The Diversity Of Symbionts In Anastatus Fulloi (Hymenoptera:Eupelmidae),and The Distribution Of Rickettsia In Reproductive System

Anastatus fulloi(Hymenoptera: Eupelmidae)is an important natural enemy parasitic wasp of the litchi stink bug,Tessaratoma papillosa(Hemiptera: Tessaratomidae).Since the 1960 s,Chinese scientists have begun to study the techniques of artificial breeding and release of this wasp and have achieved good control effects in litchi and longan orchards.The parasitic rate in orchards can be up to 85~90%,the damage of the litchi stink bug was basically controlled.This biocontrol method provides a very good and necessary protection method for the green and pollution-free litchi and longan.There are a large number of symbionts in parasitic wasps,including Wolbachia,Rickettsia,Cardinium,Spiroplasma,etc.These symbiotic bacteria not only regulate the nutrient metabolism and reproductive metabolism of host insects,but also help parasitic wasps to resist biotic and abiotic stresses,improve parasitic wasp resistance to chemical pesticides and adaptability to parasitic pests.But there are still no reports of the symbionts of Anastatus fulloi.The study of the diversity,species and distribution of symbionts of A.fulloi,will promote the understanding of its parasitic,digestant and reproductive characters,and promote the use of this parasitic wasp in the biological control of pests.In this thesis,the reproductive system structure of A.fulloi was studied firstly;then the high-throughput sequencing and 16 S rDNA cloning technology were used to detect the diversity of symbionts of A.fulloi;finally,the position of secondary symbiont Rickettsia in A.fulloi was determined by fluorescence in situ hybridization(FISH),and its function was further explored to improve the A.fulloi.artificial breeding efficiency and new ideas for controlling the growth potential of agricultural and forestry pest populations.The main findings of this paper are as follows:1.The internal physiological anatomy of male and female adults of A.fulloi was studied.The reproductive system of female consists of a pair of ovaries,a pair of lateral oviducts,a common oviduct,attached glands,and a sperma theca.The venom organ is composed of a colorless,transparent,oil-filled poison sac;a white,curly venom gland;and a Duchenite gland that is sac-like and oil-filled.The reproductive system of male consists of a pair of testes,a pair of accessory glands,a pair of vas deferens,and an ejaculatory ducts.2.Based on the 16 S rDNA sequencing with universal primers for bacteria,the diversity of symbiotic bacteria in the laboratory population of A.fulloi from Guangzhou and the wild population from Fuqing were analyzed.It was found that six kinds of symbiotic bacteria were detected in the wild population: Wolbachia,Rickettsia,Acinetobacter,Acetobacteraceae,Spiroplasma,and Gluconobacter;only Spiroplasma and Acetobacteraceae were found in laboratory populations.3.The high-throughput sequencing technology was used to detect the symbiotic bacteria in the wild population of Fuqing.The dominant bacteria in class category were Verrucomicrobiae and Spirochaetes.The dominant bacteria in order category were the Verrucomicrobiales,Spirochaetales and Enterobacteriales;the dominant bacteria in family category were Spirochaetaceae,Prevotellaceae,Enterobacteriaceae and Lachnospiraceae;The dominant bacteria in genus category were Treponema,Bacteroides and Escherichia.4.The fluorescence in situ hybridization(FISH)was used to detect the distribution of Rickettsia in the reproductive system of A.fulloi from the wild population from Fuqing.It was found that Rickettsia in adult A.fulloi were outside bacteriocytes,that was,in the mature egg of the female abdomen and the mature oocyte that were concentrated around the follicular cells.Rickettsia was also detected in the position of the egg pedicle and the stalk,and the fluorescent signal of Rickettsia was also detected in the testis of the male of the wasp.In summary,the reproductive system of A.fulloi were studied,the biodiversity of symbionts of A.fulloi was studied by using traditional molecular biology techniques and high-throughput techniques.Then,Fluorescence signals were then detected by fluorescence in situ hybridization in mature eggs,egg stalks,ovum stalks,and immature oocytes and ovarian accessory glands in the ovary of female adult larvae.It was proved that the bacteria were vertically transmitted through the eggs in the wasps.And the presence of Rickettsia was also found in the gland of the male adult sperm.The conclusions were to improve the breeding efficiency of parasitic wasps and provide evidence to reveal the ways of secondary symbiotic bacteria in the host population.