DOI: 10.5061/dryad.44j0zpcsc
title: Data from: Ame-miR-2161 affects the survival and development of honeybee larvae through the juvenile hormone acid methyltransferase gene

description: 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.
description: <strong>Experimental Procedures</strong>
 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 &lt; 0.05; **, P &lt; 0.01; ***, P &lt;
 0.001; ns, not significant. Log-rank (Mantel-Cox) test for
 survival.
description: # Data from: Ame-miR-2161 affects the survival and development of honeybee
 larvae through the juvenile hormone acid methyltransferase gene Dataset
 DOI: [10.5061/dryad.44j0zpcsc](10.5061/dryad.44j0zpcsc) ## Description of
 the data and file structure We have submitted our raw data. We organized
 the information in the order shown in the figure, including the original
 gel image (Fig. 1A), the results of gene expression detection, as well as
 the survey data such as survival rate (%) and body weight (Unit: g). ###
 Descriptions Name: Fig.1A Original image mir2161 * The original gel image
 of ame-miR-2161 Name: Fig.1B all targets and 22 targets * All the target
 genes of ame-miR-2161 and the top 22 selected target genes Name: Fig. 2A
 The expression level of miR2161 * The expression level of ame-miR-2161
 Name: Fig. 2B The expression level of jhamt * The expression level of
 juvenile hormone acid methyltransferase(jhamt) Name: Fig. 2C Jhant-wt and
 Fig. 2C Jhant-mutant * The dual luciferase assay was used to verify the
 target sequences of the relationship between mir2161 and Jhamt: wild-type
 and mutant sequences Name: Fig. 2D Dual-Luciferase assay * The dual
 luciferase assay was used to verify the target sequences of the
 relationship between mir2161 and Jhamt Name: Fig. 3A and C The expression
 level of miR2161 after miR2161 mimic or inhibitor treatment * The
 expression level of miR2161 after miR2161 mimic or inhibitor treatment
 Name: Fig. 3B andD The expression level of jhamt after miR2161 mimic or
 inhibitor treatment * The expression level of jhamt after miR2161 mimic or
 inhibitor treatment Name: Fig. 4A and C JH titer * juvenile
 hormone titer after miR2161 mimic or inhibitor treatment Name: Fig. 4B and
 D The expression level of kr-h1 after miR2161 mimic or inhibitor treatment
 * The expression level of kr-h1 after miR2161 mimic or inhibitor treatment
 Name: Fig. 5A and C survival rate * The survival rate after miR2161 mimic
 or inhibitor treatment (Unit: %) Name: Fig. 5B and D pupation rate * The
 pupation rate after miR2161 mimic or inhibitor treatment (Unit: %)
 Name: Fig. 5E body weight * The body weight after miR2161 mimic or
 inhibitor treatment  (Unit: g) ### Files and variables #### File:
 Fig.1A_Original_image_mir2161.tif **Description:** The original gel image
 of ame-miR-2161 #### File: Fig._2C_Jhant-mutant.ab1 **Description:** The
 dual luciferase assay was used to verify the target sequences of the
 relationship between mir2161 and Jhamt: wild-type and mutant sequences
 #### File: Fig._2C_Jhant-wt.ab1 **Description:** The dual luciferase assay
 was used to verify the target sequences of the relationship between
 mir2161 and Jhamt: wild-type and mutant sequences #### File:
 Fig._2A_The_expression_level_of_miR2161.csv
 **Description:** The_expression_level_of_miR2161 ##### Variables * gene:
 name miR2161 * 4 day-1: 4 days Repeat 1 * 4 day-2: 4 days Repeat 2 * 4
 day-3: 4 days Repeat 3 * 5 day-1: 5 days Repeat 1 * 5 day-2: 5 days Repeat
 2 * 5 day-3: 5 days Repeat 3 * 6 day-1: 6 days Repeat 1 * 6 day-2: 6
 days Repeat 2 * 6 day-3: 6 days Repeat 3 #### File:
 Fig._2B_The_expression_level_of_jhamt.csv
