Wolbachia Transfection And Biological Control Capability Of Trichogramma Spp.

Trichogramma spp.is the one of the egg parasitoids controlling insect pests to manage many pests from maize,rice,cottons,vegetables and forests wiedly and successfully. Wolbachia,intracellular symbiotic bacteria of arthropods,in recent years had aroused wide attention from scholars because they could regulate biological evolution and reproductive behavior of the host,and present prospects in some applications areas such as a carrier of exogenous gene.Wolbachia could infect the many species of parasitic Trichogramma, resulting in female offspring of thelytokous Trichogramma,and could be transfected vertically and horizontally among species.Wolbachia could induce thelytoky parthenogenesis of their parasitic Trichogramma and enhance female fertility characteristic,so they could be used as specific genetic tools manipulating their host to change their reproductive characteristics.The better parasitic Trichogramma without producing female offsprings could be rebuilt through infection by Wolbachia,and finally Trichogramma increase female rate and reduce costs.It was useful to improve the effectiveness of biological control.In this thesis, firstly,Trichogramma infected by Wolbachia were screened to identify the better donor to transfect other species.Secondly,we try to make horizontal transfection by artificial method and clarify the mechanism of transfection.Thirdly,the biological characteristics of the target Trichogramma with producing female were analyzed included the response capacity to external environmental factors,olfactory responses,population parameters and the fitness of Wolbachia in the Trichogramma.Finally we released Trichogramma to survey the effects to control corn borer in field with a large area.The results are as follows:The three kinds of thelytokous T.cacoeciae,T.embryophagum and T.pretiosum response to separately by hexane extracts from the eggs surface of Corcyra cephalonica and abdominal scales of female C.cephalonica were measured by using the improved Y-tube olfactometer.The results helped to evaluate the capacibility of Trichogramma's searching its host,parasitic and eclosion.To uncover the role of Wolbachia in regulating the reproductive capacity of Trichogrammas,the stability of Wolbachia in three kinds of Trichogrammas after antibiotic treatment was observed.The results indicated that the eclosion male offspring of T embryophagum are the smallest after antibiotics treatment,the relationship of Wolbachia and their symbiotic host-T.embryophagum was more stable than others.Combining with sensitive olfactory responses and strong parasitic ability,Wolbachia in T.embryophagum were the better strains to make artificial transfection,and used as donor of horizontal transfection.Taken thelytokous T.embryophagum as donor,gender reproductive T.dendrolimi,T. ostriniae,T.chilonis and T.brassicae as receptor,the horizontal artificial transfection Wolbachia among Trichogramma species was investigated through the three methods involved in sharing of food source,feeding bacteria suspension and microinjection. Wolbachia was transfected successfully from T.embryophagum to T.dendrolimi by the method of sharing of food source,and the reproductive patterns of receptor T.dendrolimi were changed to thelytoky parthenogenesis with genic stability.Furthermore,the identity of wsp sequence from Wolbachia between thelytokous T.dendrolimi and T.embryophagum reached to 99%,both belonging to"Dei"sub-group of B-group in Wolbachia,while identity between thelytokous T.dendrolimi and C.cephalonica that was classified into"Pip"sub-group of B-large group was 81%.However other Trichogrammas could not accept Wolbachia.Under microinjection,gender thelytoky of T.brassicae were transformed to thelytoky parthenogenesis,and T.dendrolimi showed partial thelytoky parthenogenesis. Horizontal transfection of Wolbachia could not be achieved between Trichogramma species by mature and young Trichogramma feeding bacteria suspension.Trichogramma species or lines embracing Wolbachia with better parasitic ability were obtained to create productive forces,and new means to regulate biological reproduction.The characteristic of host Trichogramma affected by Wolbachia was diversity and uncertainty.So it is necessary to elucidate the impact of the symbiotic bacteria Wolbachia on Trichogramma.The biological indicators of thelytokous T.dendrolimi including parasitic and eclosion number were analyzed under mating treatments,different combination treatments and antibiotic treatments at 25℃and 32℃.The results indicated that under the conditions of 25℃,the reproduction mode of thelytokous Trichogramma cultured with many generations was fixed.After thelytokous Trichogrammas mating with male,reproduction mode not be changed besides Parasitic ability decreased.Under 32℃,whether Trichogramma mating with male or not were cultured four generations continuously(about 30 days),Wolbachia could be inactivate completely and restore the way of thelytoky parthenogenesis.The results showed that reproduction mode of thelytokous T dendrolimi would not be changed for mating with male.The determination factors of changing mode of reproduction were antibiotic treatment and high temperature 32℃for 20 days at least. Antibiotics may remove Wolbachia in T.dendrolimi and restore the reproduction of gender. Most of male Trichogramma were present exposered antibiotics with middle-low concentration(25mg/mL).The impacts of ecological factors on the genetic stability of thelytokous T.dendrolimi were showed,and theoretical basis were provided releasing T. dendrolimi infected by Wolbachia in field.Olfactory response of thelytokous T.dendrolimi to volatile infochemicals of host, parasitic capacity and eclosion ability under different rate of wasps to eggs were studied.The thelytokous T.dendrolimi could be attracted in varying degrees by the eggs surface of Corcyra cephalonica and volatile infochemicals of abdominal scales of female C. cephalonica in several provided Lepidoptera pests(C.cephalonica,O.furnacalis,and A. pernyi).The T.dendrolimi response to the eggs surface of corn borer was stronger than gender lines.Under constant temperature and humidity(25℃,RH 70%),the parasitic capacity of T.dendrolimi infected by Wolbachia to several kinds of host eggs was stronger than gender T.dendrolimi's,but the difference was not significant.Focusing on different rate of wasp eggs,the parasitic capacity of thelytokous T.dendrolimi was higher than normal gender lines' when inoculating 1,3 or 4 except 2 of 30(1 piece) corn borer eggs in T. dendrolimi.In summary,the parasitic capacity of thelytokous T.dendrolimi to corn borer eggs was higher than normal gender lines' in different rate of wasp eggs.This research could provide theoretic basis for T.dendrolimi infectted by Wolbachia in controlling corn borer and other important agricultural pests in field.The reproductive parameters of thelytokous T.dendrolimi infected by Wolbachia were studied at different temperatures in order to investigate the best breeding conditions of them. The biological data including development,surviving rate and reproduction of T.dendrolimi infected by Wolbachia were observed at 16℃,20℃,24℃,28℃and 32℃respectively.The population life table was constructed under the corresponding temperature. The results suggested that the optimal reproduction temperature of thelytokous T.dendrolimi infected by Wolbachia was 24℃-28℃.Thelytokous T.dendrolimi were released into corn field in the regions of Xiuyan and Shenyang in Liaoning province for two consecutive years,to evaluate effects on controlling corn borer.Through the two-year investigation,it was found that thelytokous T.dendrolimi and gender T.dendrolimi had achieved some effect in controlling corn borer to increase yield more than 40%.Corn fresh weight and dry weight per acre in fields with T.dendrolimi were significantly higher than without T.dendrolimi's.There were not significant differences between the Thelytokous T.dendrolimi and normal T.dendrolimi lines in pest control,corn yield and other indicators.So thelytokous T.dendrolimi in controlling pests was similar to gender T.dendrolimi,and may be greater in potential applications.