MicroRNAs (miRNAs), as endogenous small non-coding RNAs, play a role in regulating the growth and development processes of insects. However, there are still few reports on the coordinated regulation of honeybee development processes by miRNAs and hormones. In this study, the regulatory network of target genes of Apis mellifera miRNA-2161 (ame-miR-2161) was constructed. The association between ame-miR-2161 and the survival and development of workers larvae was analyzed. The results showed that ame-miR-2161 potentially targets 22 mRNAs, focusing on target gene juvenile hormone acid methyltransferase (Jhamt), which is a key rate-limiting enzyme in the final step of the juvenile hormone synthesis pathway. RT-qPCR revealed that the expression of ame-miR-2161 and Jhamt had the same trend during the larval stage. Dual-luciferase assays confirmed the targeting relationship between ame-miR-2161 and Jhamt. Overexpression of ame-miR-2161 increased the mRNA level of Jhamt, which led to significantly increased juvenile hormone (JH) titer in 4- to 6-day-old larvae, and the expression of the JH signaling downstream response gene Kr-h1 also gradually increased. Conversely, inhibition of ame-miR-2161 decreased the mRNA level of jhamt, significantly reduced JH titer in larvae, and markedly declined the expression of Kr-h1. This indicates that ame-miR-2161 positively regulates the expression of Jhamt. Further findings showed that overexpression of ame-miR-2161 improved larval survival rate, while inhibition of ame-miR-2161 reduced larval survival rate. Changes in ame-miR-2161 did not affect the pupation rate of larvae but influenced changes in larval body weight. The results suggest that ame-miR-2161 affects JH levels by positively regulating the expression of Jhamt, participating in the regulation of the survival and development of honeybee larvae. These findings provide important information for interpreting the regulation of miRNA on hormone levels and metamorphic development in honeybees.

Experimental Procedures

Honeybee larvae rearing

The A. mellifera worker larvae were reared in the apiary of the bee protection group at the College of Bee Science and Biomedicine, Fujian Agriculture and Forestry University. We selected Apis mellifera ligustica colonies with strong colony vigor and healthy individual bees. The queen was restricted from laying eggs in the empty comb for 6 h and then removed. After the eggs hatched, 2-day-old larvae (determined by size) were transferred onto a 6-well plate using a clean grafting needle and fed with 50 μL of pre-prepared artificial diet (63% royal jelly, 30% purified water, 6% honey, and 1% yeast extract), then reared them under conditions of 35±0.5°C and 90% relative humidity (RH). Uniform-sized 3-day-old larvae were selected and individually transferred to a 48-well plate for cultivation, with the artificial diet replaced every 24 hours.

Sample collection and RNA extraction

The 4- to 6-day-old worker larvae were collected (three larvae as one biological sample, with three biological sample replicates). The samples were flash-frozen in liquid nitrogen and stored at –80°C for detecting the expression profiles of ame-miR-2161 and Jhamt. Additionally, 4- to 6-day-old worker larvae treated with miR-2161 mimic or miR-2161 inhibitor were collected (three larvae as one biological sample, with three biological sample replicates), rapidly frozen in liquid nitrogen and stored at –80°C for detecting the expression of ame-miR-2161, Jhamt, and Kr-h1. Total RNA was extracted using the SteadyPure Quick RNA Extraction Kit (Accurate Biotechnology, China). Briefly, frozen larvae were ground in liquid nitrogen, and 300 μL of lysis buffer was added to each sample. The homogenates were mixed thoroughly and incubated for 10 minutes to ensure complete lysis. Samples were then centrifuged at 12,000 rpm for 5 min at 4°C. The supernatant was transferred to a new tube and mixed with 500 μL of absolute ethanol, followed by gentle mixing for 2 min. The mixture was applied to Quick RNA Mini Columns and centrifuged at 12,000 rpm for 2 min. DNase I solution was added directly to the column matrix and incubated for 15 min at room temperature. After DNase digestion, the column was washed with 700 μL of Buffer QWB and centrifuged at 12,000 rpm for 1 min. The flow-through was discarded, and a second centrifugation step was performed under the same conditions to remove residual ethanol. RNA was eluted with 50 μL of RNase-free water, and the eluate was collected. RNA concentration and purity were determined using a spectrophotometer, and all RNA samples were stored at –80°C until further use.

