Explore The Relationship Between Nosema Ceranae And Gut Bifidobacteria

Honeybeesare important pollinatorsthat occupy an important niche in agricultural economy and ecology.Health status of bees affects the development of bee related industries.Intestinal microorganisms,including bacteria and pathogens,are main factors of bee health.Among the pathogens,Nosema ceranae is the most common intestinal pathogenthat infects mainly midguts.Reproduced N.cerana spores migrate to the hindgut and infect other hosts through contaminated feces.The hindgut has structural support for bacterial symbiotes and biofilms;moreover,the hindgut is the place where intestinal microorganisms are most abundant.There are no obvious symptoms of N.ceranae infection which rarely resulted in defecations within hives that used to be a conventional symptom of nosema disease;therefore,we speculated that N.cerana infection may affect the hindgut flora to benefit the survival of N.cerana.In the studies,we used field and laboratory bees and found the connection between N.cerana and gut microbes.Besides,we also observed the spatial and temporal distribution of N.cerana,lactobacteria and bifidobacteriato clarify if any direct contacts and interactions existusing CLARITY technology.The main results are as follows:1.Nosema ceranae infection enhanced the abundance of bifidobacteria in hindguts of honeybeesIn the field,we collected forage bees that were severely infected with N.ceranae and found that only the abundance of bifidobacteria was significantly increased by 16 S r RNA high-throughput sequencing(P = 0.008).The q PCR results of caged bees showed the similar trend,and the number of lactobacteriain the feces has also increased significantly.2.The relationship between N.ceranae and bee gut bifidobacteriaFeeding IMO increased the lifespan of N.ceranae infected bees and increased the infection intensities.Compared with thecontrol group,food consumption of N.ceranaeinfected groups were increased,with or without IMO feeding.In the analysis of bee feces microbial diversity,we found that the bacterial species abundance increased after feeding IMO.After inoculation with N.ceranae,one core bacterial species Snodgrassellaalvi increased significantly(P <0.01).The results of q PCR in the hindgut showed the number of core flora feeding IMO or Suc had the same trend of increased bacteria in infected bees.In the Suc group,N.ceranae infected bees had significantly(p <0.05)increased Bifidobacterium spp.,S.alvi and G.apicola.Lactobacillus spp.were in an increasing trend but not significant there was.In the IMO group,only S.alvi increased significantly.3.Dynamic analysis of the intestinal bacteria in the Apis mellifera bees infected by Nosema ceranaeWe fixed,delipidized and in situ hybridized the bee intestine and analyzed the Nosemaspores from 0 to 12 days post inoculation by confocal microscope imaging.The results showed that many spores were found in the midgut and thepyloric on the 6th day post inoculation,and the sporeswerewidely and evenly distributed in the midguts on the 9th day.There are no spores in the cardiac valve and ileum,we assumedthat Nosemaspores do not infectcardiac valve and ileum.Nosema spores have not been found in Malpighian tubes.4.Spatiotemporal distribution of lactobacillus and bifidobacteriumOligonucleotide fluorescent probes targeting bifido and lacto were used forfluorescence in situ hybridization and confocal microscopy observations on infectedbees at different infection stages.We found that lacto always present in bees,ranging from a small amount in the foregut of newly emergedbees to biofilms in adult bee.Lacto began to appear in large numbers after 6h,which mainly attached to the ileum and pyloric valve,while the amount of bifido was relatively small.From 2-day to 3-day,the bifido and lacto coexisted in the ileum.The bifido in the ileum increased on the 10-day,but the total number was far less than that of the lacto.In this experiment,we dissected the peritrophicmembrane of the midgut,which solved the effect of pollen on observation.In summary,studies have shown that intestinal microorganisms play an important role in bee health,and our study investigated the relationship between gut bacterial microbes and N.ceranae.In addition,we explored the distribution of microbes and N.ceranae.It will be the foundationfor furthering the interaction mechanism between microbes and N.ceranae whichmay result in a new control method in bee pathology.