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 RR50 = 66, adult mortality of 5.6%) than Baiyun strain (larval RR50 = 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.