp;\u0026micro;l of the filtered lysate was added to the filter paper (S\u0026amp;S903) and dried for storage. A paper circle with 3.2\u0026nbsp;mm diameter was punched from the dry larval lysate filter paper by a Panthera-Puncher TM 9 Puncher (PerkinElmer, Waltham, MA, USA)]. A NeoBase non-derivatized MS/MS kit (Perkin Elmer) was employed for extraction of amino acids and AcylCNs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMetabolomics analysis\u003c/h2\u003e \u003cp\u003eLC-MS/MS (API 2000, ABSciex, Framingham, MA, USA) platform, with stable isotopes labelled amino acid standards (Cambrige Isotope Labs) as an internal solution, was selected for quantification of 17 amino acids including alanine, arginine, aspartate, citrulline, glutamine, glutamate, glycine, histidine, leucine, methionine, ornithine, phenylalanine, proline, serine, threonine, tyrosine, valine) and 34 AcylCNs including C0, C2, C3, C3DC, C4, C4-OH, C4DC, C5, C5:1, C5-OH, C5DC, C6, C6:1, C6DC, C8, C8:1, C10, C10:1, C10:2, C12, C12:1, C14, C14-OH, C14:1, C14:2, C16, C16:1, C16-OH, C16:1-OH, C18, C18:1, C18-OH, C18:2, C18:1-OH (34AC). Analysis of 17 amino acids and 34 AcylCNs was interpreted in multiple reaction monitoring (MRM) mode with Analyst 1.6.2 software (ABSciex) and NeoBase database.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eFinally, the contents of 17 amino acids and 34 AcylCNs were scaled according to the preparation procedure of dry larval lysate filter paper. The concentration of the detected metabolites was normalized and analyzed by orthogonal pattern recognition methods principal component analysis (PCA). PCA with performed using Metaboanalyst (v3.0) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.metaboanalyst.ca/faces/home.xhtml\u003c/span\u003e\u003c/span\u003e) to display the characteristic of amino acids and AcylCNs profiles of \u003cem\u003eAe. albopictus\u003c/em\u003e with different levels of deltamethrin resistance. SPSS 17.0 was applied for the spearman correlation analysis between the deltamethrin LC\u003csub\u003e50\u003c/sub\u003e values and the contents of amino acids and AcylCNs in \u003cem\u003eAe. albopictus.\u003c/em\u003eLC\u003csub\u003e50\u003c/sub\u003e and KDT\u003csub\u003e50\u003c/sub\u003e were estimated using the log-probit models [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. For larvae bioassays, the resistant status was measured by the resistant ratio (RR\u003csub\u003e50\u003c/sub\u003e), i.e., the ratio of LC\u003csub\u003e50\u003c/sub\u003e (or IE\u003csub\u003e50\u003c/sub\u003e) for the field population over LC\u003csub\u003e50\u003c/sub\u003e (or IE\u003csub\u003e50\u003c/sub\u003e) for the laboratory-susceptible strain [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Larval resistance status was defined as susceptible if RR\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;5, moderately resistant if 5\u0026thinsp;\u0026lt;\u0026thinsp;RR\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;10, and highly resistant if RR\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;10 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. For adult bioassays, resistant status was defined by mortality rate: Resistant if mortality\u0026thinsp;\u0026lt;\u0026thinsp;90%, probably resistant if mortality was between 90 and 98%, and susceptible if mortality\u0026thinsp;\u0026gt;\u0026thinsp;98% [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The relationship between nonsynonymous mutations and resistance was verified by Fisher\u0026rsquo;s exact test or the χ2 -test (when all n\u0026thinsp;\u0026gt;\u0026thinsp;5), and the odds ratio (OR) was calculated for each mutation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eInsecticide resistance and kdr mutation of\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eAe. albopictus\u003c/span\u003e \u003cstrong\u003elarvae from Yuexiu and Baiyun districts in 2017\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAe. albopictus\u003c/em\u003e larvae from both Yuexiu and Baiyun districts had moderate to high resistance to adulticides like deltamethrin and permethrin, both of them remained susceptible to malathion (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Moreover, larvae from Yuexiu district had developed much higher level of resistance to deltamethrin than the larvae from Baiyun district, but larvae from Baiyun district was more resistant to both permethrin and DDT (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In 2017, the larvae and adult populations of \u003cem\u003eAe. albopictus\u003c/em\u003e from Baiyun/Yuexiu districts of Guangzhou city, China has developed high resistance to currently used insecticides (deltamethrin, permethrin, DDT and bendiocarb, mortality\u0026thinsp;\u0026lt;\u0026thinsp;90%) except malathion (mortality\u0026thinsp;\u0026gt;\u0026thinsp;98%) [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. The