 **Description:** The_expression_level_of_jhamt ##### Variables * Gene:
 name jhamt * 4 day-1: 4 days Repeat 1 * 4 day-2: 4 days Repeat 2 * 4
 day-3: 4 days Repeat 3 * 5 day-1: 5 days Repeat 1 * 5 day-2: 5 days Repeat
 2 * 5 day-3: 5 days Repeat 3 * 6 day-1: 6 days Repeat 1 * 6 day-2: 6
 days Repeat 2 * 6 day-3: 6 days Repeat 3 #### File:
 Fig._2D_Dual-Luciferase_assay.csv **Description:** The dual luciferase
 assay was used to verify the target sequences of the relationship between
 mir2161 and Jhamt ##### Variables * NC-jhamt-wt: mimic-NC group  jhamt WT
 squence * mimic2161-jhamt-wt: mimic2161 group  jhamt WT squence *
 NC-jhamt-mut:mimic-NC group  jhamt mutant squence *
 mimic2161-jhamt-mut: mimic2161 group  jhamt mutant squence #### File:
 Fig.1B_all_targets_and_22_targets.csv **Description:** miR2161
 all_targets_and_22 top targets ##### Variables * miRNA: name * target:
 name * target_len: DNA length * target_start:  DNA length start site *
 hairpin_energe(kcal/mol):  binding energy #### File:
 Fig._3A_and_C_The_expression_level_of_miR2161_after_miR2161_mimic_or_inhibitor_treatment.csv **Description:** The_expression_level_of_miR2161_after_miR2161_mimic_or_inhibitor_treatment ##### Variables * 6 days mimic-nc-1: 6 days Repeat 1 of mimic-nc treatment * 6 days mimic-nc-2: 6 days Repeat 2 of mimic-nc treatment * 6 days mimic-nc-3: 6 days Repeat 3 of mimic-nc treatment * 6 days 2161mimic-1: 6 days Repeat 1 of 2161mimic treatment * 6 days 2161mimic-2: 6 days Repeat 2 of 2161mimic treatment * 6 days 2161mimic-3: 6 days Repeat 3 of 2161mimic treatment * 5 days mimic-nc1: 5 days Repeat 1 of mimic-nc treatment * 5 days mimic-nc2: 5 days Repeat 2 of mimic-nc treatment * 5 days mimic-nc3: 5 days Repeat 3 of mimic-nc treatment * 5 day 2161mimic1: 5 days Repeat 1 of 2161mimic treatment * 5 day 2161mimic2: 5 days Repeat 2 of 2161mimic treatment * 5 day 2161mimic3: 5 days Repeat 3 of 2161mimic treatment * 4 days mimic-nc1: 4 days Repeat 1 of mimic-nc treatment * 4 days mimic-nc2: 4 days Repeat 2 of mimic-nc treatment * 4 days mimic-nc3: 4 days Repeat 3 of mimic-nc treatment * 4 days 2161mimic1: 4 days Repeat 1 of 2161mimic treatment * 4 days 2161mimic2: 4 days Repeat 2 of 2161mimic treatment * 4 days 2161mimic3: 4 days Repeat 3 of 2161mimic treatment #### File: Fig._3B_andD_The_expression_level_of_jhamt_after_miR2161_mimic_or_inhibitor_treatment.csv **Description:** The_expression_level_of_jhamt_after_miR2161_mimic_or_inhibitor_treatment ##### Variables: * The same as the above description #### File: Fig._4A_and_C_JH_titer.csv **Description:**  JH_titer detection ##### Variables none #### File: Fig._4B_and_D_The_expression_level_of_kr-h1_after_miR2161_mimic_or_inhibitor_treatment.csv **Description:** The_expression_level_of_kr-h1_after_miR2161_mimic_or_inhibitor_treatment ##### Variables * The same as the above description #### File: Fig._5A_and_C_survival_rate.csv **Description:** survival_rate after_miR2161_mimic_or_inhibitor_treatment (Unit: %) ##### Variables * Day-m: mimic treatment days * mimic: mimic2161 * mimiicNC: mimic-nc * Day-i: inhibitor treatment days * inhibitor: inhibitor2161 * inhibitorNC: inhibitor-NC * Unit: Percentage (%) #### File: Fig._5B_and_D_pupation_rate.csv **Description:** pupation_rate after_miR2161_mimic_or_inhibitor_treatment (Unit: %) ##### Variables * The same as the above description * Unit: Percentage (%) #### File: Fig._5E_body_weight.csv **Description:** body_weight after_miR2161_mimic_or_inhibitor_treatment (Unit: g) ##### Variables * The same as the above description * Unit: gram (g) ## Code/software Microsoft Excel Chromas ## Access information Other publicly accessible locations of the data: * none Data was derived from the following sources: * none