A Research On Immune Responses Of The Larval Guts Of Honeybees To The Stress Of Ascosphaera Apis Based On RNA-seq

Chalkbrood,which is the most representatively fungal disease of honeybee,is caused by Ascosphaera apis.A.apis specifically infects honeybee larvae,which results in great damage to the apiculture.Apis mellifera ligustica larvae is vulnerable to A.apis,while Apis cerana cerana larvae has a strong A.apis-resistance.Previously,studies on A.apis were focused on taxonomic classification,cultural method,pathogen detection,epidemiology,infection process and immune defense and so forth,but the progress in molecular study on A.apis was limited due to a lack of reference genome information.The corresponding studies were mainly involved in Apis mellifera larvae invaded by A.apis,however,studies on Apis cerana larvae challenged with A.apis were extremely lagged behind.At present,there are very few reports about the omics study of chalkbrood,and no transcriptome study on honeybee larvae is documented.In this study,A.m.ligustica and A.c.cerana larvae were fed withthe purified A.apis spores at a concentration of 107 spores/mL,followed by deep sequencing of the larval guts using the next generation sequencing technology.Finally,the immune responses of the hosts under stress of A.apis were investigated.In summary,the main findings are as follows:(1)We for the first time confirmed that the isolated pathogen from the mummy of A.c.cerana larvae is A.apis,and the larvae of A.c.cerana could be infected by A.apis and became chalkbood under laboratory conditions via morphology and molecular biology methods.(2)We successfully assembled and annotated the reference transcriptome of the larval gut of A.c.cerana,which can provide key information for molecular biology and omics studies of adult bee and larvae of A.c.cerana.Furthermore,we developed 21 novel SSR markers of A.c.cerana larvae based on the transcrptome data,which can be applied to the genetic map construction,genetic diversity analysis as well as genetic location of eastern honeybee and its larvae,and it's confirmed that to develop SSRs of non-model organisms on a large scale based on transcriptome data is an efficient and feasible menas.(3)We performed a comprehensive analysis of immune responses of the larval gut of A.c.cerana through differentially expressed genes(DEGs)analysis and trend analysis.We found that,for the DEGs enriched in cellular immune pathways,the quantity of DEGs showing up-regulated trends were much more than those displaying down-regulated trends,and all DEGs enriched in humoral immune pathways showed a up-regulated trend.It's demonstrated that both cellular and humoral immune pathways were activated by A.apis.(4)We conducted a comprehensive analysis of immune responses of the larval gut of A.m.ligustica by DEGs analysis and trend analysis,we found that a part of DEGs enriched in cellular immune pathways displayed a up-regulated trend,while more DEGs showed down-regulated trends.Additionally,the majority of the DEGs enriched in humoral immune pathways showed up-regulated profiles.It's suggested that the cellular immune pathways were partially induced,and the humoral immune pathways were greatly activated.