The Study On Pyrethroid Resistance In The Malaria Vector,Anopheles Sinensis

Malaria is one of the most important parasitic diseases in the world, and vector control is one of the most effective methods in malaria control and elimination stages. Currently, chemical control is most widely used in vector control, which mainly relies on insecticide to treat the adult mosquitoes. The pyrethroids have been widely used in public health and agriculture, and has become the first choice for indoor residual sparying (IRS), insecticide-treated net (ITN) and Long-lasting insecticidal net (LLIN), due to their low toxicity and high efficacy. However, the significant increase in insecticide-based methods has resulted in rapid spread of resistance globally. The malaria vectors show the resistance to almost all the avialble insecticides classes.In addition to the acceleration of the novel insecticide or new formulation, the delay of resistance developing is also one of the most economic and effective methods. The understanding of the mechanism of resistance is the foundation of choosing and using the insecticides effectively, which could delay their development and spreading.In this study, we collected field Anopheles sinensis (An. sinensis) mosquitoes, detected the resistance level and the kdr mutation frequencies; we further explored the potential mechanism of pyrethroids resistance in An. sinensis, through the investigation of resistance and kdr mutation level under the continuous pyrethroids selection; In addition, we analyzed the transcriptome profiling and resistance-associated genes between the pyrethroids-resistant and susceptible population, to provide the research foundation for the identification and functional study of the key candidate genes associated with insecticide resistance.Part1The detection of pyrethroids resistance level and the kdr mutation frequencies among the field An. sinensis populationObjective:To investigate the pyrethroids resistance level and the kdr mutation frequencies among the field An. sinensis population. Methods:The fourth-instar larva or pupae of Anophline mosquitoes were collected from four places in Jiangsu Province, after the morphological and molecular identification, the confirmed An. sinensis were cultured in the insectory, the WHO recommended tube bioassay method were used to detect the susceptiblity to deltamethrin among the3-4days post emergence, and the AS-PCR were used to detect the knock dowm resistance(kdr) mutation level.Results:All the tested An. sinensis mosquitoes in the four places showed lower mortality (less than10%) after24h recovery to1h exposure to deltamethrin paper, they exhibited high resistance level to deltamethrin based on the WHO criteria; the kdr mutation frequencies ranged from88.2%to100%in the field An. sinensis, three types of kdr mutations at codon position1014of the para-type sodium channel gene were detected:two mutations (from TTG to TTT and from TTG to TTC) cause nonsynonymous changes from leucine to phenylalanine (L1014F) and one mutation from TTG to TGT that cause leucine to cysteine (L1014C) substitution. We discovered that not only the insecticide resistance level decreased rapidly, but the kdr mutation disappeared, after1-2years colonization in the laboratory among the field An. sinensis.Part2Laboratory selection for pyrethroid resistance in field An. sinensisObjective:To prepare an insecticide-resistant strain in the laboratory, and analyze the changing of the kdr, cytochrome P450monoxygenases (P450s) and Glutathione S-transferases (GSTs) among the continuous generations under the pyrethroids selection, to explore the potential mechanism of pyrethroids resistance in An. sinensis.Methods:The engorged An. sinensis mosquitoes were collected, then subjected to continuous pyrethroid selection after species confirmation, and the forced copulation method was used to increase the mating rate. In addition, the kdr mutation frequencies of each generation of An. sinensis were measured via the recently established AllGlo-qPCR method; and the metabolic enzyme activities of P450s and GSTs were detected.Results:The kdr mutation frequencies reached to100%after two generations’ selection, and the TTT is the dominant mutation genotype which cause to L1014F; the resistant level increased stably with the pyrethroids treatment, however, it exhibited synchronized with kdr mutation, the female An. sinensis showed100%resistant to deltamethrin after four generations’seletion. Elevated levels of both P450s and GSTs were significantly found in field selected populations comparing with the laboratory susceptible strain.Part3The study on screening of candidate genes associated with pyrethroids resistance in An. sinensis by RNA-seq.Objective:To generate an EST database for An. sinensis, and screen the candidate genes to pyrethroids resistance, to provide research basis of understanding insecticide resistance mechanism and developing the monitoring method for insecticide resistance.Methods:The total RNA was extracted from various life stage of susceptible An. sinensis and3-4days post emergence deltamethrin-resistance and susceptible field populations. Four cDNA libraries were developed and sequenced by454GS FLX system. The de-novo assembly, gene annotation, differentially expressed genes (DEGs) analysis and qPCR verification for selected genes was performed. In addition, the molecular markers screening for microsatellite and SNP was also performed in this study.Results:The de novo assembly generated33,411contigs with average length of493bp. A total of8,057ESTs were generated with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation. A total of2,131ESTs were differentially expressed between deltamethrin resistant and susceptible mosquitoes. Furthermore, a total of2,408microsatellites and15,496single nucleotide polymorphisms (SNPs) were identified.Conclusions:I. High kdr frequencies ranged from88.2%to100%are found in field An.sinensis in Jiangsu Province, the L1014F is the dominant mutation type, in which TTT is the dominant mutation genotype, however, and it differes significantly in mutation types and genotypes from different areas.Ⅱ. We find that both the resistant level and kdr mutation in An. sinensis populations decrease rapidly after the loss of selective pressure from deltamethrin.III. We colonized a pyrethroid-resistant population of An. sinensis in the laboratory, and find the elevation of metabolic enzymes including P450and GSTs are also involved in the formulation of deltamethrin resistance in An. sinensis. Ⅳ. We use RNA-seq method to generate the transcriptome database of An. sinensis. Furthermore, the insecticide-associated screened ESTs and the molecular markers of microsatellites and SNPs, provide new basis for further study on the mechanism of pyrethroids resistance in An. sinensis.Ⅴ. The present study indicates that high resistant level to pyrethroids have been formulated from An. sinensis in Jiangsu Province, it’s possible to delay the spread of formulation and development of pyrethroids resistance in An.sinensis by adjusting the insecticide classes and choosing the appropriate using method, so as to get rid of the potential threat to achieving of eliminating malaria.