A Study On The Immune Response Of Larvae And Prepupae Of Apis Mellifera Ligustica To Nosema Ceranae Stress

Nosema ceranae is a fungal pathogen that specifically infects the epithelia cell of honeybee midgut,leading to microsporidiosis,which can result in huge losses for beekeeping industry.N.ceranae is originated from Apis cerana;however,studies showed it is more detrimental for Apis mellifera.Previous studies were mainly related to interactions between adult honeybee and N.ceranae,but knowledge of interactions between honeybee larvae and N.ceranae is extremely limited.In this work,to investigate the infectivity of N.ceranae to A.mellifera ligustica larvae,as well as host immune responses to N.ceranae stress,firstly,3-day-old larvae of A.mellifera ligustica were inoculated with purified spores of N.ceranae,followed by paraffin section and HE dying of larval and prepupal guts;secondly,the response of key immune genes of larvae and prepupae were detected with qRT-PCR;thirdly,normal and N.ceranae-stressed larvae were sequenced using RNA-seq,followed by comprehensive analyses of host highly expressed genes(HEG)and differentially expressed genes(DEG),and host cellular and humoral pathways and enriched genes were further explored.The main results obtained in this study are as follows:(1)Paraffin sections of A.mellifera ligustica larval guts inoculated with N.ceranae were prepared followed by HE dying,the microscopic observation result suggested there is no obvious infection,implying a low level infection of larvae with N.ceranae.(2)The expression levels of key immune genes in A.mellifera ligustica larvae and prepupae were detected with qRT-PCR,the results indicated the expression levels of abaecin,hymenoptaecin and defencin were significantly increased,while that of apidaecin was significantly decreased;the expression levels of vigellogenin、glucose dehydrogenase,lysosome and phenoloxidase were significantly activated,while that of eater was first activated and then inhibited;the results demonstrated that the expression levels of key immune genes differed when responding to N.ceranae stress.(3)HEG analysis result showed,to response to N.ceranae stress,specific HEG of A.mellifera ligustica larvae were mainly enriched in cellular immune pathways such as endocytosis and Lysosome,as well as humoral immune pathways such as ak-STAT signaling pathway and NF-kappa B signaling pathway;specific HEG of A.mellifera ligustica prepupae were mainly enriched in cellular immune pathways such as lysosome and apoptosis.(4)DEG analysis result suggested cellular immune response was the principal immune response for 4-and 5-day-old A.mellifera ligustica larvae;additionally,MAPK signaling pathway was involved in host immune response;cellular immune responses in 6-day-old larvae were induced;cellular and humoral immune responses in 7-day-old prepupae were induced,while cellular immune responses in 8-day-old prepupae wereinduced.Our findings provide cellular evidence for low level infection of A.mellifera ligustica larvae with N.ceranae and information about key immune genes response to N.ceranae,but also reveal host immune response to N.ceranae at transcriptome level and offer a basis for uncovering the molecular mechanism underlying host immune responses.