Study On Autophagy In Honeybee (Apis Mellifera) Challenged By Nosema Ceranae

Nosema ceranae(N.ceranae)is a worldwide honeybee fungal endoparasite threatens the apiculture.N.ceranae mainly infects the midgut of honey bees and is spread through the fecal-oral route.Infected bees turn prematurely into foraging bees,which are also less oriented and less adaptable to pollen collection than normal foraging bees,leading to a shortened life span and early decline,thus weakening the colony until it disappears.Current research focuses on the biological characteristics of N.ceranae,its genome and related molecular biology,pathological characteristics of infected bees,immune regulation,and related control studies.Previous studies in our laboratory found that N.ceranae infestation may induce an autophagic response in honey bees,so this thesis focuses on exploring the role of honey bee autophagic response in response to N.ceranae infestation.This study investigated the tissue localization of N.ceranae in infested bees using in situhybridization,analyzed differentially expressed genes before and after N.ceranae infection in bees using highthroughput sequencing technology,and analyzed related regulatory signaling pathways;RT-q PCR was used to verify the expression of genes related to the autophagic pathway,and further used transmission electron microscopy(TEM)technology to verify the occurrence of autophagy.The main results are as follows:1.In vivo bee testing experiments revealed that the lifespan of bees infected with N.ceranae was shortened and that the number of N.ceranae in the bees increased with the duration of infection.2.The tissue localization of N.ceranae in honey bees was examined using fluorescence in situ hybridization,and N.ceranae were found to be present mainly in the midgut,hypopharyngeal glands and fat body tissues of honey bees,and were not detected in muscle tissue.3.Transcriptome sequencing analysis identified 1766 up-regulated and 1038down-regulated genes in adult bees infected with N.ceranae.KEGG pathway analysis identified signaling pathways that play important roles in host cell and humoral immunity,nutrient metabolism,host cell transcription,cell cycle and hormone synthesis.4.Detection of m RNA expression levels of six autophagy-related genes revealed that the relative expression of autophagy genes were significantly up-regulated in N.ceranae infected bees.5.In order to further verify the occurrence of autophagy,the autophagosome were observed using the TEM technique,and it was found that autophagosome and autolysosomes were observed in the midgut,hypopharyngeal gland and fat body tissues of bees 6 days after infection with N.ceranae.6.Whether autophagy occurrence is beneficial to bee survival and helps bees resist N.ceranae infestation was tested by adding an autophagy inducer or inhibitor.It was found that the combination of the autophagy inducer Rapamycin and N.ceranae prolonged the life span of bees compared to the N.ceranae group alone,while the combination of the autophagy inhibitor 3-MA and N.ceranae resulted in a shorter life span of bees.