ame-miR-34 Modulates the Larval Body Weight and Immune Response of Apis mellifera Workers to Ascosphara apis Invasion

Abstract

MiRNAs are critical regulators of numerous physiological and pathological processes. Ascosphaera apis exclusively infects bee larvae and causes chalkbrood disease. However, the function and mechanism of miRNAs in the bee larval response to A. apis infection is poorly understood. Here, ame-miR-34, a previously predicted miRNA involved in the response of Apis mellifera larvae to A. apis invasion, was subjected to molecular validation, and overexpression and knockdown were then conducted to explore the regulatory functions of ame-miR-34 in larval body weight and immune response. Stem-loop RT-PCR and Sanger sequencing confirmed the authenticity of ame-miR-34 in the larval gut of A. mellifera. RT-qPCR results demonstrated that compared with that in the uninfected larval guts, the expression level of ame-miR-34 was significantly downregulated (p < 0.001) in the guts of A. apis-infected 4-, 5-, and 6-day-old larvae, indicative of the remarkable suppression of host ame-miR-34 due to A. apis infection. In comparison with the corresponding negative control (NC) groups, the expression level of ame-miR-34 in the larval guts in the mimic-miR-34 group was significantly upregulated (p < 0.001), while that in the inhibitor-miR-34 group was significantly downregulated (p < 0.01). Similarly, effective overexpression and knockdown of ame-miR-34 were achieved. In addition, the body weights of 5- and 6-day-old larvae were significantly increased compared with those in the mimic-NC group; the weights of 5-day-old larvae in the inhibitor-miR-34 group were significantly decreased in comparison with those in the inhibitor-NC group, while the weights of 4- and 6-day-old larvae in the inhibitor-miR-34 group were significantly increased, indicating the involvement of ame-miR-34 in modulating larval body weight. Furthermore, the expression levels of both hsp and abct in the guts of A. apis-infected 4-, 5-, and 6-day-old larvae were significantly upregulated after ame-miR-34 overexpression. In contrast, after ame-miR-34 knockdown, the expression levels of the aforementioned two key genes in the A. apis-infected 4-, 5-, and 6-day-old larval guts were significantly downregulated. Together, the results demonstrated that effective overexpression and knockdown of ame-miR-34 in both noninfected and A. apis-infected A. mellifera larval guts could be achieved by the feeding method, and ame-miR-34 exerted a regulatory function in the host immune response to A. apis invasion through positive regulation of the expression of hsp and abct. Our findings not only provide a valuable reference for the functional investigation of bee larval miRNAs but also reveal the regulatory role of ame-miR-34 in A. mellifera larval weight and immune response. Additionally, the results of this study may provide a promising molecular target for the treatment of chalkbrood disease.

Expression Profile, Regulatory Network, and Putative Role of microRNAs in the Developmental Process of Asian Honey Bee Larval Guts

“…In addition, we observed that miR-6001-y putatively targeted as many as 2042 mRNAs, involving Wnt, Hippo, and Jak-STAT signaling pathways, suggesting the involvement of miR-6001-y in adjusting these three signaling pathways of great importance. Additional work such as overexpression and knockdown of miR-6001-y in the larval guts via feeding method, following our previously established protocol [ 36 ], is required to decipher the function and action mechanism of miR-6001-y.…”

“…Findings from this work and our previous studies demonstrate that miR-6001 as a hub within ceRNA regulatory networks likely exerts a critical function in modulating the larval guts’ growth and development and workers’ midguts in both A. c. cerana and A. m. ligustica . Recently, we established the platforms for dissecting the functions of miRNA [ 36 ] and lncRNA [ 69 ] in bee larval guts. Hence, to identify candidate lncRNAs and circRNAs targeted by ace-miR-6001 and investigate the regulatory functions of the lncRNA-ace-miR-6001 axis and circRNA-ace-miR-6001 axis in the process of the development of larval guts is an interesting and promising direction in the near future.…”

“…Next, reverse transcription was performed with stem-loop primers, and the resulting cDNA was used as template for PCR. On basis of the method described by Wu et al [ 36 ], PCR amplification was conducted and subjected to 1.8% agarose gel electrophoresis, followed by extraction of the expected fragment, ligation to the pESI-T vector (Yeasen, China), and transformation into Escherichia coli DH5α-competent cells. After microbial PCR, 1 mL of bacterial solution with a positive signal was sent to Sangon Biotech Co., Ltd. (Shanghai, China) for single-end Sanger sequencing.…”

Systematic Characterization and Regulatory Role of lncRNAs in Asian Honey Bees Responding to Microsporidian Infestation

“…In summary, the results demonstrated that TCONS_00032996, TCONS_00032996, and TCONS_00035424 in the workers’ midguts were likely to competitively bind to miR-252 and miR-60 to modulate the mRNA surveillance pathway and DNA replication and repair, further mediating the host response to the infestation by N. ceranae . Recently, we established the technical platform for the functional investigation of honey bee lncRNAs and miRNAs [ 58 , 59 ]. In the near future, we will conduct RNAi of these promising candidate DElnRNAs mentioned above to further explore their ceRNA functions in the response of A. c. cerana workers’ midguts to N. ceranae invasion.…”

CircRNAs and their regulatory networks associated with the antioxidant enzymes and immune responses of Asian honey bee larvae to fungal infection

“…This suggested that the DEcircRNA-mediated ceRNA network is potentially engaged in regulating the response of A. c. cerana worker larvae to A. apis infection. In the near future, functional dissection of key DEcircRNAs (e.g., RNAi via feeding siRNA targeting back-splicing sites) [71] and miRNAs (overexpression and knockdown via feeding the mimic and inhibitor) [72] will be conducted to explore the ceRNA metabolism underlying the A. c. cerana larval response to A. apis invasion.…”