| Nosema ceranae(N.ceranae)is a kind of fungus,which exclusively parasitizes the midgut epithelial cells of honeybees,infects a variety of honeybees and spreads around the world.N.ceranae has no mitochondria and must reproduce using ATP of host cells..In addition,N.ceranae can infect the host immune response by secreting small RNAs and other means,carry out immune evasion,and affect the health of bees and the development of bee colonies.The honeybee gut microbiota is a complex ecosystem comprised of a diverse array of microbial organisms.It can co-evolve with the honeybee,playing an indispensable role in host health.When the pathogens infect the host,the microbial community will produce a dramatic response to enhance the host’s immunity,which in turn plays a role in resisting pathogen infection.It takes about 4 days for oriental honeybee microspores to infect host cells to spore maturation.During this process,oriental honeybee microsporidia will inevitably be affected by gut microbes.Therefore,studying the competition between honeybee gut microbes and N.ceranae has positive significance for the treatment of Nosemosis.This study investigated the differences in the infection levels of N.ceranae between different colonies of Apis cerana(A.cerana)and Apis mellifera(A.mellifera)and assessed the ability of A.cerana gut microbiota to resist N.ceranae infection of A.mellifera by fecal microbiota transplantation.In addition,the N.ceranae infection of A.mellifera colonies with different genetic backgrounds was investigated,and the gut microbial levels and metabolites of bee colonies with different infection levels were analyzed by 16 s r RNA sequencing technology,liquid chromatography mass spectrometry and other technologies,in an attempt to explore the production of Microbial and metabolic pathways of this difference.By infecting honeybees of different genetic backgrounds with the same amount of oriental honeybee microspores from the same source,combined with transcriptomic analysis,the molecular mechanism of A.mellifera inhibiting the reproduction of microsporidia was analyzed.The main findings are as follows:(1)In the natural state,the spores load in the midgut of A.mellifera was significantly higher than A.cerana,and there were also significant differences in the spore load among different A.mellifera colonies.(2)In order to study the inhibitory effect of A.cerana gut microbiota on the proliferation of N.ceranae,we inoculated gut tissue solution from A.cerana to A.mellifera,and detected the content of progeny spores and bee immune response on the14 th day condition.Immune genes remained low in honeybees that were not inoculated with gut microbes and spores.Honeybees inoculated with gut microbes showed significant differences in the expression of three immune genes(Am PPo、dorsal-1、Apidaecin),with 26% reduction in spore levels,compared with bees infected with spores only.(3)In order to evaluate the effect of A.mellifera of different genetic backgrounds on the resistance of N.ceranae,honeybees in different colonies were inoculated with N.ceranae experiments.Among them,Group A,Group B and Group C are three different bee colonies,and Group D is a newly emerged workers randomly grabbed from the three bee colonies.Spore counts and transcriptome sequencing were performed at day 12 of age.The results showed that the spore infection level of Group B was significantly lower than that of the other three Groups.Compared with Group D,a total of 473 differentially expressed genes were detected in Group B,of which 242 genes were significantly up-regulated and 231 genes were significantly down-regulated.Through GO and KEGG enrichment analysis of the differential genes,it was found that most of the differential genes were concentrated in biological processes such as the interaction between bees and microorganism,system immunity,material metabolism,and cellular processes.Combined with literature reports and functional annotation analysis,five differentially expressed genes(Vg,LOC409286,defensi-1,nkd,Jhe)related to N.ceranae infection were screened out.(4)In order to analyze the differences in the intestinal microbes of bee colonies with different levels of N.ceranae infection,12 colonies whose spore load was outside the 95%confidence interval were divided into Group H and Group L according to the number of spores.16 s r RNA sequencing was performed on each set of samples.The results showed that five microorganisms(Snodgrassella,Gilliamella,Lactobacillus,Frischella and Bifidobacterium)comprise the vast majority of the honeybee’s flora at the genus level.The results based on Anosim analysis,MRPP analysis,Adonis analysis and Amova analysis showed that there was no significant difference in community structure between the two Groups.However,the Venn diagram based on the OTUs cluster analysis results showed that 76 OTUs were shared by Group L and Group H,43 OUTs were unique to Group L,and 30 OTUs were unique to Group H.These unique OTUs only accounted for0.14% of the total bee midgut microbial.(5)In order to analyze the differences in the metabolites and metabolic pathways of different bee colonies with different levels of N.ceranae infection in the natural,liquid chromatography-tandem mass spectrometry combined with non-targeted metabolomics technology was used to analyze the differences of A.mellifera colonies from different bee colonies.Bee midgut grinding fluid for detection.Through multivariate statistical analysis of metabolites in Groups H and L,with VIP > 1.0,FC > 1.5 or FC < 0.667 and P< 0.05 as thresholds,132 differential metabolites(74 up-regulated,58 down-regulated)were screened in positive ion mode,2 metabolic pathways(Citrate cycle,Glyoxylate and dicarboxylate metabolism)with differences.In the negative ion mode,115 differential metabolites(48 up-regulated,67 down-regulated)were screened,and 1 differential metabolic pathway(Biosynthesis of unsaturated fatty acids).Three metabolites closely related to sugar metabolism(alpha-Ketoglutaric acid,Citric acid,cis-Aconitic acid)were significantly lower in Group L than Group H,that were located in the Citrate cycle,Glyoxylate and dicarboxylate metabolism pathway.Three metabolites closely related to fatty acid metabolism(Arachidonic acid,Docosahexaenoic Acid,Erucic acid)were significantly higher in Group L than Group H,and they were involved in Biosynthesis of unsaturated fatty acids pathway.In conclusion,this study found that the number of spores in the intestinal tract of the A.mellifera was significantly lower than that of the A.cerana in the natural state through the sampling survey of the A.mellifera and A.cerana.Among the different colonies of A.cerana,A.mellifera have different degrees of resistance to N.ceranae infection.Combined with fecal bacteria transplantation technology,16 s r RNA sequencing technology,transcriptome sequencing technology and non-targeted metabolomics technology,the inhibitory effects of gut microbes and honeybees on N.ceranae were evaluated,and the possible effects on N.ceranae were screened and compared.Sporozoites have inhibitory gut microbes,immune genes and metabolic pathways.The results of this study have important reference value for further exploration of the control methods of N.ceranae. |
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