mortality rate against deltamethrin, permethrin, DDT and bendiocarb was 5.6%, 55.2%, 52.1%, 42.4% in the adult populations from Yuexiu district, and 38.4%, 68.8%, 74.7% and 51.2 in the adult population from Baiyun district, respectively [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. \u003cem\u003eAe. albopictus\u003c/em\u003e larvae from both districts were still sensitive to Bti and hexaflumuron (RR\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;5), but high resistance to pyriproxyfen (RR\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;10) [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eThe larval resistance of \u003cem\u003eAe. albopictus\u003c/em\u003e from both Yuexiu and Baiyun districts to five adulticides (deltamethrin, permethrin, DDT, bendiocarb, malathion) in 2017\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003edeltamethrin\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003ePermethrin\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eDDT\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eBendiocarb\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eMalathion\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLC\u003csub\u003e50\u003c/sub\u003e(95%)(mg/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRR\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLC\u003csub\u003e50\u003c/sub\u003e(95%)(mg/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRR\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLC\u003csub\u003e50\u003c/sub\u003e(95%)(mg/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRR\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLC\u003csub\u003e50\u003c/sub\u003e(95%)(mg/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRR\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLC\u003csub\u003e50\u003c/sub\u003e(95%)(mg/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRR\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eBaiyun\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.027(0.020,0.049)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e27\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.628(0.527,0.730)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.281(5.073,5.538)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.460(0.995,1.745)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.77\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.073(0.068,0.080)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eYuexiu\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.066(0.061,0.094)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e66\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.356(0.113,0.533)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.47\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.912(1.560,2.227)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.37\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.37(4.826,6.031)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.54\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.092(0.081,0.109)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.14\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eFoshan\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.001(0.001,0.006)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.042(0.031,0.053)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.438(0.389,0.484)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.969(0.908,1.038)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.043(0.039,0.046)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eKdr mutation associates with deltamethrin resistance in R18 strain\u003c/h2\u003e\n\u003cp\u003eThe larvae and adult populations of \u003cem\u003eAe. albopictus\u003c/em\u003e from R18 has developed high resistance to deltamethrin (larval LC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.039, RR\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;13, adult mortality\u0026thinsp;=\u0026thinsp;85%). Sequences of domains II (480\u0026nbsp;bp), III (346\u0026nbsp;bp) and IV (280\u0026nbsp;bp) of the VGSC gene were also obtained from the resistant and susceptible mosquitoes after deltamethrin adult bioassays for R18 [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. Kdr genotyping showed non-synonymous mutations of F1534S could be detected at codon 1534 in domain III of R18, where wild-type TTC (Phe) was changed to TCC (Ser) at a high frequency of 78.38% (29/37), indicating a significant correlation with deltamethrin resistance (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003e\u003cstrong\u003eKdr mutations at position F1534 of the VGSC gene in adults of\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eAe. albopictus\u003c/span\u003e \u003cstrong\u003efrom R18 strain and its parental susceptible R0 