| Wolbachia is an intracellular bacterium carried by thousands of arthropods.Wolbachia’s success is due to the effective vertical transmission of the host’s maternal line.Wolbachia’s behavior is evident during the host’s oogenesis period.However,it is not clear that the vertical transmission of.Wolbachia WSTRI strain in the gray planthopper is very effective during embryo development.In order to understand the cause,we studied its localization and vitality in the embryo of Small Brown Plant Hopper(SBPH).Microscopic observation showed that Wolbachia was mainly distributed in the anterior part of the embryo.The distribution of prezone was formed during oogenesis,and phylogenetic analysis suggested that it might be affected by the intrinsic factors of Wolbachia.An expression profile of embryonic development genes infected with Wolbachia showed that Ddxl mRNAs aggregated in the posterior zone of the third day embryo,while other developmental genes mRNAs accumulated in the posterior zone of the sixth day embryo.As a result,these genes appear to be associated with the anterior localization of Wolbachia wStri,although their function is not yet clear.We found that wStri moved to the gonadal formation region at the late stage of embryogenesis.This discovery may explain its high prevalence among Small Brown Plant Hopper(SBPH).In this paper,the effects of Wolbachia on embryo development,the relationship between Wolbachia and embryonic development genes,and the localization of Wolbachia in the laying stage of gray planthopper were studied by feeding infected and non-infected Wolbachia Small Brown Plant Hopper(SBPH).Fluorescence quantitative PCR,tissue immunohistochemistry and electrophoresis were used to detect the effect of symbiotic Wolbachia on the oogenesis of Small Brown Plant Hopper(SBPH).The results were as follows:1.Morphology of embryogenesisThe embryos of the planthopper need 7 days to complete their development at a temperature of 26±1℃.There was no significant difference in embryo development between infected and uninfected B.planthopper.Confocal observation showed that the embryo skin was formed within 24 h and the embryo band formation map was completed at 48 H.The frontal lobe and chest of the embryo changed from backward to forward during blastocyst division and began to divide within 72H.The blastocyst was differentiated and segmented before 96H,and the blastocyst formation and dorsal lamina closure were performed at the same time.On the 5th day,a complete movement was formed from the back to the ventral side from the spot of the eye.Along with the embryo development,the eye spot gradually increases,the chest film development is completed on the 6th day.Embryo The fetus completed its development on the 7th day,and cracks appeared in the anterior micropores,which was favorable for hatching.At any stage of development,Wolbachia has no significant effect on embryo size(length,width,perimeter)2.Wolbachia localization during embryogenesisWe used confocal analysis to detect the presence of Wolbachia.in early(1-4-day-old)and late-stage(5-7-day-old)embryos.In the early stage of embryogenesis,we observed that the Wolbachia signal was more in the anterior part of the embryo and less in the posterior part of the embryo,which remained unchanged at the age of 1-3 days after embryogenesis We then found that the weak Wolbachia signal had moved to the middle of the 4-day-old embryo.Wolbachia in the late embryonic stage is also clustered in the prezone and distributed to the back end of the embryo.These results suggest that Wolbachia The location of bachia in the preoogenesis region of the Small Brown Plant Hopper(SBPH)was conservative,and the content of Wolbachia in the head of one day old nymphs was higher than that in the abdomen,which proved that the titers of Wolbachia in the preembryonic region were higher.Interestingly,Wolbachia is located in the anterior region with the Wolbachia classification of Drosophila based on the Wolbachia MLST gene sequence Phylogenetic analysis showed that the localization of Wolbachia in different host backgrounds was conservative.The distribution of Wolbachia in group A was mainly concentrated in the posterior part of embryo,and the distribution of wRi was obvious.The Wolbachia in group B was more distributed in the anterior part of embryo than that in group B.These results suggest that the intrinsic factors of,Wolbachia wStri may determine its distribution in host embryos.The density of Wolbachia at different stages of embryo development was measured by qPCR.The titers of Wolbachia increased with the development of embryos,but no significant increase was observed during embryonic development.The same results were also observed in Drosophila melanogaster.These results indicated that there was no significant correlation between the titers of Wolbachia and embryo development.3.Embryonic development genes in Wolbachia localizationIn the embryos infected with Wolbachia,the expression level of developmental genes in most embryos increased significantly in the anterior part and in the posterior part of the embryo,while the expression level of Ddxl increased significantly in the posterior part of the embryo.The expression of Atp α,α Tub67C,CtBP,Eip63E,and Dorsal genes in 6-day-old infected Wolbachia embryos was significantly increased.These results suggest that the expansion of Wolbachia to the back of the embryo is related to the host embryonic development gene,and most of the embryonic development genes expressed in uninfected embryos are stronger than those in infected embryos at 3 and 6 days of age.There was no significant difference in the expression of 5 genes(Eip63E,Dorsal,fd64A,CPO,and Ddxl)between infected and uninfected embryos.4.Wolbachia localization during oogenesisIn order to determine whether the distribution of Wolbachia in embryos was determined by the mother,we observed the distribution of Wolbachia-infected oocytes at different developmental stages.The location of different developmental oocytes in the ovary and the representative position of Wolbachia in this area.Wolbachia is dispersed in the cumulus of the arrested oocytes in the ovary trophozoites,where early infection occurs.Many Wolbachia have also accumulated irregularly in early developing oocytes,which connect the trophoblast and yolk layers of the ovary.Hia also aggregates in developing oocytes and in the anterior part of mature oocytes. |
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