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

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 eld 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) proles by LC-MS/MS spectrometry. Results: Heatmap and PCA analysis of amino acids and AcylCNs prole 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 ve amino acids (Pro, Val, Arg, Glu, His) and two AcylCNs (C0, C2). Five amino acids (Tyr, Cit, Pro, Thr, Gly) were signicantly 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 prole from its parental R0 strain. Conclusion: This study displayed a distinction of amino acids and AcylCNs proles between insecticide-resistant and geographically or genetically-close insecticide-susceptible strain of Ae. albopictus, suggesting a possible application of amino acids and AcylCNs proles for rapid diagnosis of deltamethrin resistance in Ae. albopictus in the future. L after exposure to chlorpyrifos, temephos and permethrin [19]. In this study, amino acids and AcylCNs proles of deltamethrin-resistant and susceptible strains of Ae. albopictus were compared to observe their alterations in accordance with deltamethrin resistance.


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][5][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 speci c detoxi cation 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 identi ed to be the top-two differential pathway categories between deltamethrin-resistant and susceptible individuals of Ae. albopictus [9]. Metabolomics could re ect 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 pro le has become is a powerful and popular tool for diagnostic of clinical metabolic alterations [14][15][16][17][18]. However, little is known of the characteristic of amino acids and AcylCNs pro les in mosquitoes. Altered pro le 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 pro les 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 eld 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, hexa umuron; as well as their adult resistance to ve 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, moderately resistant; RR 50 > 10, highly resistant), for adult bioassays, resistant status was de ned by mortality rate: resistant if mortality < 90%, probably resistant if mortality was between 90 and 98%, and susceptible 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 eld 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 ltration. Finally, 30 µl of the ltered lysate was added to the lter paper (S&S903) and dried for storage. A paper circle with 3.2 mm diameter was punched from the dry larval lysate lter paper by a Panthera-Puncher TM 9 Puncher 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 eld population over LC 50 (or IE 50 ) for the laboratorysusceptible strain [7]. Larval resistance status was de ned as susceptible if RR 50 < 5, moderately resistant if 5 < RR 50 < 10, and highly resistant if RR 50 > 10 [7].
For adult bioassays, resistant status was de ned by mortality rate: Resistant if mortality < 90%, probably resistant if mortality was between 90 and 98%, and susceptible if mortality > 98% [7,9]. The relationship between nonsynonymous mutations and resistance was veri ed 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 < 90%) except malathion (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 hexa umuron (RR 50 < 5), but high resistance to pyriproxyfen (RR 50 > 10) [7].  Amino acids and AcylCNs pro le analysis demonstrated that Pro, Val, Arg, Glu and His were the ve 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 uctuation in comparison to Foshan larvae. T test analysis showed the concentration of ve amino acids Tyr, Cit, Pro, Thr, Gly ( Fig. 2A) and ve AcylCNs C3DC, C6-1, C10, C10-1,C10-2 (Fig. 2B) were signi cantly 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 signi cant discrepancy of amino acids and AcylCNs pro le between Foshan and Baiyun/Yuexiu larvae (Figs. 3A and 3B). However, pro le of Yuexiu larvae was partly overlapped with that of Baiyun larvae (Fig. 3B).

Differential larval amino acids and AcylCNs pro les between deltamethrin-resistant (R18) and its parental susceptible (R0) strains
Amino acids and AcylCNs pro le analysis demonstrated that Pro, Val, Arg, Glu and His were the ve 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 signi cant 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 pro le (Figs. 4A and 4B).

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 laborconsuming, di cult 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 pro le have been popular for diagnostic of clinical metabolic diseases. In this study, we rst tried to explore the characteristic of amino acids and AcylCNs pro le 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 signi cant difference of insecticide resistance between the larvae and adults from different districts were their differential resistance to deltamethrin, such as signi cantly 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 signi cantly 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 ve 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 signi cant 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 pro les among different strains demonstrated that Ae. albopictus larvae with differential deltamethrin resistant level displaced differential amino acids and AcylCNs pro les, including clear distinction of amino acids and AcylCNs pro le 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 insecticideresistant larvae from relative geographically-close Baiyun and Yuexiu districts. This may suggest a potential role of amino acids and AcylCNs pro le in predicting larval insecticide resistance of Ae. albopictus.
Moreover, ve amino acids (Tyr, Cit, Pro, Thr, Gly) were signi cantly 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 signi cant 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 detoxi cation of Culex quinquefasciatus L [19], elevated P450s and esterases (EST) in the insecticide-resistant house y 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 detoxi cation 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 ve strains. The changes of free carnitine (C0) and acetyl-carnitine (C2) were regarded to be involved in energy production and insecticide detoxi cation of Culex quinquefasciatus L [19]. In this study, ve AcylCNs (C3DC, C6-1, C10, C10-1, C10-2) were found to be signi cantly 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 rst captured the characteristic of amino acids and AcylCNs pro le of Ae. albopictus larvae with differential resistance to insecticides especially deltamethrin, indicating the possibility of LC-MS/MS based amino acids and AcylCNs pro le 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.