strain\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eInsecticide\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eN\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"5\" align=\"left\"\u003e\n\u003cp\u003eGenotype\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eOdds Ratio(95%Cl)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eFF\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eFS\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eSS\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eFL\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eLL\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eF1534S\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eF1534L\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eSusceptible strain (R0)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e---\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eR18\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e37\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e123.00 (14.30,1013.39)**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e--\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"9\"\u003e**:\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eDifferential larval amino acids and AcylCNs profiles between insecticide-resistant\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eAe. albopictus\u003c/span\u003e \u003cstrong\u003efrom Baiyun/Yuexiu and susceptible Foshan strain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmino acids and AcylCNs profile analysis demonstrated that Pro, Val, Arg, Glu and His were the five most abundant amino acids (Fig.\u0026nbsp;1A) whereas C0, C2 were the richest AcylCNs in the larvae of \u003cem\u003eAe. albopictus\u003c/em\u003e (Table S2) \u0026amp; (Figs.\u0026nbsp;1B and 1C). The metabolites in Baiyun/Yuexiu larvae displaced fluctuation in comparison to Foshan larvae. T test analysis showed the concentration of five amino acids Tyr, Cit, Pro, Thr, Gly (Fig.\u0026nbsp;2A) and five AcylCNs C3DC, C6-1, C10, C10-1,C10-2 (Fig.\u0026nbsp;2B) were significantly higher, while the levels of C2 (Fig.\u0026nbsp;2B) and four amino acids His, Val, Glu, Ala (Fig.\u0026nbsp;2A) were obviously lower in Yuexiu strain in comparison to Foshan strain. Similarly, Cit and Arg were increased while His was decreased in Baiyun strain (Fig.\u0026nbsp;2C). Heatmap and PCA analysis of 17 amino acids and 34 AcylCNs contents indicated significant discrepancy of amino acids and AcylCNs profile between Foshan and Baiyun/Yuexiu larvae (Figs.\u0026nbsp;3A and 3B). However, profile of Yuexiu larvae was partly overlapped with that of Baiyun larvae (Fig.\u0026nbsp;3B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferential larval amino acids and AcylCNs profiles between deltamethrin-resistant (R18) and its parental susceptible (R0) strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmino acids and AcylCNs profile analysis demonstrated that Pro, Val, Arg, Glu and His were the five most abundant amino acids whereas C0, C2 were the richest AcylCNs in the deltamethrin-resistant (R18) and -susceptible (R0) larvae of \u003cem\u003eAe. albopictus\u003c/em\u003e (Table S2). Between deltamethrin-resistant R18 and susceptible (R0) strains, no significant difference of amino acids and AcylCNs were found, but the heatmap and PCA analysis of 17 amino acids and 34 AcylCNs contents indicated discrepancy of amino acids and AcylCNs profile (Figs.\u0026nbsp;4A and 4B).\u003c/p\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003e\u003cstrong\u003eKdr mutations at position F1534 of the VGSC gene in resistant and susceptible adults of\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eAe. albopictus\u003c/span\u003e \u003cstrong\u003eagainst deltamethrin, permethrin and DDT from both Yuexiu and Baiyun districts in 2017\u003c/strong\u003e [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eInsecticide\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003ePhenotype\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eN\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"5\" align=\"left\"\u003e\n\u003cp\u003eGenotype\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eOdds Ratio(95%Cl)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eFF\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eFS\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eSS\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eFL\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eLL\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eF1534S\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eF1534L\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eDeltamethrin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e41\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.44(1.12,17.52)*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNA\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003epermethrin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e42\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.55(1.38,15.05)*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e27.20(4.60,160.69)**\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eDDT\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e44\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.00(1.27,19.74)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNA**\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"10\"\u003eNA: