Analysis Of MicroRNAs In Different Castes Of The Bumblebee Bombus Lantschouensis(Hymenoptera:Apidae)

Bumblebees are important insect pollinators of many wild plants and crops,and play a key role in natural and agricultural ecosystems.The functions of the three castes of bumblebees,male,queen,and worker,are significantly different in colonies.Bombus lantschouensis is one of the most important local bumblebee species used commercially in China due to its beneficial characteristics such as easy rearing in captivity,including large colony size,large number of queens,high mating rate,and pollination services.However,there are still many problems in the commercial production of native bumblebees in China,such as caste differentiation and low spawning rate of queens.This affects both quality and efficiency of artificial scale breeding of bumblebee and determines whether bumblebee can be successfully used in production.It has been demonstrated that miRNAs play regulatory roles in many biological processes in animals,including embryonic development,tissue differentiation,cell proliferation,apoptosis,and morphogenesis.These miRNAs are ubiquitous in eukaryotes and are highly conserved in related species.The miRNAs are endogenous,single-stranded,and non-coding small RNAs approximately 22 nt in length that induce translational inhibition or target gene degradation by binding to complementary mRNA sequences.In this study,the miRNAs expressed in the three castes of B.lantschouensis were identified and characterized based on high-throughput sequencing.The RT-qPCR was used to validate the significant differences in expression levels of miRNAs between the castes.The target genes of miRNAs were predicted,and their regulatory pathways were analyzed.The main results were as follows:(1)Analysis of miRNAs in the heads of different castes of B.lantschouensis.The miRNAs in the heads of bumblebees from the three castes of B.lantschouensis were identified and characterized by small RNA deep sequencing.The significant differences in the expression of miRNAs and their target genes were analyzed.The results showed that length of the small RNA reads from males,queens,and workers was distributed between 18 and 30 nt,with a peak at 22 nt.A total of 364 known and 89 novel miRNAs were identified from the heads of the three castes.Eight miRNAs with the highest expressed levels in males,queens,and workers were identical,although the order of these miRNAs based on expression differed.The male vs.queen,male vs.worker,and worker vs.queen comparisons identified nine,fourteen,and four miRNAs with significant differences in expression,respectively.Different castes were clustered based on the differentially expressed miRNAs(DE miRNAs),and the expression levels of DE miRNAs obtained by RT-qPCR were consistent with the read counts obtained through Solexa sequencing.The putative target genes of these DE miRNAs were enriched in 29 Gene Ontology(GO)terms,catalytic activity was the most enriched GO term,as demonstrated by its association with 2837 target genes in the male vs.queen comparison,3535 target genes in the male vs.worker comparison,and 2185 target genes in the worker vs.queen comparison.(2)Analysis of miRNAs in the ovaries of egg-laying queen and non-egg-laying queen of B.lantschouensis.The miRNAs in the ovaries of egg-laying queen and non-egg-laying queen of the bumblebee B.lantschouensis were identified and characterized by small RNA deep sequencing.The significant differences in the expression of miRNAs were analyzed.The results showed that length of the small RNA reads from males,queens,and workers was distributed between 18 and 30 nt,with a peak at 22 nt.A total of 296 known and 65 novel miRNAs were identified from the ovaries of the different status queens.The egg-laying queen vs.non-egg-laying queen comparisons identified fifteen miRNAs with significant differences in expression,included five up-regulated miRNAs and ten down-regulated miRNAs in the egg-laying queen.The expression levels of DE miRNAs obtained by RT-qPCR were consistent with the read counts obtained through Solexa sequencing.In this study,the analysis of miRNAs in the three castes of B.lantschouensis head and queen of B.lantschouensis ovary were predicted.There were significant differences in the expression of miRNAs among different castes or queens of different reproductive states.These differentially expressed miRNAs were mainly involved in the regulation of developmental and reproductive pathways.The results of this study lay a foundation for further study on the function of miRNAs of B.lantschouensis and the molecular regulation mechanism.