The Role Of Wolbachia In The Oogenesis Of Small Brown Planthopper,Laodelphax Striatellus(Hemiptera:Delphacidae)

Apoptosis,a common form of programmed cell death,is needed in several physiological processes and also a classic feature of female germline development.Apoptosis causes highly conserved changes in cellular morphology and structure.Morphological changes are accompanied with apoptosis such as chromatin and cytoplasm condensation,chromosomal DNA fragmentation,breaking up of nuelei,and disintegration of the actin cytoskeleton.The main effectors in the apoptosis pathway are cysteine proteases called caspases.Caspases are divided into initiation caspases and effector caspases.Initiator and effector caspases are the core components in the apoptosis pathway.One function of apoptosis in insects involves the process of oogenesis.At the late stages of oogenesis,nutrients and cytoplasm are provided by apoptosis of the nurse cells,which allows rapid oocyte growth.Apoptosis of the nurse cell cluster is essential for oocyte maturation.Wolbachia are intracellular,maternally inherited bacteria in numerous arthropod species that have a wide range of effects on host physiology and reproduction.Because Wolbachia are dependent on their host for survival,they have developed intimate associations with their hosts,ranging from parasitism to mutualism.Where fitness benefits of Wolbachia on hosts have been demonstrated,they tend to involve an increase in egg production,which may reflect a link between the presence of Wolbachia and ovarian cells apoptosis.The small brown planthopper,Laodelphax striatellus,is 100%infected with Wolbachia strain wStri in China.Wolbachia are abundant in L.striatellus ovarian cells where they are vertically transmitted.While the influence of Wolbachia on host oogenesis and the processes associated are currently unknown.We are interested in investigating the responses of the ovaries to Wolbachia infection because this organ provides the essential link for understanding Wolbachia transmission.In this study,the Wolbachia-induced L.striatellus ovarian cells changes were studied through situ hybridization(FISH),RT-qPCR,TUNEL,and RACE approach.The possible role of Wolbachia-induced apoptosis in L.striatellus oogenesis was also investigated.At the same time,we used immunohistochemistry to reveal characteristic localization patterns of Wolbachia in the ovariole.The main results were as follow:1.Effects of Wolbachia on L.striatellus fitnessEgg production in symbiotic females(137±7(mean±SE)eggs per female)was significantly greater(P<0.001,two-tailed t test)than in aposymbiotic females.Additionally,the number of oppspring that reached maturity was greater for symbiotic females(70±3 per female)than for aposymbiotic females(58±3 per female).However,Wolbachia had no significant effect on either the proportion of offspring females or the proportion of eggs that hatched.Wolbachia infection also did not significantly affect development time or adult female longevity.2.Effect of Wolbachia on apoptosis in ovariesTUNEL staining was used to detective signs of apoptosis in both aposymbiotic and symbiotic ovarioles.The percentage of TUNEL-positive cells in four-day-old symbiotic females(11.8%)was somewhat higher than in aposymbiotic females(7.5%).These percentages increased proportionately in 10-day-old adults.At this age,apoptotic cells in the tropharia were present in both aposymbiotic and symbiotic L.striatellus ovarioles,but the frequency of apoptosis was significantly higher in symbiotic females.In both 4-and 10-day-old females,caspase-3-like protease activity and the total amount of inactive and cleaved caspase-3-like protein were significantly higher in symbiotic females than in aposymbiotic females.3.Effect of caspase-3 tetrapeptide inhibitor on L.striatellusEgg production in symbiotic females fed with L.striatellus artificial diets was also significantly greater than that in aposymbiotic females.Egg production decreased with increasing concentration of z-DEVD-fink fed to symbiotic females.The egg number of L.striatellus fed with z-DEVD-fink in artificial diets(1.50 μg/μl)was only 11±3 per female.The levels of caspase-3-like mRNA and Vg mRNA were lower in symbiotic females fed with different concentrations of z-DEVD-fink than in the control.Similar results were obtained for caspase-3-like protease activity.4.Caspase genes in Wolbachia-indnced apoptosisFour caspase homolog genes were identified.Two genes(Lscaspase-1a and Lscaspase-1c)were highly homologous to each other and shared significant sequence similarities with insect caspase-1s.Two long prodomain-carrying caspase homolog genes were also identified in L.striatellus.One(Lscaspase-Nc)has significant sequence identities with insect caspase-Nc genes,the other homolog(Lscaspase-8)shares similar characteristics with caspase-8 genes of several insect species.Four caspase genes and Vg expression levels increased in Wolbachia-symbiotie L.striatellus females emerged different time.The results of dscas-RNAi indieated that apoptosis had a role in oogenesis of L.striatellus.5.Wolbachia transferred into oocyte from trophariumImmunohistochemistry using a Wolbacehia Hsp-60 antibody revealed characteristic localization patterns of Wolbachia in the ovariole,which were suggestive of the route and mechanism of its vertical transmission.Wolbachia bind to Vg outside the ovarioles and then enter into ovarioles through terminal filament and are centrally located in the stem cells at the anterior pole of tropharium.As stem cells divide into oocytes and nurse cells,Wolbachia disperse throughout all of the cells in the tropharium.Some Wolbachia enter the arrested oocyte and establish an early infection.Wolbachia in the nurse cells spread into the growing oocytes through the nutritive cords as Vg is transported.However,Wolbachia transmission is prevented when the oocyte is compeletly developed and the nutritive cords disappear.Wolbachia distribution in the ovariole of dsVg-injected L.striatellus supports our results.Compared with the dsGFP-microinjected insects,dsVg injection obviously affected Wolbachia accumulation in the ovaries.Wolbachia were irregularly dispersed throughout the whole ovariole,and Wolbachia titer in ovarioles was greatly decreased.These results further indicated that Vg was specifically involved in Wolbachia invasion of the insect ovaries.