oriental fruit fly, Bactrocera dorsalis
(Hendel) belonging to the order Diptera is an economically important pest of
several fruits and vegetables throughout the world. The fruit fly is one of the
most destructive pests in several Asian countries, can cause severe economic
loss to more than 117 fruit crops (Alwood et
al 1999) and is considered as one of the 
five most damaging and aggressive fruit flies in the world (Vargas et al 2007). India being a major
tropical and sub-tropical fruit producer faces a considerable yield and quality
losses due to fruit fly. It causes around 5-80%
loss in mango and 10-80 % in guava (Vergese et al 2002). Since it is a polyphagous, multivoltine and highly
mobile fly, its management is relatively difficult (Sharma et al 2011). Various cultural practices (sanitation, tillage), bagging,
postharvest techniques and methyl eugenol baiting techniques are commonly
employed. Often a collective use of the management practices are being used to
control its infestation. Despite to these efforts, there is 100% infestation of
guava fruits in Punjab during rainy season (Singh
and Kaur 2016). There
is a need for dsRNA-mediated gene silencing approach in managing the fruit flies which can further strengthen the
management of fruit fly.     

RNA interference (RNAi) is a post-transcriptional
gene silencing technique that can specifically target a gene of interest by
cleaving the corresponding mRNA in plants. Although, it was first discovered in
Caenorhabditis elegans (Fire et al 1991) it has been widely used in plant
functional genomics. Due to the specificity and ensured effects in closely
related species, RNAi has generated wide interests for pest management (Whyard et al 2009). The silencing is based on
the recognition and cleaving of dsRNA by Dicer (in case of insects, fungi and
animals) and Dicer like elements (in case of plants), a member of endonuclease
family to generate short interfering RNA (siRNA) of 21-26 nucleotides
(Mohanpuria et al 2015). Dicer has a
PAZ (Piwi/Argonaute/Zwille) protein-protein interaction domain, putative RNA
helicase domain, two ribonuclease III (RNaseIII) domains and one or two
dsRNA-binding domains. The function of Dicer
(siRNA) biogenesis was found to be localized in the cytoplasm of insects
however it has also been observed in the nucleus. The siRNAs produced are recognized by Ago protein in the RNA
induced silencing complex (RISC) which is ribonucleoprotein complex (Tomari et al 2004). Using siRNA, RISC guides the cleavage of
complementary mRNA of the corresponding gene (Agrawal et al 2003). Drosophila
melanogastor has been reported to express two isoforms of Dicer where Dicer 2 binds and cleaves dsRNA into siRNA (Lee
et al 2004, Tomari et al 2007).

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RNAi can be highly efficient in strengthening the management techniques against the fruit
fly except its important genes which are required for its survival and growth, are
to be targeted. It is potential to target the genes involved in
sex-determination pathway in insect pests. The sex determination pathway has
been well characterized in Drosophila melanogaster.
The doublesex (dsx) gene which is the
bottom most in the sex determination cascade is the best characterized (Shukla
2010). The topmost in the hierarchy of sexual determination in Drosophila is sxl (sex lethal), followed by tra1
and tra2 (transformer). In insects sex is determined by the ratio of X
chromosomes to autosomes (X:A) by activating the sxl but recently in contrary
to this Erickson and Quientero (2007) has proposed that the gene is activated
by early promoter (Sxl-pe) in
response to X-signaling elements (XSEs) which is in response to the double
dosage of X- linked genes in females. This early pulse is required for the
processing of late sxl (Sxl-pm) transcript
which are present in both the sexes. The processed transcript leads to
functional SXL proteins in females. In males, XSEs cannot activate the Sxl-pe and the early pulse is absent
therefore, default processing occurs to produce nonfunctional SXL protein (Erickson
and Quientero 2007). The SXL protein in females produces a functional TRA
protein which along with constitutive tra2
leads to female specific splicing producing the female specific dsx mRNA and subsequently to produce
female specific dsx proteins, DSXF (Hoshijima et al 1991). In males default splicing of sxl-pm leads to nonfunctional TRA which in turn leads to default
splicing of dsx mRNA producing DSXM (Shukla 2010).

The dsx gene as reviewed above was first
identified in Drosophila. This acts as a double switch gene that serves as a
link between the upstream sexual determination hierarchy and the down-stream genes
that perform the sex-specific functions in male and female insects (Permpoon
and Thanaphum 2010). In Drosophila, the primary transcriptional product of dsx gene contains six exons which are
differentially regulated at transcriptional level by sex specific alternative
splicing. The female specific splicing results in a mature mRNA
containing exons 1,2,3 and 4 while the male specific splicing results in mRNA
containing exons 1,2,3,5 and 6.(Hedley and Maniatis 1991). The male and female
specifically spliced mRNA produces DSXM (Male) and DSXF (Female)
proteins respectively (Baker and Wolfner 1988, Burtis and Baker 1989). These
proteins have a common N terminal region (DM or OD1 2domain) which is involved
in DNA binding and protein oligomerization, and different C terminal region
(OD2) the dimerisation domain (Shukla 2010) which is the result of the sex
specific alternative splicing. The DSXM
is shown to repress the transcription of yp1 and yp2 (yolk
protein) and DSXF enhances it in Drosophila (An et al
1996, Erdman et al 1996). The
female specific DSXF proteins showed 98% identity to Anaestrpha obliqua, B. oleae and B. tyroni showing that it is conserved
(Chen et al 2008). In Drosophila, these
proteins control the sexual differentiation characters like genitalia, sex
combs, courtship behavior (Villela and Hall 1996) and yolk protein (Bownes
1994). The primary sexual determination genes in Bactrocera are not that of the
Drosophilids and are not well known. 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.

 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

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. 


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