In a number ofDipterans such as Ceratis capitata, B. tyroni, B. oleae, Musca domestica,the sex is determined by a gene called Dominant Male Determiner (M) which is Ylinked determines the male sex (Nothinger and Steinman-Zwichy 1985, Bedo andFoster 1985, Lifschitz and Cladera 1989, Shearmann and Frommer 1988 and Morrow et al 2014). The sxl ortholog in Bactroceraoleae is found to have no link with sexual determination (Lagos et al 2005). The analysis of the dsx homolog in B. dorsalis revealed four 13 nucleotide conserved elements that actas tra binding sites (Hedley andManiatis 1991).
The molecular organization i.e. the production of sexspecific transcripts of the dsx genewas found to be similar to that of the Drosophila in Dipterans like B. tyroni (Shearmann and Frommer 1988), B.
oleae (Lagos et al 2005), Musca scalaris(Kuhn et al 2000) and M.domestica (Hediger et al 2004).This is in accordance with the concept of Wilkins1995 that, the sexual determination has evolved from bottom to top. Theintroduction of dsRNA specific to female dsxfgene through abdominal injection in B. dorsalis showed ovary underdevelopment, reduction in mature eggsand egg laying rate reduction up to 10 times (Chen et al 2008).
There are number of factorswhich affect the efficiency of RNAi such as length of dsRNA, life stages ofinsect, variation among different tissues and organisms and dsRNA deliverymethods etc. The response to RNAi may be cell autonomous i.e. dsRNA isexpressed within the cells or intracellular i.e. dsRNA is directly picked up fromthe immediate environment.
The other type is the non-cell autonomous which canbe environmental RNAi (eRNAi), which occurs when the gene silencing is due tothe signal from extracellular environment or systemic RNAi when theextracellular signal spreads from one cell to cell (Darrington et al 2017). Systemic RNAi is reportedin plants and C. elegans where RNA-DependentRNA polymerases (RdRP) are present which amplify the siRNA signals in terms ofsecondary siRNA and transfer these signal to the entire organism (Mohanpuria et al 2015, Mamta and Rajam 2017). AlthoughRdRP is absent in insects but high gene silencing efficiency through RNAi hasbeen reported in Triboliun castaneum (Tomoyasuet al 2008) which shows that someother mechanism is involved in insects that are responsible for systemiceffects of dsRNA. There are two types of dsRNA uptake mechanisms trans-membranechannel mediated and endocytosis mediated uptake mechanism (Xue etal 2012, Mamta and Rajam 2017). In C. elegans the uptake happens through an intestinal transmembrane proteinSID (Systemic Interference Defective), the SID1 is proposed to have function intransmitting the signal in systemic RNAi by passive transport ofdsRNA and SID2allows the uptake of dsRNA from the gut lumen.
SID1 is also shown to play rolein the secondary step of taking it into cytoplasm (Cappelle et al 2016, Mamta and Rajam 2017). In silico analysis showed that sid1 homologsare absent in Tribolium castaneum buthigh levels of systemic RNAi was observed (Tomoyasu et al 2008). This indicates that another mechanism is involved in dsRNA uptakein insects. The sid1 homologs werealso absent in Drosophila and study of Drosophila S2 cells showed thatreceptor-mediated endocytosis plays role in uptake of dsRNA (Saleh et al 2006, Joga et al 2016).
B. dorsalis showed refractorinessto RNAi when they were fed with dsRNA of endogenous genes and the blockage ofRNAi mechanism required the Clathrin mediated endocytic pathway. It was alsoshown that increasing the endocytic capacity disrupted the RNAi refrectoriness (Liet al 2015). These evidences suggestthe presence of an alternate method (receptor mediated endocytosis) is involvedin dsRNA uptake in case of insects. Efficiency of RNAi also depends upon the delivery of intact dsRNAinto insect body and thereafter up to its cleavage into siRNAs. The maggots canbe fed with transgenic bacteria expressing dsRNA or the purified dsRNA can be incorporatedin the feed.
