ame-miR-5119- Eth axis modulates larval-pupal transition of western honeybee worker

Abstract

The miRNA plays a key role in the regulation of hormone signaling in insects. The pathways by which miRNAs affect hormone levels are unclear in the honeybee (Apis mellifera), an indispensable pollinator in nature. In this study, ame-miR-5119 was overexpressed and knocked down in larvae by feeding mimics and inhibitors, respectively, and we determined that ame-miR-5119 regulates hormone signaling through the target gene ecdysis triggering hormone (Eth), which affects the larval-pupal transition of workers. The results showed that ame-miR-5119 with a length of 19 nt targets six genes related to the hormone pathway. We focused on Eth and found that ame-miR-5119 and Eth exhibited reverse expression patterns during the transition from larval to pupal stages in workers. Dual luciferase assay confirmed the negative regulatory between ame-miR-5119 and Eth. Overexpression of ame-miR-5119 decreased the mRNA level of Eth, and the Eth receptor (Ethr) expression was not significantly affected, but the expression levels of juvenile hormone (JH) pathway related genes juvenile hormone acid methyltransferase (Jhamt) and Krüppel homolog 1 (Kr-h1) were significantly reduced. In contrast, knockdown of ame-miR-5119 increased the mRNA level of Eth, and the expression of Ethr, Jhamt and Kr-h1 was significantly upregulated. ame-miR-5119 did not affect larval body weight. The number of larvae overexpressing ame-miR-5119 survived in the prepupal stage was lower than that in the control group, and the number of pupations reduced at 11-day-old. The number of larvae that knocked down ame-miR-5119 survived in the prepupal stage was significantly higher than that in the control group, and the number of pupations increased at 11-day-old. These results indicated that ame-miR-5119 negatively regulates the expression of Eth, indirectly inhibits the expression of Ethr, Jhamt, and Kr-h1, and affects the JH biosynthesis, thereby preventing the metamorphic transition from larva to pupa in worker bees. These findings provide evidence that the miRNA regulation of hormone levels in honey bees.

Keywords: Apis mellifera; ame-miR-5119; hormone; metamorphosis development; microRNA.

FIGURE 1

Molecular validation and regulatory network of ame-miR-5119. (A) Detection of the amplification products from ame-miR-5119 by stem-loop RT-PCR, Lane M: DNA marker, Lane1-3: 4-, 5-, and 6-day-old larvae; (B) Peak diagram of the signal fragment amplified from stem-loop RT-PCR; (C) Regulatory network between ame-miR-5119 and 6 target genes. The mRNAs XM_006564318.2, XM_006564319.2, XM_006564320.2 and XM_016914663.1 are ecdysone-induced protein 75 gene (E75); The mRNA NM_001242435.1 are ecdysoneless gene (ecd); NM_001142607.1 are Eth gene.

FIGURE 2

Expression pattern determination and binding relationship confirmation of ame-miR-5119 and Eth. Relative expression levels of ame-miR-5119 (A) and Eth gene (B) in A. mellifera worker larva (3-day-old), prepupa (7-day-old and 8-day-old), and pupa (12-day-old). Two-way ANOVA, Tukey’s multiple comparisons, n = 3. (C) pmirGLO vector construction model for dual luciferase analysis, and Sanger sequencing of the Eth mutated binding sites. (D) Dual-luciferase reporter assay of the binding relationship between ame-miR-5119 and Eth. One-way ANOVA, n = 3. Different letters above bars indicate groups that are statistically significant (p < 0.05).

FIGURE 3

Effect of ame-miR-5119 overexpression and knockdown on Eth expression in A. mellifera worker larvae. (A, B) Relative expression level of ame-miR-5119 in the larva after feeding mimic-miR-5119 and inhibitior-miR-5119. (C) A schematic diagram of the target binding site between ame-miR-5119 and Eth. (D, E) RT-qPCR detection of the expression level of Eth in the 4-, 5-, and 6-day-old larvae after ame-miR-5119 overexpression or knockdown. Two-way ANOVA, Tukey’s multiple comparisons, n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, non-significant.

FIGURE 4

Effect of overexpression and knockdown of ame-miR-5119 on the (A) mellifera larvae. (A) The body weight of A. mellifera worker after ame-miR-5119 overexpression and knockdown. Multiple t tests, n = 3, **, p < 0.01. (B, C) The number of pupae after ame-miR-5119 overexpression and knockdown (n = 34–50). (D, E) The survival number of pupae after ame-miR-5119 overexpression and knockdown (n = 72). Two-way ANOVA, Tukey’s multiple comparisons. *, p < 0.05; ns, non-significant.

FIGURE 5

Relative expression levels of Ethr (A, B), Jhamt (C, D) and Kr-h1 (E, F) genes in A. mellifera worker larvae after ame-miR-5119 overexpression and knockdown. Unpaired t-test, n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, non-significant.

FIGURE 6

A speculated model of ame-miR-5119 regulating larval metamorphosis through Eth. The ame-miR-5119 negatively regulates the expression of Eth, indirectly inhibits the expression of Ethr, Jhamt, and Kr-h1, affects the JH biosynthesis, thereby prevents the metamorphosis of A. mellifera larvae.