The Effect Of Knockdown Of Ocnus On Reproduction In Drosophila

Wolbachia is a common gram-negative bacterium in arthropods, which is passed to the offspring through maternal cytoplasm, it can regulate the reproduction ways of the host through various machanisms, such as, cytoplasmic incompatibility (CI), parthenogenesis, feminization and male-killing. CI is the common cell phenotype:when infected females or with different strain of Wolbachia mated with infected females, having low hatching rate. Some studies indicated that male pronucleus development was lower than that of female pronucleus at the first of fertilized eggs when infected males mated with normal females. Although many research have shown that Wolbachia might modify sperm, the molecular mechanism of cytoplasmic incompatibility induced by Wolbachia has not been studied clearly.Our previous study shown that Wolbachia could induce many kinds of genes varied in Drosophila 3st larvae testis infected wMel, specially related to reproduction. Among these genes, we found that ocnus(ocn) expression level was down-regulative significantly in testis from both 3st larvae. We detected the expression level of ocn in 1 day old testes infected by Wolbachia, finding that the expression level was significantly reduced (p<0.01). Our data shown that ocn was related to male reproduction in Drosophila.Another study demonstrated that ocn was widespread in Drosophila testes, suggesting that ocn might be important to male reproduction. To study the function of ocn further, we used NosGal4 and BamGal4 to knock down ocn in gonad and testis, respectively, through UAS-RNAi/Gal4 system. We found that the expression level of ocn in both experimental male was remarkably declined (p<0.01), accounting for excellent knock down effect.To study the effect of ocn knock down on male flies reproduction, we used 1 day old males(NosGal4;ocn-hp and BamGal4;ocn-hp) mated with 3-4 day old normal virgin flies to statistics the hatching rate of offspring embryo. Our data shown that the both hatching rate were significantly declined (p<0.01), illustrating that ocn knock down damaged male flied reproduction seriously. So, we inferred that ocn participated in the process of spermatogenesis.Next, we dissected NosGal4; ocn-hp, BamGal4; ocn-hp and W1118 lday old testes and stained with DAPI. We found that testis from ocn knock down male was extremely abnormal. The abnormal testis was smaller, the top of the testis became narrow, and GSCs were loose. What is more, there were fewer cysts, sperm bundles and mature sperm in abnormal testis. So, the knockdown of ocn had a strong impact on the development of testis and the process of spermatogenesis in Drosophila. We guessed that it might be one reason of the lowered hatching rate.Because of the extremely high expression level of ocn in male flies, we detected the male mating activity to study the effect of ocn knock down on male courtship and mating behavior. Our data stated that the mating rate of both ocn knock down male flies and normal female flies (Dmel wMel T) remarkably reduced, respectively, in 30min; and the mating duration of abnormal males was also significantly declined. These data shown that ocn could also influence mating activity of male flies.We know that there are many factors have an influence on mating activity, such as, olfactory and movement ability, and so on. So, we first detected the ability responding to food trap, finding that the cumulative capture rate was remarkably declined in the same time (p<0.01). On the one hand, the movement ability of ocn knock down males can influence the ability responding to food trap; on the other hand, the response capability was may also affected by abnormal olfactory ability. Then, we tested the movement ability and olfactory respond capability of both ocn knock down males,10 day old, through unbaited trap and female trap, respectively. We found that the movement ability and olfactory capability to female were remarkably reduced. In addition, we also detected the expression level of olfactory-related genes(PBP1、PBP2、PBP3、orco) and motility-related genes(mlc2、fln、myo61F、how、mef2、actn、Imd). Our data indicated that only the expression level of mlc2 had highly significant differences (p<0.05), becoming 0.35 times as the control. Research has shown that mlc2 mutation could have a strong impact on flight mechanism and "love song". So, we speculated that ocn knock down had negative effects movement ability, and then reduced mating activity.We also found that knockdown of ocn influenced reproduction of ocn knock down female flies. The egg laying amount was notably reduced when BamGal4; ocn-hp 3-4 day old females crossed with Dmel wMel T 1 day old males.From the above, ocn has an important role in spermatogenesis, and it also has a notably impact on movement ability.