Not Available. Significance level. *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05,**P\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eFor insecticide resistance monitoring, the bioassays like WHO susceptibility test [22] and CDC bottle assay [23], molecular detection of knock-down resistant gene mutations, analysis of metabolic enzymes GSTs, p450s and CCEs activities have been frequently used, but limitations existed, such as time and labor-consuming, difficult for standardization with complicated procedures, requirement of special insectary laboratory to rear mosquitoes and subjective judgement of larval and adult death, etc. Amino acids and AcylCNs profile have been popular for diagnostic of clinical metabolic diseases. In this study, we first tried to explore the characteristic of amino acids and AcylCNs profile among geographically or genetically-close strains of \u003cem\u003eAe. albopictus\u003c/em\u003e with differential resistance to insecticides especially deltamethrin, and compared their differences. The insecticide resistance status and the corresponding possible genetic mechanism of the test strains was analyzed thoroughly before their metabolomic analysis. Deltamethrin was the most popular adulticide for vector control in urban areas of Guangzhou city [7]. Accordingly, the most significant difference of insecticide resistance between the larvae and adults from different districts were their differential resistance to deltamethrin, such as significantly higher larval and adult resistance to deltamethrin in Yuexiu strain (larval RR\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;66, adult mortality of 5.6%) than Baiyun strain (larval RR\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;27, adult mortality of 38.4%) (Table\u0026nbsp;1) [7]. In \u003cem\u003eAe. albopictus\u003c/em\u003e adults from both districts, the resistance to pyrethroids (deltamethrin, permethrin) and DDT has been proved to be significantly associated with non-synonymous 1534 codon mutations from homozygote wild-type TTC (Phe) (F1534F) to homozygote TCC (Ser) (F1534S) or CTC (Leu) (F1534L) to at domain III (346\u0026nbsp;bp) of the VGSC gene, with five detected kdr genotype (FF, FS, SS, FL, LL) in the resistant and susceptible individuals of \u003cem\u003eAe. albopictus\u003c/em\u003e adults for three insecticides (deltamethrin, permethrin and DDT) from both districts (Table\u0026nbsp;3) [7] as previous report [8]. Similarly, Kdr genotyping detected F1534S mutation at codon 1534 in domain III of R18 strain at a frequency of 78.38% (29/37), indicating a significant correlation of F1534S mutation with laboratory-induced deltamethrin resistance in \u003cem\u003eAe. albopictus\u003c/em\u003e (Table\u0026nbsp;2, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\n\u003cp\u003eThe PCA analysis of amino acids and AcylCNs profiles among different strains demonstrated that \u003cem\u003eAe. albopictus\u003c/em\u003e larvae with differential deltamethrin resistant level displaced differential amino acids and AcylCNs profiles, including clear distinction of amino acids and AcylCNs profile between insecticide-susceptible larvae (Foshan strain) and larvae collected from geographically-close and insecticide-resistant Baiyun/Yuexiu districts, or obvious difference between a laboratory-induced deltamethrin-resistant strain (R18) and its parental susceptible Shanghai strain (R0), whereas it was relatively similar between insecticide-resistant larvae from relative geographically-close Baiyun and Yuexiu districts. This may suggest a potential role of amino acids and AcylCNs profile in predicting larval insecticide resistance of \u003cem\u003eAe. albopictus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eMoreover, five amino acids (Tyr, Cit, Pro, Thr, Gly) were significantly higher, whereas four amino acids (His, Val, Glu, Ala) were obviously lower in larvae from Yuexiu district than the susceptible Foshan strain. However, only Cit and Arg were increased while His was decreased in larvae from Baiyun district than the susceptible Foshan strain, this phenomenon was probably due to their differential resistance to insecticides especially deltamethrin. Amino acids are metabolites required for protein synthesis, and have roles in metabolism as energy sources and nucleic acid precursors, they could be transformed into carbon-hydrates and fatty acids. The significant difference of amino acids