miRNA cDNA synthesis and cloning

According to the nucleic acid sequence of ame-miR-2161 (Xiong et al., 2018), specific stem-loop primers were designed (Table S2). Refer to the method of Dong et al. (2024). Briefly, the total RNA of 4- to 6-day-old worker larvae was extracted using an RNA extraction kit (Promega, Madison, USA). Then, cDNA was obtained through reverse transcription with stem-loop primers. With the cDNA as a template, amplification was carried out using PCR mix (Vazyme, Nanjing, China). The reaction conditions were set as follows: pre-denaturation at 95°C for 1 min, denaturation at 95°C for 10 s, annealing at 61°C for 30 s, and extension at 72°C for 30 s, with a total of 35 cycles. The PCR products were detected by 1.5 % agarose gel electrophoresis. The recovered target fragments were ligated to the pClone 007 vector (Tsingke, Tianjing, China) and then transformed into E. coli DH5α (Tiangen, Beijing, China). Cultured at 37°C for 10 h on LB agar medium containing 50 μg/mL ampicillin. Subsequently, single colonies were picked and transferred to LB liquid medium containing 50 μg/mL ampicillin for shaking culture. The bacterial cultures were taken for PCR identification, and the positive clone samples were sent to Shanghai Sangon Biotech Co., Ltd (Shanghai, China). for sanger sequencing.

RT-qPCR

Part of the total RNA of 4- to 6-day-old worker larvae samples was reverse transcribed using stem-loop primers, and the obtained cDNA templates were used to detect the expression of ame-miR-2161. Another part was reverse transcribed using the HiScript® III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China), and the obtained cDNA was used as a template for detecting the expression of other genes. According to the instructions of the Hifair® qPCR SYBR Green Master Mix (Low Rox Plus) (Yeasen, Shanghai, China) kit, qPCR was performed on the QuantStudio 3 (ABI, Florida, USA). The reaction system (20 μL) consisted of 10 μL of Hifair® qPCR SYBR Green Master Mix, 1 μL each of the forward and reverse primers (2.5 μmol/L), 1 μL of the cDNA template and 7 μL of DEPC water. The reaction conditions were set as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 10 s, annealing and extension at 60°C for 30 s, with a total of 42 cycles. The melting curve program is the default setting of the system. The relative expression levels of genes were calculated using the 2−ΔΔCt method. The experiment was repeated three times using three independent biological samples. U6 (GenBank ID: LOC725641) was used as an internal reference for ame-miR-2161, while Actin (GenBank ID: NM_001185145) was used as an internal reference for the Jhamt and Kr-h1 genes. The primers for quantitative PCR are shown in Table S2.

Dual-Luciferase assay

The potential binding site between Jhamt and ame-miR-2161 was predicted using RNA hybridization software (v.2.1.2), located at positions 1189 to 1208 (20 bp) in the 3'UTR of Jhamt mRNA. We amplified the region spanning positions 1180 to 1217 (38 bp) of the Jhamt mRNA 3'UTR, which includes the binding site, and named Jhamt-wt. The design principle for the mutant fragment involved mutating all bases of the target site to non-complementary bases (e.g., G to T, T to C), and then synthesized by PCR amplification and named Jhamt-mutant. The binding site-specific primers are shown in Table S2. The amplified fragments were cloned into the pmirGLO vector and named Jhamt-wt and Jhamt-mutant (Jhamt-mt), respectively. For the bacterial liquid with correct sequencing results, plasmids were extracted using the Endotoxin Removal Plasmid Extraction Kit (Zongjin, Beijing, China).

Mammalian HEK-293T cells were cultured until the cell density reached 90–95%. Cell transfection experiments were carried out by following the instructions for the Hieff Trans™ Liposomal Nucleic Acid Transfection Reagent (Yeasen, Shanghai, China), and 4 transfection groups were set up at the same time: (1) Co-transfection of miR-2161 mimic and Jhamt-wt; (2) Co-transfection of mimic-NC and Jhamt-wt; (3) Co-transfection of miR-2161 mimic and Jhamt-mut; (4) Co-transfection of mimic-NC and Jhamt-mut. After the transfection was completed, the cell culture plates were incubated in a 37°C incubator for 24 h. According to the methods described by Matsumura et al. (2022) and Wang et al. (2023), the activities of firefly luciferase and Renilla luciferase were detected using the Dual-Luciferase Reporter Assay System (Promega, Madison, USA) and the Dual-Luciferase Detection Kit (Yeasen, Shanghai, China). The relative expression folds were obtained by calculating the ratio of firefly fluoresceinase/Renilla fluoresceinase. The experiment was repeated three times.

