| Objective:Investigation of the species composion and population dynamics of the main mosquito-borne population,the virus species of mosquito vectors,and the polymorphism of insecticide resistance related genes in different habitats in Zichuan District,Zibo City,Shandong Province,to understand the seasonal population density,the virus species and virus carrying rate of mosquito vectors.The distribution of resistance gene polymorphism was analyzed from gene level.The results would provide basic data for local vector control strategies.Methods:1 According to the topographical characteristics of Zichuan district,the mosquito vector samples were collected at one random sampling point in the hilly and plain terrain,Taihe Town selected as investigation point in hilly area and Luocun Town selected as investigation point in plain area,with 15.8 km of the straight line distance between the two investigation points.From August 2017 to July 2018,during the peak period of mosquito-borne activities,we collect mosquito vector samples by mosquito trapping lamp,according to "the National Plan for the Surveillance of Medical Vector",to investigate mosquito vector population and density,and mosquito density monitoring was carried out once a month at intervals of more than 15 days,and 2 mosquito traps were placed at each point.The collection time was from 1 hour after sunset to 1 hour to sunrise.After the mosquitoes were collected,the samples were placed on the ice and the identification was completed quickly with morphological identification of adult mosquito reference to "the Classification and Identification of important Medical Insects in China".Keep the female mosquito,according to the type of mosquito vector,sampling time,sampling location,lamp number and other information to be divided into frozen storage.Excel software was used to analyze the population density of mosquitoes at each survey time.2 Culex pipiens pollens was selected to detect the species and density of mosquito pathogen.DNA was extracted from Cx.pipiens pallens by high-throughput automatic nucleic acid purification system(MagNA Pure 96 System).Reverse Transcription-Polymerase Chain Reaction(RT-PCR)was used to amplify the target fragments of Alphacirus virus,Flavivirus virus,Orthobunyavirus virus and Liaoning virus.We detected the type and number of viruses carried by mosquitoes.The obtained PCR positive products were sent to Biotechnology Co.,Ltd to sequence the genome.The successful sequencing showed that the samples were positive for the pathogen.The nucleotide sequence of RNA non-structural proteins gene NS5 gene and encode structural proteins gene E gene with each known Culex Flavivirus in Genbank were analyzed by MEGA7.0 software.Phylogenetic tree construction and genetic distance calculation were carried out to determine the genotype of the virus.The Excel software PooledInfRate v4 was used to estimate the mosquito infection rate by the maximum likelihood method.3 35 sampling tubes were sampled by stratified sampling according to the sampling time and location of mosquitoes,from the collected samples of Cx.pipiens pallens,and then one Cx.pipiens pallens was randomly selected from each sampling tube.DNA was extracted from Cx.pipiens pallens by high-throughput automatic nucleic acid purification system.Kdr gene fragment of sodium channel in Cx.pipiens pallens was amplificated by PCR.The positive products were sent to Biotechnology Company for sequencing.The kdr allele sequence was detected.The sequence of PCR products was compared and analyzed by Bioedit software to determine the mutation type of knockdown resistance-associated sodium channel gene mutation site.The frequency of kdr gene mutation of mosquitoes in Luocun town and Taihe town was statistically analyzed by IBM SPSS Statistics 25 software with x2,with the statistically significant was P<0.05.Results:1 A total of 830 female adult mosquitoes were collected in Zichuan district,including 690 Cx.pipiens pallens,,114 Armigeres subalbatus,21 Aedes albopictus,and 2 Ae.Vexans,2 Anopheles sinensis and 1 Cx.Tritaeniorhynchus,and Cx.pipiens pallens was the local dominant mosquito species.The population density of mosquitoes in Zichuan area was 14.82/(per unit,night).The year-round monitoring curve of mosquito density showed a double-peak trend,the peak appeared in June and September,and a temporary downward trend appeared