between insecticide-susceptible larvae (Foshan strain) and insecticide-resistant larvae collected from geographically-close Baiyun/Yuexiu districts, was probably associated with the amino acid changes in correspondence with the deltamethrin-resistance correlated genetic changes [7] and altered expression of P450s, cuticle proteins, glutathione S transferase, serine proteases, heat shock protein cytochrome s, esterase [9], as well as P450 gene SNPs such as Arg226Ser in CYP6A8, Pro175Gln in CYP9B2, His877Tyr in CYP9B2, Cys212Ser in CYP1A1 in \u003cem\u003eAe. albopictus\u003c/em\u003e [9]. The change of amino acids and proteins was also reported to be involved in insecticide resistance of other mosquitoes, such as arginine (ARG) changes for energy production and insecticide detoxification of \u003cem\u003eCulex quinquefasciatus L\u003c/em\u003e [19], elevated P450s and esterases (EST) in the insecticide-resistant housefly \u003cem\u003eM. domestica\u003c/em\u003e [24], more proteins involved in oxidoreductase, transferase, hydrolase, transporter and catalytic activity, cuticle formation in the pyrethroid resistant strain of \u003cem\u003eculex pipiens pallens\u003c/em\u003e [25, 26]. Changes of amino acids and insect proteases also occurred under insecticide exposure for protein metabolism, such as for re-synthesis of up-regulated detoxification enzymes, suggesting a role of protein metabolism under insecticide pressure [24, 27].\u003c/p\u003e\n\u003cp\u003eCarnitine is derived from amino acids lysine and methionine, AcylCNs are metabolites of organic acids and beta oxidation of fats. In this study, C0 and C2 were the two most abundant AcylCNs in larvae of all five strains. The changes of free carnitine (C0) and acetyl-carnitine (C2) were regarded to be involved in energy production and insecticide detoxification of \u003cem\u003eCulex quinquefasciatus L\u003c/em\u003e [19]. In this study, five AcylCNs (C3DC, C6-1, C10, C10-1, C10-2) were found to be significantly higher in larvae from Yuexiu strain than the susceptible Foshan strain, while the levels of C2 were obviously lower, suggesting a probable variation of these AcylCNs in association with the development of insecticide resistance in \u003cem\u003eAe. albopictus\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study first captured the characteristic of amino acids and AcylCNs profile of \u003cem\u003eAe. albopictus\u003c/em\u003e larvae with differential resistance to insecticides especially deltamethrin, indicating the possibility of LC-MS/MS based amino acids and AcylCNs profile as a tool for fast discrimination of deltamethrin-resistant strain from -susceptible strain of \u003cem\u003eAe. albopictus\u003c/em\u003e, as well as a potential way for seeking potential biomarkers for early diagnosis of insecticide resistance like deltamethrin in \u003cem\u003eAe. albopictus\u003c/em\u003e.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAA, amino acids. AC, acylcarnitine. AcylCNs, acylcarnitine. \u003cem\u003eAe. albopictus, Aedes albopictus\u003c/em\u003e. PCA, principal component analysis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Hongxia Liu from Shanghai CDC for assistance in induction of laboratory deltamethrin-resistant strains of \u003cem\u003eAe. albopictus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Lianfen Huang, Ruili Xie, Xiao-Guang Chen, Hua Li. Data curation: Lianfen Huang, Xinhua Su, Chenfang Tang. Formal analysis: Lianfen Huang, Ruili Xie, Lilan Peng, Chenfang Tang, Jun Li, Xinhua Su. Funding acquisition: Xiao-Guang Chen, Hua Li. Investigation: Lianfen Huang, Ruili Xie, Lilan Peng, Xinhua Su. Methodology: Lianfen Huang, Ruili Xie, Lilan Peng, Chenfang Tang, Yonglan Huang, Xinhua Su. Project administration: Lianfen Huang, Ruili Xie, Jun Li. Supervision: Xiao-Guang Chen, Hua Li, Yonglan Huang. Final approval: all authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Institutes of Health, USA(R01AI083202), Guangzhou Science and technology project, China (202002030491) and the Medical Scientific Research Foundation of Guangdong Province, China (A2019512).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cbr /\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u003cbr /\u003e\u003c/strong\u003e\u003cstrong\u003eAvailability of supporting data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe insecticide resistance and Kdr mutation analysis data of the adults of Ae. albopictus from Yuexiu/Baiyun districts of Guangzhou city in 2017 is available from the paper (Su X, Guo Y, Deng J, Xu J, Zhou G, Zhou T, et al. (2019) Fast emerging insecticide resistance in Aedes albopictus in Guangzhou, China: Alarm to the dengue epidemic. PLoS Negl Trop Dis 13(9): e0007665.) at the website (https://doi.org/10.1371/journal. pntd.0007665).