miRNA-mimic/inhibitor feeding

The miR-2161 mimic and miR-2161 inhibitor as well as the corresponding negative controls (mimic-NC and inhibitor-NC) were synthesized by GenePharma company in Shanghai. The primers are shown in Table S3. According to the method described previously (Dong et al., 2024), 2-day-old larvae of A. mellifera were carefully transferred to 6-well culture plates with 800 μL of artificial diet, 40 larvae per well, and then the plates were placed in a constant temperature and humidity incubator (35 ± 0.5°C, RH 90 %) for 24 h. 3-day-old larvae were transferred to 48-well culture plates, each larva was fed 50 μL of artificial diet containing miR-2161 mimic, mimic-NC, miR-2161 inhibitor or inhibitor-NC (40 pmol/g). Every 24 h thereafter, 50 μL of diet was added to each well until 6 days old. 4-, 5-, and 6-day-old larvae (n=3) were collected into sterile and RNA-free microcentrifuge tubes. They were immediately frozen in liquid nitrogen and subsequently stored at –80°C for future use. The effects of overexpressing and inhibiting ame-miR-2161 in 4- to 6-day-old larvae were evaluated by RT-qPCR.

Juvenile hormone titers assay

The 4- to 6-day-old worker larvae samples treated with miR-2161 mimic or miR-2161 inhibitor were collected (three larvae as one biological sample, with three biological sample replicates), flash-frozen in liquid nitrogen, and stored them at −80°C. Samples were mashed with 500 µL of phosphate-buffered saline (PBS), centrifuged at 3000 rpm for 10 min, and the supernatant was collected for JH determination. JH determination used JH III Elisa kit (mlbio, ml062708, China) according to the instructions. Briefly, the samples, standards and HRP (Horseradish Peroxidase)-labeled antibodies were added in turn to the coating micropores of the pre-coated juvenile hormone (JH) antibody, and then incubated in a 37°C water bath for 60 min. Subsequently, discarded the liquid, dried it on absorbent paper, added 200 µL of washing solution for 1 min, removed the washing solution, patted dry on absorbent paper, and repeated five times. After thorough washing, TMB (Tetramethylbenzidine) substrate was added for color development, and incubated at 37°C in the dark for 15 min. then added 50 μL of the stop solution. The absorbance was measured by a microplate reader (Molecular Devices, CMax Plus, China) at a wavelength of 450 nm, and the concentration of insect juvenile hormone (JH) in the sample was calculated by the standard curve. The level of juvenile hormone (JH) in the specimens was determined by the double antibody sandwich method.

Monitoring the pupation rate, survival, and weight of in vitro reared honeybee workers

The 2-day old larvae (n=27) were fed with diets containing miR-2161 mimic, mimic-NC, miR-2161 inhibitor and inhibitor-NC (40 pmol/g). The diets were replaced every 24 h until the 12th day of age. The number of pupae and surviving larvae was continuously observed and counted.

Larvae aged 4- to 6-day-old from the miR-2161 mimic, mimic-NC, miR-2161 inhibitor, and inhibitor-NC groups were collected, respectively. The larvae were repeatedly rinsed with PBS to remove the residual feed on their body surfaces, and the liquid on the larvae's body surfaces was carefully blotted with filter paper. The larvae were weighed using the FA2004 ultra-precise electronic balance (Sunny Hengping, Shanghai, China). Three larvae were grouped, and the weighing was biologically repeated three times.

Construction of Regulatory Network

Based on the obtained high-quality transcriptome data (Fan et al., 2022), the target mRNA of ame-miR-2161 was predicted by combining RNAhybrid (v2.1.2)+svm-light (v6.01), Miranda (v3.3a) and TargetScan (v7.0) software (All are default parameters). Cytoscape (v3.6.1) software was used to construct and visualize the regulatory network between ame-miR-2161 and target genes.

Data analysis

GraphPad Prism version 8 (GraphPad, San Diego, USA) was used for graph construction and statistical analysis. Data are presented as the mean ± SD. Statistical analysis was performed using the one-way ANOVA or Two-way ANOVA, and Tukey’s multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. Log-rank (Mantel-Cox) test for survival.