in July.Ae.albopictus was found locally for the first time as a new recorded species.The seasonal fluctuation trend of mosquito population density in Luocun town and Taihe town was consistent with that in Zichuan district.The peak of the density of the mosquito in Luocun Town was earlier than that of the Taihe town.Moreover,there were more mosquito species in Luochun town than in Taihe town.Cx.pipiens pallens accounted for 83.13%of the total number of mosquitoes collected in Zichuan district,and the population density was 12.32/(per unit,night).The year-round density monitoring curve also showed a double peak trend,and the peak also appeared in June and September.It was consistent with the seasonal fluctuation trend of mosquito population density in Zichuan district.2 A total of 690 Cx.pipiens pallens were collected from 33 groups.Insect-specific Flavivirus(ISFVs)was amplified from three groups of Cx.pipiens pallens,with the positive rate was 9.09%.The virus-carrying rate was 5.04 ‰,with 95%CI was 1.35 ‰-13.78 ‰,during the peak period of mosquito-borne activities.No viruses of Alphacirus virus,Orthobunyavirus virus and Liaoning virus were isolated.The three groups of Cx.pipiens pallens were respectively amplified by NS5 gene and E gene specific primers to obtain specific bands.The phylogenetic analysis of the NS5 gene found that the genetic distance of the three groups was less than 0.008,and the genetic relationship with the virus strain isolated from Shanghai(MG602497.1)was the most recent,the similarity was 96.83%-97.23%;the phylogenetic analysis of E gene found that the genetic distance of 3 groups was less than 0.002,and the genetic relationship with the virus strain isolated from Tokyo(AB262759.2,AB377213.1)was the most recent,the similarity was 97.55%-97.72%.The virus was identified as Culex Flavivirus(CxFV),and the nucleic acid typing was presumed to be type I.Three strains of Culex Flavivirus were submitted into GenBank database.The GenBank numbers of the NS5 gene sequence were MK422516,MK422517,MK422518,and E gene sequence was MK609521,MK609522,MK609523.3 A total of 35 Cx.pipiens pallens sodium channel kdr gene mutations were detected in this study,including 19 samples in Luocun town and 16 samples in Taihe town.Five kdr alleles were detected at 1014 site of kdr gene,including TTA(75.71%),TTT(10.00%),CTA(5.71%),TCA(4.29%),TTC(4.29%).At the same time,two non-synonymous mutations were found,namely leucine(L1014)to phenylalanine(L1014F)and serine(L101HS).A total of 7 kdr genotypes were detected in Zichuan area.The kdr gene of Cx.pipiens pallens in Luocun town includes 4 genotypes,including L1014AL1014(TTA/TTA),L1014F/L1014(TTT/TTA),L1014L/L1014S(TTA/TCA),L1014S/L1014(TCA/TTA).The kdr gene of Cx.pipiens pallens in Taihe town includes 6 genotypes,including L1014/,1014(CTA/CTA),L1014Ff/L1014F(TTT/TTT),L1014S/L1014F(TCA/TTC),L1014/L1014(TTA/TTA),L1014F/L1014(TTT/TTA),L1014S/L1014(TCA/TTA).The CTA,TTC mutation type of kdr gene was detected for the first time in Cx.pipiens pallens in Taihe town.The mutation frequency of kdr gene of Cx.pipiens pallens in Luocun town and Taihe town was 10.53%and 40.63%respectively,the difference was statistically significant(x2 = 8.559,P = 0.003).The frequency of kdr allele mutation in Cx.pipiens pallens in Zichuan region was 24.29%(17/70),kdr genotype mutation frequency was 37.14%(13/35).Conclusion:Six kinds of mosquitoes,such as Cx.pipiens pallens,Armigeres subalbatus,Ae.albopictus,Ae.vexans,Anopheles sinensis,and Cx.tritaeniorpiens,were collected in the region of Zichuan district,with Cx.pipiens pallens was the dominant species in the area.Ae.albopictus was found locally for the first time as a new recorded species.The seasonal fluctuation curve of mosquito density in Zichuan district showed a double peak trend.The species of mosquitoes in Luocun Town were richer than those in Taihe Town.It was found that Cx.pipiens pallens carried Culex Flavivirus in Zichuan district,but no other vector viruses were found.Therefore the pathogen surveillance of mosquito carrier should be carried out continuously.In this study,the resistance-related sodium channel kdr allele mutation was found in Cx.pipiens pallens,and the mutations of the two kdr alleles of CTA and TTC were detected for the first time in Cx.pipiens pallens,with genotype polymorphism. |
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