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest in this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' information\u003cbr /\u003e\u003c/strong\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of Pathogen Biology and Experimental Teaching Center of Preventive Medicine, Guangdong Provincial Key Laboratory of Tropical Disease, School of Public Health, Southern Medical University, Guangzhou, China. \u003csup\u003e2\u003c/sup\u003eClinical Laboratory, Guangzhou Women and Children\u0026rsquo;s Medical center, Guangzhou Medical University, Guangzhou, China. \u003csup\u003e3\u003c/sup\u003eNewborn Screening Center, Guangzhou Women and Children\u0026rsquo;s Medical center, Guangzhou Medical University, Guangzhou, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eManni M, Guglielmino CR, Scolari F, Vega-Rua A, Failloux AB, Somboon P, Lisa A, Savini G, Bonizzoni M, Gomulski LM, et al. Genetic evidence for a worldwide chaotic dispersion pattern of the arbovirus vector, \u003cem\u003eAedes albopictus\u003c/em\u003e. PLoS Negl Trop Dis. 2017;11(1):e5332.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBohers C, Mousson L, Madec Y, Vazeille M, Rhim A, M'Ghirbi Y, Bouattour A, Failloux AB. The recently introduced \u003cem\u003eAedes albopictus\u003c/em\u003e in Tunisia has the potential to transmit chikungunya, dengue and Zika viruses. PLoS Negl Trop Dis. 2020;14(10):e8475.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaldacchino F, Caputo B, Chandre F, Drago A, Della TA, Montarsi F, Rizzoli A. Control methods against invasive \u003cem\u003eAedes mosquitoes\u003c/em\u003e in Europe: a review. PEST MANAG SCI. 2015;71(11):1471\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbramides GC, Roiz D, Guitart R, Quintana S, Guerrero I, Gimenez N. Effectiveness of a multiple intervention strategy for the control of the tiger mosquito (\u003cem\u003eAedes albopictus\u003c/em\u003e) in Spain. Trans R Soc Trop Med Hyg. 2011;105(5):281\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWorld Health Organization. Dengue: Guidelines for diagnosis, treatment, prevention and control: new edition. Geneva: World Health Organization; 2009.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWorld Health Organization. Global insecticide use for vector-borne disease control, a 10 year assessment (2000\u0026ndash;2009). 2011.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu X, Guo Y, Deng J, Xu J, Zhou G, Zhou T, Li Y, Zhong D, Kong L, Wang X, et al. Fast emerging insecticide resistance in \u003cem\u003eAedes albopictus\u003c/em\u003e in Guangzhou, China: Alarm to the dengue epidemic. PLoS Negl Trop Dis. 2019;13(9):e7665.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu J, Bonizzoni M, Zhong D, Zhou G, Cai S, Li Y, Wang X, Lo E, Lee R, Sheen R, et al. Multi-country survey revealed prevalent and novel F1534S mutation in voltage-gated sodium channel (VGSC) gene in \u003cem\u003eAedes albopictus\u003c/em\u003e. PLoS Negl Trop Dis. 2016;10(5):e4696.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu J, Su X, Bonizzoni M, Zhong D, Li Y, Zhou G, Nguyen H, Tong S, Yan G, Chen XG. Comparative transcriptome analysis and RNA interference reveal CYP6A8 and SNPs related to pyrethroid resistance in \u003cem\u003eAedes albopictu\u003c/em\u003es. PLoS Negl Trop Dis. 2018;12(11):e6828.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang T, Liu N. Genome analysis of cytochrome P450s and their expression profiles in insecticide resistant mosquitoes, \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e. 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Altered serum amino acid and acylcarnitine profiles in hyperinsulinemic hypoglycemia and ketotic hypoglycemia. Front Endocrinol (Lausanne). 2020;11:577373.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGunther SH, Khoo CM, Tai ES, Sim X, Kovalik JP, Ching J, Lee JJ, van Dam RM. Serum acylcarnitines and amino acids and risk of type 2 diabetes in a multiethnic Asian population. BMJ Open Diabetes Res Care 2020, 8(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKasakin MF, Rogachev AD, Predtechenskaya EV, Zaigraev VJ, Koval VV, Pokrovsky AG: Changes in amino acid and acylcarnitine plasma profiles for distinguish in patients with multiple sclerosis from healthy controls. Mult Scler Int 2020, 2020:9010937.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartin-Park A, Gomez-Govea MA, Lopez-Monroy B, Trevino-Alvarado VM, Torres-Sepulveda MD, Lopez-Uriarte GA, Villanueva-Segura OK, Ruiz-Herrera MD, Martinez-Fierro ML, Delgado-Enciso I, et al. Profiles of amino acids and acylcarnitines related with insecticide exposure in \u003cem\u003eCulex quinquefasciatus (Say)\u003c/em\u003e. Plos One. 2017;12(1):e169514.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWorld Health Organization. Guidelines for laboratory and field testing of mosquito larvicides. Geneva: World Health Organization; 2005.