Differential expression patterns and potential regulatory function of piRNAs in western honey bee larval response toAscosphaera apisinfestation
Authors: Sun Minghui , Long Qi , Fan Xiaoxue , Wang Zixin , Zhang Kaiyao , Wang Siyi , Gao Xuze , Guo Sijia , Niu Qingsheng , Jiang Haibing , Chen Dafu , Fu Zhongmin , Guo Rui 
Published: December 2022

piRNAs are a class of small non-coding RNAs that play an essential part in genomic defense, as well as in modulation of gene expression and diverse biological processes such as host–pathogen interaction. However, little is known about the expression pattern of the regulatory function of piRNAs in interactions between honey bees and pathogens. In this work, on the basis of our previously obtained high-quality small RNA-seq datasets from western honey bee (Apis mellifera) larval guts, the differential expression profile of piRNAs inA. melliferalarval guts afterAscosphaera apisinfestation was analyzed, followed by structural characterization, target prediction, and regulatory network investigation. The potential roles of differentially expressed piRNAs (DEpiRNAs) in regulating host response, especially immune response, were further discussed. In this study, 504, 657, and 587 piRNAs were respectively identified in the 4-, 5-, and 6-day-old larval guts infected byA. apis, with 411 (53.24%) piRNAs shared. The length distribution of these piRNAs ranged from 24 nt to 33 nt and their first base had a “C” bias, similar to piRNAs discovered in other mammals and insects. Additionally, 96, 103, and 143 DEpiRNAs were detected in the 4-, 5-, and 6-day-old comparison groups; among these, piR-ame-149736, piR-ame-1066173, and piR-ame-1125190 were the most up-regulated, while piR-ame-1202932 was the most down-regulated. There were 68 DEpiRNAs shared between these three comparison groups. The targets of the DEpiRNAs in the three comparison groups were engaged in a suite of crucial functions associated with biological processes, molecular function, and cellular components, including molecular transducer activity, biological regulation, and membrane part. These targets were also relevant to diverse vital pathways such as the phosphatidylinositol signaling system, inositol phosphate metabolism, and Wnt signaling pathway. Further investigation demonstrated that targets of DEpiRNAs were involved in three energy metabolism-related pathways, seven development-associated signaling pathways, and seven immune-relevant pathways, including lysosome and endocytosis, as well as the MAPK and Jak-STAT signaling pathways. The expression trends of five randomly selected DEpiRNAs were verified using a combination of RT-PCR and RT-qPCR. Moreover, the expression levels of six genes targeted by piR-ame-945760 were detected by RT-qPCR, with the results showing that their expression trends were the same as the expression trend of piR-ame-945760, indicative of the positive correlation between piR-ame-945760 and these targets. These results suggest thatA.apisinfestation increased the overall expression level of piRNAs and altered the expression pattern of piRNAs inA. melliferalarval guts. DEpiRNAs potentially participate in theA. apisresponse of the host by modulating the expression of target genes associated with energy metabolism and development, as well as cellular and humoral immune response. Our findings not only offer novel insights intoA. melliferalarva–A. apisinteraction, but also lay the groundwork for clarifying the mechanisms underlying DEpiRNA-regulated larval response.