In that of the Drosophila in Dipterans like

In a number of
Dipterans such as Ceratis capitata, B. tyroni, B. oleae, Musca domestica,
the sex is determined by a gene called Dominant Male Determiner (M) which is Y
linked determines the male sex (Nothinger and Steinman-Zwichy 1985, Bedo and
Foster 1985, Lifschitz and Cladera 1989, Shearmann and Frommer 1988 and Morrow et al 2014). The sxl ortholog in Bactrocera
oleae 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 act
as tra binding sites (Hedley and
Maniatis 1991). The molecular organization i.e. the production of sex
specific transcripts of the dsx gene
was 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 Wilkins
1995 that, the sexual determination has evolved from bottom to top. The
introduction of dsRNA specific to female dsxf
gene through abdominal injection in B. dorsalis showed ovary underdevelopment, reduction in mature eggs
and egg laying rate reduction up to 10 times (Chen et al 2008).

There are number of factors
which affect the efficiency of RNAi such as length of dsRNA, life stages of
insect, variation among different tissues and organisms and dsRNA delivery
methods etc. The response to RNAi may be cell autonomous i.e. dsRNA is
expressed within the cells or intracellular i.e. dsRNA is directly picked up from
the immediate environment. The other type is the non-cell autonomous which can
be environmental RNAi (eRNAi), which occurs when the gene silencing is due to
the signal from extracellular environment or systemic RNAi when the
extracellular signal spreads from one cell to cell (Darrington et al 2017). Systemic RNAi is reported
in plants and C. elegans where RNA-Dependent
RNA polymerases (RdRP) are present which amplify the siRNA signals in terms of
secondary siRNA and transfer these signal to the entire organism (Mohanpuria et al 2015, Mamta and Rajam 2017). Although
RdRP is absent in insects but high gene silencing efficiency through RNAi has
been reported in Triboliun castaneum (Tomoyasu
et al 2008) which shows that some
other mechanism is involved in insects that are responsible for systemic
effects of dsRNA. There are two types of dsRNA uptake mechanisms trans-membrane
channel mediated and endocytosis mediated uptake mechanism (Xue et
al 2012, Mamta and Rajam 2017). In C. elegans the uptake happens through an intestinal transmembrane protein
SID (Systemic Interference Defective), the SID1 is proposed to have function in
transmitting the signal in systemic RNAi by passive transport of
dsRNA and SID2
allows the uptake of dsRNA from the gut lumen. SID1 is also shown to play role
in the secondary step of taking it into cytoplasm (Cappelle et al 2016, Mamta and Rajam 2017). In silico analysis showed that sid1 homologs
are absent in Tribolium castaneum but
high levels of systemic RNAi was observed (Tomoyasu et al 2008). This indicates that another mechanism is involved in dsRNA uptake
in insects. The sid1 homologs were
also absent in Drosophila and study of Drosophila S2 cells showed that
receptor-mediated endocytosis plays role in uptake of dsRNA (Saleh et al 2006, Joga et al 2016). B. dorsalis showed refractoriness
to RNAi when they were fed with dsRNA of endogenous genes and the blockage of
RNAi mechanism required the Clathrin mediated endocytic pathway. It was also
shown that increasing the endocytic capacity disrupted the RNAi refrectoriness (Li
et al 2015). These evidences suggest
the presence of an alternate method (receptor mediated endocytosis) is involved
in dsRNA uptake in case of insects.

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 Efficiency of RNAi also depends upon the delivery of intact dsRNA
into insect body and thereafter up to its cleavage into siRNAs. The maggots can
be fed with transgenic bacteria expressing dsRNA or the purified dsRNA can be incorporated
in the feed.
Delivery by oral means was first reported in
C.elegans and was successful in eliciting the RNAi response (Timmons and Fire 1998). Other non-feeding
methods include topical application, spraying or soaking of the larvae (from review article Mamta and
Rajam 2017sir, each method is being said in the next lines). Uptake of dsRNA by
soaking method was reported in C. elegans
(Tabara et al 1998) and in flatworms
(Orii et al 2003). Spraying of dsRNA
against Colorado potato beetle in leaves and plants showed no infestation for up
to 28 days (Miguel and Scott 2016) which indicates the stability of dsRNA. Microinjection
of dsRNA directly into the target tissue proves to be efficient and was first
reported in C. elegans (Fire et al 1998).  But this method has the disadvantages that it
is expensive, can damage the insect and requires skilled personnel. Hence,
feeding is comparatively simple, natural and feasible methods of dsRNA delivery for large
scale applications. The purified dsRNA, or recombinant bacteria expressing
dsRNA or dsRNA complexed with liposomes or nanoparticles can be fed to the
insect pests (Whyard et al 2009, Zhang
et al 2010, He et al 2013 and Taning et al
2016).

Few RNAi-mediated gene
silencing work has been reported in Bactrocera
dorsalis. The adult fruit flies were fed with dsRNA of spr (sex peptide receptor) incorporated in diet showed 52 % gene
knockdown 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 yolk
protein gene (Bdyp1) expression with
27% 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 results
like 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 the
adult 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 affected
the egg production while dsRNA of rab11 caused
20% mortality. It has been shown that sperm less males for sterile insect
technique can be produced by targeting the genes involved in cell
differentiation and azoospermia formation. In context to this recently, genes
like boul, zpg, dsxm, fzo and
gas8 (testis specific) were
targeted and the oral feeding of corresponding dsRNA showed significant
reduction in their expression and caused reduced reproductive ability of males (Ali et al 2017).

The dsRNA can be
produced by both in vitro using in vitro transcription kits and in vivo by using T7 RNA polymerase under
the lac promoter and HT115 E. coli strain (which is deficient in
RNaseIII). The target genes are generally cloned into L4440 plasmid vectors
which has two T7 RNA promoters on either side, producing complementary strands
that bind to form the dsRNA in host bacteria.

RNAi
technology and its efficiency has been greatly explored and its application as
a pest control strategy has made an outstanding growth (Joga et al 2016, Mamta and Rajam 2017). It
has been proven that gene silencing through RNAi affected the growth and
development 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 the
mechanism of systemic response of dsRNA inside the insect’s body. Various methods are already
under development like generating sterile males using RNAi for sterile insect
technique (SIT), spray able RNAi-based products for field level application and
dsRNA expressing transgenic plants etc. However, the research is still ongoing
and has some limitations like requirement of large scale cheap production of
dsRNA, development of novel and efficient delivery methods for pests of
different Order and identifying potential target genes. Through this review we
have come to know that double sex gene will be a very potential target against B. dorsalis and detailed studies of its
dsRNA-mediated silencing effect will prove to be essential to provide the solutions
to the existing problem of fruit fly in our country. This will play a pivotal
role in generating the management strategies against this notorious pest.