The as one of the five most damaging and

The
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.The
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).

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.