| There are a variety of interactions between symbiotic fungi and insects,which affect the growth and development,food digestion and absorption,defense,and various behaviors of the host.The potential use of fungi as biological control agents has been preliminarily confirmed.It can control agricultural and vector pests or protect beneficial insects by targeting or using symbiotic fungi.However,the current research on the interaction between fungi and insects is still in their initial stage,and most of the research only focuses on model insects.This research systematically studied the community composition and probiotic effects of the intestinal fungi of the important agricultural pest Bactrocera dorsalis through high-throughput sequencing technology,classical microbial pure culture and aseptic biology methods.This research provide a scientific basis to clarify the interaction mechanism between symbiotic fungi and insects and provide new strategies and targets for the control of Bactrocera dorsalis.The main results are as follows:1 Community structure and diversity of intestinal fungi in laboratory population of B.dorsalisHigh-throughput sequencing was used to investigate the community structure and diversity of the first,second and third instar larvae and adult intestinal fungi of B.dorsalis.The results showed that the diversity index of the first instar larvae was the lowest,the fungal structure was relatively simple.The second-instar larval stage has the highest diversity index,and the fungal species richness in the intestine is higher.Ascomycota(74.84%)dominates the intestinal fungal community at different developmental stages of B.dorsalis.Yeast exists stably in the intestinal tract,especially Pichia and Candida.The structure of the fungus changes with the development of the host.Candida dominates in first instar larval gut(67.81%),Pichia has the highest abundance in the intestines of second instar larvae of 40.39%;Pichia and Candida are the main group of third-instar larvae,with abundance of 29.28% and 24.51%,Starmerella(20.13%)mainly exists in the intestines of adults.The qPCR results showed that most of the fungi were concentrated in the larval stage,and the abundance of total fungi and yeast in the intestinal tract of the second instar larvae was the highest.A total of 364 fungal strains were obtained from larvae larval intestines,these strains were assigned to 13 species,12 of them are ascomycetous yeast,and only one basidiomycetous yeast Trichosporon asahii was isolated.Surprisingly,no fungus was isolated from the adult’s gut.2.Community structure and diversity of intestinal fungi in larvae of B.dorsalis from different geographic populationsBased on the above research results,the second instar larvae with the highest fungal abundance and diversity were selected as the test subjects,and the intestinal fungi of the second instar larvae from 15 different geographic populations of B.dorsalis were studied using high-throughput sequencing and pure fungal culture methods.Community composition and diversity.The sequencing results showed that the diversity of the intestinal fungal community of B.dorsalis larvae in different geographical populations was significantly different.Among them,the diversity of JXMHT(Larvae in kiwi from Guangxi province)and Hu NGJ(Larvae in citrus from Hunan province)was the lowest,and the diversity index of FJLW(Larvae in wax-apple from Fujian province)and GXLW(Larvae in wax-apple from Guangxi province)was the highest.In the composition of fungi,Ascomycota occupies an absolute advantage(93.67%).The prevalence of yeast was observed in all larval intestinal samples.At the genus level,Pichia(52.1%)and Candida(12.1%)have higher abundance,Saturnispora(1.3%),Starmerella(1.1%),Wickerhamomyces(0.27%)and Hanseniaspora(0.06%)have lower abundance.A total of 691 yeasts were isolated from the larval intestines of different geographic populations,these strains were assigned to 8 genera and 25 species.Among them,Pichia(57.51%)and Hanseniaspora(26.75%)are the dominant genus,some other ascomycetous yeast species were isolated at lower frequency and included Saturnispora(5.51%),Yamadazyma.sp(3.58%),Wickerhamomyces(2.58%),Starmerella(2.29%),Candida(2.0%)and Zygosaccharomyces(0.14%).A comparative analysis of the structure of intestinal fungi of different populations shows that the host and geographic location significantly influence the composition of intestinal fungi.Although the composition of the intestinal fungal community of different B.dorsalis populations is highly variable,the function of the larval intestinal mycobiota of different populations is stable and mainly related to energy metabolism,amino acid synthesis metabolism and carbohydrate metabolism,except for the JXMHT and Hu NGJ populations.3 The effect of intestinal fungi on the growth and development of B.dorsalis larvaeOur research results found that feeding antifungal agents to B.dorsalis larvae eliminates intestinal fungi and significantly affects the growth and development of the host,reducing the body length of the larvae by 29.90% compared with the control group.Furthermore,the aseptic biological research method of feeding sterile larvae with single fungi,studied the effect of the above-mentioned 32 species of B.dorsalis intestinal fungi on the growth and development of B.dorsalis larvae,including 7 species of Candida.akabanensis,C.ethanolica,C.humilis,C.orthopsilosis,C.quercitrusa,C.sorboxylosa,C.stellimalicola,4 species of Hanseniaspora guilliermondii,H.opuntiae,H.thailandica,H.uvarum,10 species of Pichia deserticola,P.fermentans,P.galeiformis,P.kluyveri,P.kudriavzevii,P.manshurica,P.membranifaciens,P.occidentalis,P.sporocuriosa,P.terricola,as well as Wickerhamomyces anomalus,W.pijperi,Saturnispora bothae,S.diversa,Wickerhamiella pararugosa,Schwanniomyces etchellsii,Starmerella bacillaris,Torulaspora delbrueckii、Trichosporon asahii、Yamadazyma sp.and Zygosaccharomyces bailii.Among them,in the sterile banana matrix,the larvae of the sterile control group(GF)cannot develop normally,and the replenishment inoculation of all 32 kinds of yeasts significantly accelerated the growth and development of the larvae to varying degrees.29 kinds of yeast were replenished and inoculated,the growth and development of the larvae were completely restored,and their body length reached the level of the control group(CR)of normal feeding larvae.Adding test fungi to artificial feed with restricted N sources,most fungi supplementation promoted the growth and development of larvae,while the growth rate of larvae in the sterile control group was significantly lower than that of the normal control group.Feeding of 13 fungi significantly increased the growth of the host and restored the larvae to the same growth rate as the normal feeding larval control group.The feeding of the 6 strains has no significant effect on the growth of larvae,while the feeding of P.kudriavzevii slowed down the growth and development of the larvae,and the body length of the larvae was significantly lower than that of the sterile control larvae.The H.uvarum strain with better growth promoting effect was selected to further study its influence on the growth and development,body weight,wing length and flight ability of the host.The results showed that adding H.uvarum to artificial feed with limited N source significantly increased the weight of pupae and adults,wing length and flight ability of B.dorsalis,and shortened the development time.These results reveal the potential probiotic role of H.uvarum in B.dorsalis from enhancing systemic growth to improving ecological fitness.4 The application potential of probiotic fungi in the large-scale propagation of B.dorsalisResponse Surface Methodology(RSM)was used to preliminarily study the application potential of probiotic fungi in the large-scale propagation of B.dorsalis and optimize the larval feed formula for large-scale rearing of B.dorsalis.The results showed that adding H.uvarum into the diet could reduce the amount of dry yeast and the feeding cost under the precondition that the larval development duration,pupa weight and adult emergence rate were not affected,and an optimal feed formula(375 g)was obtained.3.13 g of inactive dry yeast,87.45 g of wheat bran,4.42 g of H.uvarum liquid,40 g of corn meal,40 g of sucrose,and 200 mLof water.Compared with the traditional feed,the cost of each artificial feed could be reduced by 2.35 yuan. |
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