Study On The Expression Profile Of Mosquito Resistance PiRNA

Mosquitoes are the important medical insects owing to their function of virus transmitting diseases,such as malaria,dengue fever,West Nile virus(WNV),Japanese encephalitis virus(JEV),Yellow fever and so on,which bring serious harm to human health.Mosquito prevention and treatment usually take comprehensive measures,chemical control is always the main method of comprehensive management of mosquito vectors because of its high efficiency.Unfortunately,with the large-scale and continuous use of pesticides,mosquito resistance to pesticides produced and developed,which bring a major challenge to the prevention and control of mosquito borne diseases.It is accepted that three major mechanisms leading to pesticide resistance,namely target-site resistance,metabolic resistance and cuticular resistance.It is still not fully understood the mechanism of pesticide resistance presently,although studies have reported a series of pesticide resistance related genes at home and abroad,which makes effective resistance management become impossible.A tough nut to crack now is to isolate and identify new pesticide resistance genes and explore resistance mechanism behind this.Piwi-interacting RNAs(piRNAs),26-3 1nt,are a new class small non-coding RNA(sncRNA)with a tendency of uracil in its 5' end that was found in 2006.Previously studies focus on reproductive regulation,besides that,latest researches bring to light that it involved in tumor growth,neural regulation and immune response to mosquito-borne virus.Genome studies in Aedes aegypti discover that piRNA could repress transposons' activity to up-regulation genes that related to resistance to pesticide.PiRNAs can also play a role in combination of target genes 3'UTR in a way similar to miRNA,and miRNA is involved in pesticide resistance of mosquitoes.Whether piRNAs leading to insecticide resistance in a manner similar to miRNA regulation or not,at present,have not been reported.In this research,based on laboratory relevant work,the China's most wide distributed,highest population density mosquito species---Culex pipiens pallens and representatives pyrethroid insecticide---deltamethrin,were selected as materials,then deltamethrin sensitivity(DS)and deltamethrin resistance(DR)mosquito strain were undertaken deep sequencing for piRNAs.We use k-mer scheme to detect piRNA molecules according to their sequence and structure features.112608 piRNA unique clean reads were gotten using Illumina Hiseq 2000 technology,and 50445(44.8%)clean reads mapping to Cx.pipiens quinquefasciatus.The unique piRNA typle number of DS and DR strain is 40456 and 15519 respectively.We got 515198 piRNA reads in DS and the number in DR is 222160.Meanwhile,the aligned piRNA number in repetitive sequence of DS,DR and pooled sample are 20343,10889 and 16090 respectively.Matched piRNA show no tendency in DS,DR and mixed sample.PiRNAs from the genome show the similar result.Interestingly,the MITEs/m4bp mapping the most number of piRNA in both DS group and DR group.Then we found piRNAs with 1U and 10A identity widespread express in genes,no matter where they are.Those mapping piRNA show two peaks in 28nt and 31nt in length.We compered piRNA expression differences of DS andDR by high-throughput sequencing.Quantitative PCR further validated piRNA expression differences between the two strains.Our further screening criteria are difference of 2 times and above(|1og2Ratio| ? 1)and false discovery rate(FDR)FDR ^ 0.001 piRNA molecules.The total differentially expressed piRNA molecular is 470 and high expression piRNA is 449 in DS and low expression piRNA is 21 in DS.Expression data of piRNAs were validated with quantitative PCR and combined with the field lines for further screening.We got the deltamethrin resistance associated candidate piRNA-piRNA-1-TF001556.piRNA-1-TF001556 in DS express 2.23 times higher than that in DR in laboratory strains(p<0.01).At the same time,piRNA-1-TF001556 high express 11.36?10 and 11.90 times in DS than in DR in Gudao,Huiming and Dongping field strain,respectively,which have significant differences(p<0.001).PiRNA-1-TF001556 express with order in each phase.Then we inject mimic or inhibitor sequence into mosquito archicoele to increase or decrease piRNA-1-TF001556 expression.We surprisingly found that DR strain became more sensitive to deltamethrin and the DS strain became more resistant to deltamethrin after injection.Those results suggest that pesticide phenotype as a consequence of piRNA-1-TF001556 expression.It is imply that piRNA-1-TF001556 involved in insecticide resistance.Our study reported the piRNA expression profiling in Cx.pipiens pallens,and found piRNA-1-TF001556 is involved in pesticide resistance,which provides new scientific knowledge for elucidating the molecular mechanism of pesticide resistance.