Journal: Insects

Article Title: MicroRNA-281-X Modulates Self-Grooming Behavior in Honeybees by Targeting Tyrosine Decarboxylase 2 in the Octopaminergic Pathway

doi: 10.3390/insects17050522

Figure Lengend Snippet: Tissue-specific expression profile and interactions between miR-281-x and its target tdc2 in honeybees. ( A ) Relative expression levels of miR-281-x and tdc2 from eclosion to 21-day-old adult bees. The smooth curve was fitted using the LOWESS spline method to visualize the dynamic expression of miR-281-x and tdc2 ( n = 3 per group). ( B ) Distribution of miR-281-x and tdc2 gene expression in different tissues of honeybees ( n = 3 per group, Kruskal–Wallis test). Different lowercase letters (a, b, c) represent significant differences ( p < 0.05); ns denotes not significant ( p ≥ 0.05). ( C ) Dual luciferase reporter assays in wild and mutant types of tdc2 in vitro ( n = 6 per group). Statistical comparisons were performed using the Kruskal–Wallis test, followed by post hoc pairwise comparisons. Effect sizes (r) for pairwise comparisons. ** p < 0.01. ( D ) RNA immunoprecipitation (RIP) was performed with an anti-Ago-1 antibody, and fold enrichment was quantified relative to rabbit IgG control. qPCR analysis was performed to amplify tdc2 mRNA from the Ago-1 immunoprecipitates in brain tissue extracts treated with agomir-281-x compared with agomir-NC. Significant differences were determined by the Student’s t -test ( n = 3 per group, * p < 0.05; ** p < 0.01). ( E ) miR-281-x and tdc2 were co-labeled to determine the co-localization in the honeybee brain by fluorescence in situ hybridization (FISH). Where green ( tdc2 ) and red signals (miR-281-x) overlap, a yellow signal is observed, indicating the co-localization of miR-281-x and tdc2 . The images were visualized using a laser confocal microscope (Zeiss) at magnifications of ×12 (the left column) and ×40 (the right column), scale bars: 200 µm (left column) and 50 µm (right column).

Article Snippet: Briefly, digoxigenin-labeled miR-281-x and tdc2 DNA probes were designed using miRBase and NCBI BLASTN (performed by Servicebio, China) and synthesized by Servicebio.

Techniques: Expressing, Gene Expression, Luciferase, Mutagenesis, In Vitro, RNA Immunoprecipitation, Control, Labeling, Fluorescence, In Situ Hybridization, Microscopy

Journal: Insects

Article Title: MicroRNA-281-X Modulates Self-Grooming Behavior in Honeybees by Targeting Tyrosine Decarboxylase 2 in the Octopaminergic Pathway

doi: 10.3390/insects17050522

Figure Lengend Snippet: miR-281-x negatively regulates the expression of tdc2 . ( A ) Relative mRNA level ( n = 5 per group) and protein level ( n = 5 per group) of tdc2 in the brains of bees injected with agomir-281-x. ( B ) Relative mRNA level ( n = 5 per group) and protein level ( n = 5 per group) of tdc2 in the brains of bees injected with antagomir-281-x. ( C ) Relative mRNA level ( n = 6 per group) and protein level ( n = 5 per group) of tdc2 in the brains of MS and MW bees. The data are presented as the mean ± SEM; The asterisks outside the strip indicate the significant difference between controls and the treatments by Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001. Protein: Tdc2: 72 kDa; GAPDH: 36 kDa.

Article Snippet: Briefly, digoxigenin-labeled miR-281-x and tdc2 DNA probes were designed using miRBase and NCBI BLASTN (performed by Servicebio, China) and synthesized by Servicebio.

Techniques: Expressing, Injection, Stripping Membranes

Journal: Insects

Article Title: MicroRNA-281-X Modulates Self-Grooming Behavior in Honeybees by Targeting Tyrosine Decarboxylase 2 in the Octopaminergic Pathway

doi: 10.3390/insects17050522

Figure Lengend Snippet: miR-281-x controls octopamine signaling by targeting tdc2 modulates self-grooming behavior. ( A ) First grooming time (s) and total grooming bouts after injecting dstdc2 . Group differences among dsEGFP ( n = 88) and dstdc2 ( n = 83) bees were tested by the Mann–Whitney U test, ** p < 0.01. ( B ) Behavioral rescue assay was performed by injecting sitdc2 into honeybees pretreated with antagomir-281-x (the Mann–Whitney U test * p < 0.05, antagomir-281-x + siNC : n = 60; antagomir-281-x + sitdc2 : n = 49). ( C , D ) Concentrations of octopamine in the brains of bees ( n = 18 per group) injected with agomir-281-x and antagomir-281-x. ( E ) Concentrations of octopamine in the brains of bees ( n = 20 per group) with tdc2 RNAi knockdown. The data are presented as the mean ± SEM; The asterisks outside the strip indicate the significant difference between controls and the treatments by Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Briefly, digoxigenin-labeled miR-281-x and tdc2 DNA probes were designed using miRBase and NCBI BLASTN (performed by Servicebio, China) and synthesized by Servicebio.

Techniques: MANN-WHITNEY, Rescue Assay, Injection, Knockdown, Stripping Membranes

Journal: Insects

Article Title: MicroRNA-281-X Modulates Self-Grooming Behavior in Honeybees by Targeting Tyrosine Decarboxylase 2 in the Octopaminergic Pathway

doi: 10.3390/insects17050522

Figure Lengend Snippet: The miR-281-x– tdc2 –octopamine axis regulates self-grooming in A. mellifera . miR-281-x acts as a negative regulator by directly targeting tdc2 , a key enzyme for octopamine synthesis. High miR-281-x levels inhibit tdc2 translation and reduce octopamine, resulting in weak grooming. Conversely, low miR-281-x levels facilitate tdc2 expression and octopamine accumulation, driving intense self-grooming behavior.

Article Snippet: Briefly, digoxigenin-labeled miR-281-x and tdc2 DNA probes were designed using miRBase and NCBI BLASTN (performed by Servicebio, China) and synthesized by Servicebio.

Techniques: Expressing