| BackgroundIn recent years,with the proliferation of mosquito-borne diseases,mosquito-control has become a hot spots in public health.And the chemical control is still the main strategy for mosquito control.However,with the frequent application of chemical pesticides,the resistance of mosquitoes is increasing.Presently,it has been found that metabolic resistance and target resistance is potentially related to the alteration of amino acids levels in the mechanism of the development of mosquito resistance.In previous work,we found that there are significant differences in the metabolites between laboratory-resistant larvae and laboratory-susceptible larvae,and that many amino acids may be related to the development of resistance.In this study,we established a method for the detection of amino acids metabolites in Aedes albopictus larvae by using liquid chromatography-tandem mass spectrometry(LC-MS/MS),to explore amino acids biomarkers associated with development of deltamethrin resistance and toxicity of deltamethrin in Aedes albopictus larvae.Objectives1.Establish LC-MS/MS detection method for amino acids of Aedes albopictus;2.Compare amino acids levels between different strains and different resistant levels of Aedes albopictus and provide reference for screening Aedes albopictus resistant markers;3.Observe alterations of amino acids levels in resistant Aedes albopictus larvae before and after exposure to deltamethrin,and explore the amino acids markers which related to deltamethrin toxicity.Methods1.Establishment of LC-MS/MS detection systemThe basic detection system was established by referring to the relevant parameters of amino acid detection,and the parameters were calibrated by pre-experiment of laboratory sensitive mosquito.Adding the internal standard to control the system error,6 parallel samples were set to control the random error,and 6 kinds of amino acids(standard concentration for sample recovery rates detection)to ensure the sample recovery rates.2.Determination of population resistance of mosquitoPopulation resistance of mosquito was determined using the mosquito resistance test(larvae dipping method)recommended by the World Health Organization(WHO)to determine the lethal concentration 50(LC50)of the population,which was calculated based on the ratio of laboratory-sensitive strain LC50,and the multiple of resistance determine the degree of resistance.3.Collection and rearing of wild mosquitoesAccording to the differences in the distribution of urban and suburban areas,main urban area and suburbs were selected as wild Aedes albopictus larvae collection area.Groups were breed to F1 generation.4.Resistant population drug exposure testAccording to the larvae impregnation method recommended by WHO,drug exposure experiments were conducted on the F1 wild mosquitoes of the Yuexiu wild-type strains which identified as resistant population.It was divided into 3 groups include the drug exposure group,acetone exposure group,and blank control group.After 24 hours,each group of mosquitoes was recovered for testing.5.Detection of larval amino acid levelsAmino acids signals of Aedes albopictus larvae were detected by LC-MS/MS.Amino acids ion signal peak was introduced into Chemview software for quantitative analysis to obtain the target amino acid level.6.Statistical Analysis and MappingThe statistical analysis was performed using SPSS 19.0.The alteration of amino acid levels of different groups were compared using one-way anova analysis(p<0.05).The mapping was performed using R and Graphad5.01 software.Results1.Concentrations of 2 amino acids(citrulline and proline)in laboratory-resistant strains Aedes albopictus were significantly higher than lab-sensitive strains(p<0.05).2.Compared with the laboratory susceptible strains,concentrations of 7 amino acids(alanine,citrulline,glutamic acid,histidine,proline,valine and methionine)were significantly different in the wild resistant strains(p<0.05).Concentrations of three amino acids(alanine,citrulline,proline)in the two wild strains were all up-regulated,concentrations of three amino acids(glutamic acid,histidine,valine)were down-regulated,and there was no consistent change in methionine among the two wild strains.3.12 amino acids(alanine,citrulline,tyrosine,glycine,leucine,ornithine,phenylalanine,proline,serine,histidine,threonine and methionine)were found in deltamethrin exposure group.Concentrations of 11 amino acids decreased and only histidine's increased.The level reduction of 4 amino acids(alanine,citrulline,leucine and phenylalanine)were attributed to deltamethrin exposure(p<0.05)eliminated the effect of acetone.Conclusion1.LC-MS/MS for the detection of amino acid levels in Aedes albopictus larvae is rapid,simple,and accurate.2.7 amino acids(lanine,citrulline,glutamic acid,histidine,valine,valine,methionine)were found to be associated with deltamethrin resistance.3.Exposure to deltamethrin acetone solution causes changes of concentration in 12 amino acids in Aedes albopictus larvae,the concentration decreased of alanine,leucine,phenylalanine,and citrulline associated with deltamethrin exposure. |
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