Abstract: 【Aim】 The objective of this study is to elucidate the regulatory function of piR-ame-1186994 of Apis mellifera ligustica, so as to offer a scientific basis for further investigation of the underlying regulatory mechanism of piR-ame-1186994. 【Methods】 The expression and sequence authenticity of piR-ame-1186994 in the 6-day-old adult worker’s midgut, 12-day-old adult drone’s testis and 7-day-old adult queen’s ovary of A. m. ligustica were verified by Stem-loop RT-PCR and Sanger sequencing, respectively. Relevant software was utilized to predict the target mRNAs of piR-ame-1186994 followed by GO and KEGG database annotation. Regulatory networks related to developmental signaling pathways, energy metabolism pathways and cellular and humoral immune pathways were further constructed. Newly emerged adult workers were fed with mimic and mimic-NC (negative control) of piR-ame-1186994, followed by the detection of the relative expression levels of piR-ame-1186994 and its key target genes (YAP1 and PLD2) in the midguts of adult workers using RT-qPCR. 【Results】 The specific fragment of piR-ame-1186994 was amplified from the 6-day-old adult worker’s midgut, 12-day-old adult drone’s testis and 7-day-old adult queen’s ovary of A. m. ligustica. piR-ame-1186994 targeted 1 097 mRNAs, which could be annotated to 30 GO terms involved in metabolic process, binding, cell, etc., and 182 KEGG pathways including Wnt signaling pathway, endocytosis and oxytocin signaling pathway. Thirty-six and 16 target mRNAs were respectively involved in five developmental-related signaling pathways (mTOR, Wnt, Hippo, AMPK and Notch signaling pathways) and four pathways related to energy metabolism (amino sugar and nucleotide sugar metabolism, fructose and mannose metabolism, glycolysis/gluconeogenesis, and pentose phosphate pathway), respectively. Additionally, 29 and eight target mRNAs were engaged in four cellular immune pathways (lysosome, endocytosis, phagosome and ubiquitin-mediated protein degradation) and three humoral immune pathways (PI3K-Akt, MAPK and FoxO signaling pathways), respectively. In the mimic-piR group, the expression level of piR-ame-1186994 was significantly up-regulated in the 1-, 3- and 5-day-old workers’ midguts and up-regulated extremely significantly in the 2- and 4-day-old workers’ midguts, the expression level of the target gene YAP1 was extremely significantly down-regulated in the 1-, 3-, 4- and 5-day-old workers’ midguts and significantly down-regulated in the 2-day-old worker’s midgut, and the expression level of target gene PLD2 was significantly down-regulated in the 2-, 3- and 5-day-old workers’ midguts and downregulated extremely significantly in the 4-day-old worker’s midguts as compared with those in the mimic-NC group. 【Conclusion】 piR-ame-1186994 exists and expresses in the worker’s midgut, drone’s testes and queen’s ovary of A. m. ligustica. piR-ame-1186994 potentially modulates the development and immunity of worker’s midgut through targeting and negatively regulating the expression of YAP1 and PLD2.

Key words: Apis mellifera ligustica, midgut, ovary, testis, piRNA