| Insects intestine inhabits various of commensal bacteria,which coevolve with their host and formed a symbiotic relationship.A variety of symbiotic bacteria in gut of Apis mellifera were reported,and some exhibited inhibitive effects against honey bee pathogenic bacteria.A.cerana have been studied on the intestinal bacteria too,however,very few studies purified the intestinal beneficial bacteria,elucidated their characteristics,and tested their effects on bee.In this paper,we used selective media for Lactobacillus and Bifidobacterium separation from A.c.cerana adult worker bee midguts,and classified by 16S rDNA sequencing and biochemistrical methods.Some isolates were evaluated for their safety by feeding adults.The influences of different feed accompanied with Nosema ceranae inoculation were also conducted.The main results are as followings:1.From A.c.cerana worker bees' midgut,24 isolates were obtained,designated as Acc-1-Acc-24,of which 13 isolates(Acc-1-Acc-13)were picked up from the lactobacillus selective medium,and 11 isolates(Acc-14-Acc-24)were picked up from the Bifidobacterium selective medium.Through phylogenetic analysis and physiological and biochemical,identification,isolates Acc-3,Acc-4,Acc-5,Acc-6,Acc-7 and Acc-9 were assigned to Leuconostoc mesenteroides subsp.dextranicum;Acc-10,Acc-11 and Acc-12 were assigned to L.pseudomesenteroides;isolates Acc-1,Acc-2,Acc-8 and Acc-13 were assigned to Lactobacillus rhamnosus;isolates Acc-14 to Acc-24 were assigned to Serratia marcescens,isolates Acc-14 and Acc-15 were assigned to the S.m.subsp.sakuensis.2.When A.c.cerana workers were inoculated orally with 1×107 cell/bee of S.marcescens,isolates Acc-19 and Acc-24,respectively,the cumulative mortality reached nearly 80%on the fourth day,significantly higher than that of control group(P<0.05).while that of bees inoculated with lactic acid bacteria(LAB)isolates Acc-11 and Acc-13 were less than 20%even on the eighth post inoculation,similar to control group,no significant(P>0.05)differencethis indicated with the control group.S.marcescens were toxic to A.c.cerana adult workers,while isolates LAB were safe to the bees.3.Anaerobic environment created by using vacuum bag,5%CO2 incubation and anaerobic bag,for better culture of eight Lactobaterial isolates(Acc-1,Acc-2,Acc-3,Acc-4,Acc-8,Acc-10,Acc-11 and Acc-13)were conducted.Two culture media,MRS and YGPB were used.The results showed that MRS medium was better for these bacteria proliferation,and the higher growth rate were obtained in the vacuum bag,then anaerobic culture,All tested isolates did not grow under 5%CO2 incubation condition.4.Different feed(50%sucrose,bee bread)and dosage of Nosema ceranae(5000 spores/bee,50000 spores/bee)administrated to newly emerged worker bees,the cumulative mortality and the richness and species of bacteria in midguts of worker bee were measured,the results showed that,the cumulative mortality of worker bees fed with 50%sucrose was higher than that of bees fed with bee bread,found that 15-day-old worker inoculation the same dosage of N.ceranae(5000 spores/bee or 50000 spores/bee)the spore load in the midguts of bee fed with bee bread were significant higher than those fed with 50%sucrose syrup(P=0.007,P=0.021).High througput sequencing data revealed that A.c.cerana adult worker bee gut flora is mainly composed of Bacteroidetes,Proteobacteria and Firmicutes and the midgut microbial communities of A.c.cerana adult worker bee fed with 50%sucrose syrup or 50%sucrose+beebread was different.The gut microbial diversity in midguts of bees fed with beebread bee is higher than those fed with 50%sucrose,The infection of N.ceranae resulted in higher diversity of intestinal microbial communities than those uninfected.Different feed and infect N.ceranae can influnence A.c.cerana adult worker bees' midgut lactobacillus.With the proliferation of N.ceranae,A.c.cerana adult worker bees' midgut lactobacillus decrease,in the feed is bee bread of worker bees is particularly evident. |
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