Genetically Enhanced Sterile Insect Technique(Sit) Based On Rnai Against Bactrocera Dorsalis (Hendel)

The oriental fruit fly,Bactrocera dorsalis(Hendel),one of the most destructive pests throughout South East Asia,causes severe economic loss to over 250 fruit crops.Sterile insect technique(SIT)is a species-specific and environmental friendly method to control B.dorsalis,but irradiation or chemosterilants used to sterilize male insects decrease the mating ability of males,which ultimately affects pest control efficiency.Recently,genetics-enhanced SIT has shown large potential for the management of a few horticultural pests,such as olive fruit fly.RNA interference(RNAi)is a conserved regulatory mechanism that is triggered by ds RNA;it functions in a remarkable variety of organisms.As RNAi can be achieved easily in many eukaryote species,it has great potential in many areas of scientific research such as functional genetic,medical and agricultural studies.Even though a number of progress of RNAi has been achieved in the studies of agricultural pests,there still remains many problems need to be solved till this technology could be applied in production.In this study we screened out the most potent spermatogenesis-related genes and check the effect of target ds RNA effect of male sterility.Afterwards we enhance the stability of ds RNA by incorporating with nanoparticles with different method,different mass ratio as well with different p H.We also explored the gene function of key gene on male sterility with nano-particles.After this we checked the mechanism of selected gene ds RNA with finalize p H(6.5)as well as most potent mass ratio(1:4)on sf9 cells.1.The target genes for producing Bactrocera dorsalis sterile males based on RNAiIn the first part of this study we developed dead sperm males by interference of germ cell differentiation and azoospermia related genes.Our data demonstrate the significant reduction of expression level of target genes(Boul,ZPG,dsxM,FZO and Gas8)in gene silencing in response to orally administration of the ds RNAs.Knocking down of target genes(Boul,ZPG and dsxM)harshly affects reproduction ability of males and reduces the eggs hatching rate by 60-75% as compared to control group(ds Egfp).More over different combinations of selective genes ds RNA(Boul+ZPG,Boul+dsxM and ZPG+dsxM)were made and assessed on male?s fertility,resulted that up to 86% males are sterilized.The best combination was selected and male fetility assay was done in response of different concentration(250,500,750 and 1000 ng/ul)and found 20.1%,39.63%,59.24% and 86.28% male sterility respectively.Right after by using 1000ng/ul of same combination of ds RNAs was used at different days old flies like(1st,5th,7th and 10th)days old reproductive assay was conducted and found 86.28%,34.14%,22.60%,and 10.83% male sterility respectively.The feeding of best combination of ds RNA all significantly reduces the number of spermatozoa and number of live sperm in treated flies.Our study helps to develop SIT in B.dorsalis through genetically control method(RNAi).This study also provides sufficient data of novel genes to minimize the population through SIT.2.Delivery techniques of ds Boul /chitosan nano-particles in Bactrocera dorsalisIn second part of experiments we used the best screened gene(Boul)ds RNA with two different methods of preparation: 1)Embedding method 2)Absorbing method.We explored the ds RNA stability with both methods with in three different p H medium of 6.5,7.5 and 8.5.Series of q RT-PCR results revealed that change in p H also affect the RNAi efficiency.In this regard we checked the gene expression until 25 th day of treatment and p H 6.5 with embedding method found to be best and showed expression at 5th day of treatment 0.22 fold as compared to control,among 3 different p H mediums and different methods.Compared to the embedding method,the absorbing method showed non-significant results after 15 th,20th and 25 th of treatment.Right after we used the p H 6.5 with different mass ratio of ds RNA 1:1,1:2,1:3 and 1:4(Chitosan:ds RNA)with both embedding and absorbing method.After feeding trail,1:4 mass ratio of embedding method showed highly significant difference(0.60 fold)as compared to control after 25 th day of.In case of absorbing method showed to be non-significant different in gene expression as compared to control.We explored the ds RNA effect under different temperature(20 °C,28 °C and 38°C)and check the ds RNA stabilty with nanoparticles.We also explored the effect of different preparation method on chitosan nano-particles size.In this regard embedding method showed almost 80 nm,which is better than that in absorbing method(more than one hundred,about 110 nm).So,at p H=6.5,1:4 mass ratio with embedding method,we conducted a series of bioassays and check the gene function on the behalf of SIT.We found more than 77% reduction in eggs hatching while the difference in eggs laying was non-significant as compared to control group(Chitosan/ds Egfp)and solely ds RNAs.After that we directly quantified the total number of spermatozoa of Chitosan/ds RNA treated male of oriental fruit flies after 14 days old.Comparison with the control males(Chitosan/ds Egfp treated flies)revealed the significant reduction in average number of sperm in seminal vesicles as of treated(Chitosan/ds Boul)males flies.We also have done sperm viability assay and found the significant difference between live and dead sperm in treated and control flies.The percentage of live sperm in treated flies was only about 21% as compare to control flies.Nanoparticles enchance the ds RNA efficiency and stability as compared to the sloly ds RNA.3.Effects of Chitosan/ds Boul treatment on sf9 cell lineIn the third series of experiment,we used the finalized best mass ratio of Chitosan/ds Boul which is prepared with embedding method and check the efficacy on sf9 cell line.We observed apoptosis induction because of Chitosan/ds Boul treatment.We noticed cell cycle arrest in G2 phase induced by Chitosan/ds Boul by Flow cytometric analysis.Moreover,we demonstrated that Chitosan/ds Boul induced deformities in sf9 cells by use of various assays including live-dead assay and transmission electron microscopy(TEM).Overall nano-particle with ds RNA showed a great impact on the sf9 cell line and arrest the cell in G2 phase.