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Subject: Entomology × Author: Brar, Gagandeep Singh × End date: 2019 × Start date: 2017 ×
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    ThesisItemOpen Access
    Structural and functional characterization of double sex (dsx) gene in Bemisia tabaci (Gennadius)
    (Punjab Agricultural University, Ludhiana, 2019) Brar, Gagandeep Singh; Satnam Singh
    Double sex (dsx) homologues in Bemisia tabaci were identified and the identification of these dsx homologues in B. tabaci assisted in characterizing several sex-specific transcripts in both male and female separately. These transcripts show that Btdsx consists of six exons and five introns, which are sex-specifically spliced to produce male and female BtdsxM and BtdsxF transcripts. The pre-mRNA of Btdsx produces four male (BtdsxM1, BtdsxM2, BtdsxM3 and BtdsxM4) and one female-specific (BtdsxF) isoforms. All the four male-specific and one female specific BtDsx proteins share common OD1 domains and differ in their OD2 domains at C-terminal sequences. These differences in the exon/intron organization propose that the splicing of Btdsx results from a different functional mechanisms of double sex gene. Btdsx shared a close phylogenetic relationship with dsx of insects belonging to order Hemiptera and Diptera. Further, RT-qPCR analysis revealed that the expression of Btdsx was higher in female adults than male adults as well as other developmental stages including egg, crawlers, second instar nymphs, third instar nymphs and male and female red-eyed pupa. The functional characterization of Btdsx was performed through RNAi using membrane feeding of dsRNA (dsdsx) against dsx in comparison to dsRNA against green fluorescent protein gene (dsGFP). The knockdown effect of Btdsx on other sex related (Vitellogenin (vg) and Vitellogenin receptor (vgr)) and autosomal genes (cytochrome p450 (CYP450) and alkaline phosphatase (ALP)) was also performed using RT-qPCR. Four different combinations (dsdsx fed ♂+ dsdsx fed♀; dsdsx fed ♂ + dsGFP fed ♀; dsGFP fed ♂ + dsdsx fed ♀; dsGFP fed ♂ + dsGFP fed ♀ (control)) of male and female whitefly were used to feed 400 ng dsRNA of Btdsx and GFP (control) per μl of artificial diet for 48 hours. Effect on fecundity, hatching percentage and male: female ratio was observed. Knockdown of Btdsx resulted in significant reduction in expression of dsx in both male and female as compare to control. The expression of vg and vgr showed upregulation in dsdsx fed males. However, it was significantly reduced in dsdsx females as compare to control. The knockdown of Btdsx caused significant reduction in the expression CYP450 and ALP as compared to GFP control. The least fecundity (8.27 eggs/pair) was recorded where dsx was knockdown in both male and female whitefly compared to all other combinations. It was also observed that the knockdown of dsx only in females (12.3 eggs/pair had more impact on fecundity compared to alone males (15.06 eggs /pair). Per cent egg hatching was also least where dsx was knockdown in both the sexes (68.52) or either in females (61.60) or males (68.83) compared to GFP control (85.46). The knockdown of dsx did not had any effect on female: male ratio in any of the combinations.