The Effects Of Knockdown Of ATPsyn-b On Spermiogenesis And Larval Growth In Drosophila

Wolbachia are kinds of intracellular symbiotic bacterial which exist in a wide range of arthropods and nematodes. Wolbachia can affect the reproductive modes of their hosts which includes cytoplasmic incompatibility(CI), feminization and male-killing. While, CI is the most common phenotype:the cross between Wolbachia-infected males and uninfected females or females infected with different strains of Wolbachia results in low even zero hatch rate. However, when Wolbachia-infected males mate with females infected with the same strain of Wolbachia producing embryos that develop normally. The molecular mechanisms of CI are currently unclear.ATPsyn-b gene encoding for subunit b of ATP synthase in Drosophila melanogaster has been suggested to be involved in the process of oxidative phosphorylation and ATP synthesis, thus providing energy for Drosophila life activities. To study the mechanisms of CI, we have previously found that the expression level of ATPsyn-b gene in the3rd instar larval testes which infected by Wolbachia shows down-regulated significantly. Additionally, we microinjected dsRNA of the gene to males, the embryo hatch rate reduced significantly, which indicated that the knockdown of ATPsyn-b affected the reproduction of Drosophila significantly.To investigate the function of ATPsyn-b in Drosophila development and male fertility, we purchased ATPsyn-b RNAi transgene flies from Fly modle center of Tsinghua University and knocked down ATPsyn-b ubiquitously or in testes by use of Drosophila strains expressing inducible hairpin RNAi constructs under the control of Gal4lines. Firstly, the transgenic RNAi line males were used to cross with Bam-Gal4vp16females. At the same time, the cross of W1118(W-) and Bam-Gal4vp16females was proceed as control. Then one day old Bam-Gal4; ATPsyn-b-hp males were arranged to mate with Dmel T virgin females. We found that no embryos from the crosses with ATPsyn-b knockdown males(Bam-Gal4; ATPsyn-b-hp) hatched out, while89.81%of embryos from the crosses with control (Bam-Gal4; W-) males hatched. Therefore, we concluded that knockdown of ATPsyn-b in testes resulted in complete sterility of male Drosophila. To examine whether ATPsyn-b was knocked down in testes by Bam-Gal4driver, we also assayed the expression level of ATPsyn-b by qRT-PCR. The result indicated that ATPsyn-b was significantly down regulated (0.13fold) in Bam-Gal4; ATPsyn-b-hp fly testes relative to control (Bam-Gal4; W-) testes which resulted in male infertility. To further investigate the cause that ATPsyn-b knockdown in testes induced sterility in male Drosophila, the Bam-Gal4; ATPsyn-b-hp and Bam-Gal4; W’fly testes were dissected and stained with DAPI. We observed that the nucleis failed to keep nuclei clustered during spermatid elongation and were completely scattered in ATPsyn-b knocked down testes. What’s more, each scattered nucleus appeared to be shorter and smaller. Additionally, while we observed the control seminal vesicle was full of mature sperm, there were no any mature sperm in the seminal vesicle of ATPsyn-b knocked down testis. Consequently, we concluded that abnormal spermatogenesis and lack of mature sperm could be the cause of male infertility resulted from ATPsyn-b knockdown in testis.Next, the transgenic RNAi line males crossed with ActGal4/Cyo-PscGFP females to knock down ATPsyn-b ubiquitously. We observed that all emerged offspring flies had curly wings.This indicated that the flies ATPsyn-b knocked down ubiquitously(Act-Gal4; ATPsyn-b-hp) couldn’t develop to adults while their sibling controls(Cyo-PscGFP; ATP syn-b) could. To investigate at which stage these flies died, the first instar larvae were selected under the fluorescence stereomicroscope and incubated separately. The larvae without GFP fluorescence (Act-Gal4; ATPsyn-b-hp) should be gene knockdown. At the same time, the cross of wild type (W1118, W-) males and ActGal4/Cyo-PscGFP females is proceed as control. We found that when control larvae developed to the3rd instar stage and even began to pupate, emergence, the mutant larvae still had the body size like the2nd instar larvae. They could live for more than15days and never became pupae. Finally, they gradually died. To test whether the larval growth defect is associated with ATPsyn-b, we analyzed the expression level of ATPsyn-b by qRT-PCR. QRT-PCR showed that ATPsyn-b expression level in Act-Gal4;ATPsyn-b-hp larvae was significantly lower (0.05fold) than that in either Act-Gal4; W’controls or their sibling controls(Cyo-PscGFP; ATPsyn-b larvae).These results suggest that ubiquitous knockdown of ATPsyn-b may result in arrest of larval growth in Drosophila. To further investigate the cause that ATPsyn-b knockdown ubiquitously induced development defect in Drosophila, we proceeded Brdu incorporation through feeding the larvae of mutants and controls. From the result of Brdu incorporation test, we found that the foregut cell nucleus and cells of mutants were significantly smaller than that of controls. Since cell nucleus of gut were growing with DNA duplicating, the size of cell nucleus could’t become bigger when DNA duplication exist defects. In that way, the size of foregut cell also could’t become bigger. So we concluded that the size of cell restricted the body size of larvae and it may be result from the DNA duplication defects.Our results suggested that the knockdown of ATPsyn-b in testes could produce living adult flies, but the male adults had severe defects in spermatogenesis, thus failed to produce progenies. These results suggested that ATPsyn-b is essential to male fecundity in Drosophila. Additionally, larvae with ATPsyn-b extensively knocked down had developmental defection because the size of cell restricted the body size of larvae and it maybe resulting from the DNA duplication defects. The results described above indicated that ATPsyn-b gene may play an important role in the process of Drosophila spermatogenesis and larvae development. Since ATPsyn-b had been identified to be Drosophila melanogaster sperm proteome (DmSP), what’s more, it’s also one of the sperm proteins in mouse and human. Therefore, investigation of the molecular function ATPsyn-b has in Drosophila spermatogenesis may become an useful model and provide a new clue for further insighting into the role ATPsyn-b plays in human male reproduction.