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang LF, Li J, Peng L. The differential metabolic profiles between deltamethrin resistant and -susceptible strains of \u003cem\u003eAedes albopictus\u003c/em\u003e. J Med Entomol 2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWorld Health Organization. Test procedures for insecticide resistance monitoring in malaria vectors, bio-efficacy and persistence of insecticides on treated surfaces: report of the WHO informal consultation, Geneva, 28\u0026ndash;30 September 1998. Geneva World Health Organization 1998.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrogdon WG, McAllister JC. Simplification of adult mosquito bioassays through use of time-mortality determinations in glass bottles. J Am Mosq Control Assoc. 1998;14(2):159\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi M, Reid WR, Zhang L, Scott JG, Gao X, Kristensen M, Liu N. A whole transcriptomal linkage analysis of gene co-regulation in insecticide resistant house flies, \u003cem\u003eMusca domestica\u003c/em\u003e. BMC GENOMICS. 2013;14:803.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLv Y, Wang W, Hong S, Lei Z, Fang F, Guo Q, Hu S, Tian M, Liu B, Zhang D, et al. Comparative transcriptome analyses of deltamethrin-susceptible and -resistant \u003cem\u003eCulex pipiens pallens\u003c/em\u003e by RNA-sEq. Mol Genet Genomics. 2016;291(1):309\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang W, Lv Y, Fang F, Hong S, Guo Q, Hu S, Zou F, Shi L, Lei Z, Ma K, et al. Identification of proteins associated with pyrethroid resistance by iTRAQ-based quantitative proteomic analysis in Culex pipiens pallens. Parasit Vectors. 2015;8:95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilkins RM. Insecticide resistance and intracellular proteases. Pest Manag Sci. 2017;73(12):2403\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"Amino acids and acylcarnitines profiles, Deltamethrin resistance, Aedes albopictus","lastPublishedDoi":"10.21203/rs.3.rs-135058/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-135058/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Fast emerging and increasing insecticide resistance in Asian tiger mosquito, \u003cem\u003eAedes albopictus\u003c/em\u003e brings alarm to the dengue epidemic over the world. The genetic and transcriptional sequencing has greatly advanced our understanding of the molecular basis for this crucial adaptation, but less is known about the regulation of insecticide resistance physiology at the metabolomic level. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eWe captured the relative abundance of 17 amino acids and 34 AcylCNs in the 3\u003csup\u003erd\u003c/sup\u003e to 4\u003csup\u003eth \u003c/sup\u003einstar larvae of three geographically close strains of \u003cem\u003eAe. albopictus\u003c/em\u003e, including a laboratory insecticide susceptible strain (Foshan) and two field deltamethrin-resistant strains (Yuexiu/Baiyun), as well as a laboratory induced deltamethrin-resistant (R18) and its parental insecticide susceptible strains (R0) to characterize their amino acids and acylcarnitines (AcylCNs) profiles by LC-MS/MS spectrometry. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Heatmap and PCA analysis of amino acids and AcylCNs profile indicated a clear separation of Foshan from Yuexiu/Baiyun populations, as well as a partly overlapping between Yuexiu and Baiyun populations. All strains were abundant in five amino acids (Pro, Val, Arg, Glu, His) and two AcylCNs (C0, C2). Five amino acids (Tyr, Cit, Pro, Thr, Gly) were significantly higher, while four amino acids (His, Val, Glu, Ala) were obviously lower in Yuexiu strain than Baiyun population. R18 displayed a differential amino acid and AcylCNs profile from its parental R0 strain. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e This study displayed a distinction of amino acids and AcylCNs profiles between insecticide-resistant and geographically or genetically-close insecticide-susceptible strain of\u003cem\u003e Ae. albopictus\u003c/em\u003e, suggesting a possible application of amino acids and AcylCNs profiles for rapid diagnosis of deltamethrin resistance in \u003cem\u003eAe. albopictus\u003c/em\u003e in the future.\u003c/p\u003e","manuscriptTitle":"Profiles of Amino Acids and Acylcarnitines Related with Deltamethrin Resistance in Aedes Albopictus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-12-28 22:10:06","doi":"10.21203/rs.3.rs-135058/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":"186dce30-860e-4cd2-9d93-a2d35d4311c0","owner":[],"postedDate":"December 28th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":1639129,"name":"Infectious Diseases"}],"tags":[],"updatedAt":"2021-01-28T12:03:18+00:00","versionOfRecord":[],"versionCreatedAt":"2020-12-28 22:10:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-135058","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-135058","identity":"rs-135058","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}