Diversity And Functional Analysis Of Gut Microorganism In Hepialus Xiaojinensis Larvae

Hepialus xiaojinensis is the predominant host of Cordyceps sinensis,mainly distributed in the Tibet Plateau of 3000 ~ 5000 m.H.xiaojinensis larvae feed on the delicate rhizome of plants.The gut microorganisms play an important role in the interaction between host insects and plants.It is even more valuable to study this topic with H.xiaojinensis whether diversity of gut microorganism is effected by food diversity.We analyzed the diversity and function of gut microorganisms of H.xiaojinensis 6th instar larvae which were wild-living or fed on different diets(Daucus carota,Potentilla anserine,Polygolum viviparum)by using metagenomic sequencing technology and the traditional microbial cultivation.It is expected to provide a theoretical basis for optimizing the artificial cultivation of H.xiaojinensis.The main results are as follow:1.By comparing the survival rate and the weight growth rate of H.xiaojinensis 6th instar larvae fed on P.anserine and then fed on D.carota,P.anserine,P.viviparum for one month,and fed on three antibiotics-treated foods.We obtained the results as follows: 1)When fed on D.carota,the larvae had a significant weight gain on 14 d and 28 d,while the weight growth rates of larvae with P.viviparum were negative on both 14 d and 28 d.The larvae in the control showed a negative growth after feeding 14 d,but a significant increase in weight after 28 d.2)For the groups feeding on the diets treated with antibiotics,the growth rates of H.xiaojinensis larvae with D.carota or P.viviparum treated with antibiotics were significantly lower than those without antibiotics on 14 d and 28 d.3)The survival rates of the groups feeding on the D.carota were significantly higher than those feeding on D.carota treated with antibiotics.2.The diversity of gut microorganism from the wild-living H.xiaojinensis and artificially fed on D.carota,P.viviparum and P.anserine was analyzed by using thetraditional microbial cultures and metagenomic sequencing technology.Following results were obtained: 1)By the traditional microbial cultures,8 species of bacteria and 8 species of fungi were isolated from the wild-living insects.The bacteria included Escherichia coli,Carnobacterium maltaromaticum,Chryseobacterium polytrichastri,Arthrobacter arilaitensis,Bacillus thuningiensis,Rahnell aaquatilis,Psedomonas fragi,C.divergens.The fungi included Cladosporium halotolerans,C.cldosporioides,Penicillium raistrickii,P.brevicompactum,P.nothofagi,Mucor hiemalis,P.commune,Trichoderma koningiopsis 4 species of bacteria were isolated from D.carota,P.anserine and P.viviparum,including Serratia plymuthica,R.aquatilis,C.polytrichastri and E.coli,but no fungi were isolated from all these artificially rearing larvae.2)Comparision of the gut microorganism of H.xiaojinensis 6th instar wild-living larvae and larvae fed on P.anserine then fed on D.carota,P.anserine,and P.viviparum showed that in the top 10 of the identified microbes of four treatments,Raoultella terrigena,R.ornithinolytica,Klebsiella pneumonia,K.michiganensis and Klebsiella sp.T4-11 were unique to the wild-living larvae,and E.coli,Rhizophagus irregularis and Wolbachia endosymbiont were unique to the artificial cultivated larvae.In guts of the artificial cultivated larvae of H.xiaojinensis,the predominated species were E.coli,R.irregularis and Wolbachia endosymbiont.Compared with the larvae fed on D.carota,P.anserine or P.viviparum.Serratia sp.Leaf50 was the predominate species in the guts of the larvae fed on D.carota.3.The functional genes of gut microorganism in H.xiaojinensis larvae were analyzed by using metegenomic sequencing.The results showed that: 1)The abundance of the glycosylase(GH)and glycosyltransferase(GT)involved in digestion of microflora in wild-living larvae was significantly higher than that of these two genes from the larvae fed on D.carota,P.anserine or P.viviparum.2)The genes involved in amino acid and vitamin metabolisms in wild-living larvae were significantly more than those from the larvae fed on D.carota,P.anserine or P.viviparum.The abundance of the genes involved in amino acid and vitamins metabolisms for the larvae fed on D.carota was significantly higher than that of the larvae fed on P.viviparum and P.anserine.The genes involved inphenylalanine biosynthesis were not detected in the larvae feeding on P.viviparum or P.anserine.3)Several secondary metabolites were synthesized in the wild-living larvae,such as Streptomycin,Betalain,Monobactam,Penicillin,etc,but not detected from the larvae feding on D.carota,P.anserine or P.viviparum.4)There were More xenobiotics biodegradation related genes were found in wild-living larval guts,compared with those feeding on D.carota,P.anserine or P.viviparum.