Delivery by oral means was first reported inC.elegans and was successful in eliciting the RNAi response (Timmons and Fire 1998). Other non-feedingmethods include topical application, spraying or soaking of the larvae (from review article Mamta andRajam 2017sir, each method is being said in the next lines). Uptake of dsRNA bysoaking method was reported in C. elegans(Tabara et al 1998) and in flatworms(Orii et al 2003). Spraying of dsRNAagainst Colorado potato beetle in leaves and plants showed no infestation for upto 28 days (Miguel and Scott 2016) which indicates the stability of dsRNA. Microinjectionof dsRNA directly into the target tissue proves to be efficient and was firstreported in C. elegans (Fire et al 1998).
But this method has the disadvantages that itis expensive, can damage the insect and requires skilled personnel. Hence,feeding is comparatively simple, natural and feasible methods of dsRNA delivery for largescale applications. The purified dsRNA, or recombinant bacteria expressingdsRNA or dsRNA complexed with liposomes or nanoparticles can be fed to theinsect pests (Whyard et al 2009, Zhanget al 2010, He et al 2013 and Taning et al2016).Few RNAi-mediated genesilencing work has been reported in Bactroceradorsalis. The adult fruit flies were fed with dsRNA of spr (sex peptide receptor) incorporated in diet showed 52 % geneknockdown and reduction in mean life span by 26 days (Zheng et al 2015). Similarly dsRNA of Bdor (odorant receptor) and Orco (odorant co-receptor) produced 70%knockdown on its simultaneous application (Yi et al 2014). The abdominal injection of dsRNA of female specific Bddsxf showed inhibition of yolkprotein gene (Bdyp1) expression with27% female flies reported to have deformed ovipositors, and also affected ovary development (Chen et al 2008). Transformed lines of B.
dorsalis expressing the Bddsxf dsRNA showed similar resultslike delayed egg maturation and change in mating behavior as that of in vitro delivery methods (Chen et al 2011). The yolk protein gene (Bdyp1) was affected in response to theadult abdominal injection of dsRNA of transformer gene (Bdtra) showing the positive effect of TRA in sexual differentiation(Peng et al 2015). Li et al (2011) used four genes rpl19, a ribosomal protein, Noa,fatty acid elongase, V type ATPase D subunit and Rab11,a GTPase for RNAi-mediated gene silencing in B. dorsalis. DsRNA of noa affectedthe egg production while dsRNA of rab11 caused20% mortality. It has been shown that sperm less males for sterile insecttechnique can be produced by targeting the genes involved in celldifferentiation and azoospermia formation.
In context to this recently, geneslike boul, zpg, dsxm, fzo andgas8 (testis specific) weretargeted and the oral feeding of corresponding dsRNA showed significantreduction in their expression and caused reduced reproductive ability of males (Ali et al 2017). The dsRNA can beproduced by both in vitro using in vitro transcription kits and in vivo by using T7 RNA polymerase underthe lac promoter and HT115 E. coli strain (which is deficient inRNaseIII). The target genes are generally cloned into L4440 plasmid vectorswhich has two T7 RNA promoters on either side, producing complementary strandsthat bind to form the dsRNA in host bacteria.
RNAitechnology and its efficiency has been greatly explored and its application asa pest control strategy has made an outstanding growth (Joga et al 2016, Mamta and Rajam 2017). Ithas been proven that gene silencing through RNAi affected the growth anddevelopment and survival of insect pests (Xu et al 2016). This method is sequence specific and has potential.But it is still in infancy and many questions are to be answered like themechanism of systemic response of dsRNA inside the insect’s body. Various methods are alreadyunder development like generating sterile males using RNAi for sterile insecttechnique (SIT), spray able RNAi-based products for field level application anddsRNA expressing transgenic plants etc. However, the research is still ongoingand has some limitations like requirement of large scale cheap production ofdsRNA, development of novel and efficient delivery methods for pests ofdifferent Order and identifying potential target genes.
Through this review wehave come to know that double sex gene will be a very potential target against B. dorsalis and detailed studies of itsdsRNA-mediated silencing effect will prove to be essential to provide the solutionsto the existing problem of fruit fly in our country. This will play a pivotalrole in generating the management strategies against this notorious pest.