The Role Of MicroRNAs In Spermatogenesis Of Oriental Fruit Fly Bactrocera Dorsalis

The oriental fruit fly Bactrocera dorsalis is one of the most destructive horticultural pests in the East Asia and Pacific region. 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. On the other hand, microRNAs(miRNAs) are the small non-coding RNAs that repress the target gene expression at post-transcriptional level, and are crucial regulators of various diverse biological processes, including insect spermatogenesis. Therefore, they are considered to be valuable for conducting mi RNA-based genetics-enhanced SIT in B. dorsalis. However, the miRNAs for spermatogenesis are still largely unknown in B. dorsalis.In the first part of this study, the miRNAs from B. dorsalis testes were firstly identified by deep sequencing, which provided an overview of miRNAs expression during spermatogenesis. Small RNA libraries were constructed from the testes of fully mature(FM), immature(IM), and middle aged(MA) adult flies of B. dorsalis. Small RNA sequencing and data analysis revealed 172 known and 78 novel mi RNAs among these libraries. Subsequently, the small RNA libraries of IM, MA and FM testes were pairwise compared, which identified 24, 15 and14 differentially expressed miRNAs in FM versus IM, FM versus MA and IM versus MA stages, respectively. Using a bioinformatics approach, 124 target genes against 13 most differentially expressed mi RNAs were predicted. In addition, the expression patterns of 6 randomly selected miRNAs(from 13 most differentially expressed miRNAs) and their putative target genes(from 124 predicted target genes) in the testis of B. dorsalis were analyzed by qRT-PCR, which showed that 4 tested miRNAs-mRNAs presented an inverse expression pattern and were likely co-regulated.inthesecondpartofthestudy,theregulatoryroleofspecificmirnainmalereproductivesystemofb.dorsaliswasdetermined.bioinformaticanalysispredictedthatfivemirnas(mir-279-3p,mir-8-3-p,mir-275-3p,mir-34-3pandmir-304-5p)bindtothe3′utrofputativeb.dorsalismitoferrin(bmfrn),theorthologindrosophilaisessentialformalefertility.theresultsshowedthatbmfrnhasallconservedaminoacidresiduesofmitoferrinsandismostabundantlyexpressedinb.dorsalistestes,makingmitoferrinhighlyinterestingcandidateforgenetics-enhancedsit.adual-luciferasereporterassaysysteminhelacellsshowedthatmir-8-3pinteractswiththe3′utrofbmfrn.dietarytreatmentsofadultmaleflieswithmir-8-3pmimics,antagomirorbmfrndsrnaalteredmitoferrinexpressioninthetestesandresultedinreducedmalereproductivecapacityduetoreducednumbersandviabilityofspermatozoa.takentogether,thisstudyprovidedthefirstcomparativeprofileofthemirnatranscriptomeinthreedevelopmentalstagesofthetestis.furthermore,itrevealedthatanappropriateexpressionofmitoferrininthetestesisindispensibleformalefertilityofb.dorsaliswhichisregulatedbymir-8-3p,andbothmitoferrinandmir-8-3parepromisingnovelagentsthatcouldbeusedforgenetics-enhancedsit.