Transcriptional dynamics and regulatory function of milRNAs in Ascosphaera apis invading Apis mellifera larvae

Abstract

In the present study, small RNA (sRNA) data from Ascosphaera apis were filtered from sRNA-seq datasets from the gut tissues of A. apis-infected Apis mellifera ligustica worker larvae, which were combined with the previously gained sRNA-seq data from A. apis spores to screen differentially expressed milRNAs (DEmilRNAs), followed by trend analysis and investigation of the DEmilRNAs in relation to significant trends. Additionally, the interactions between the DEmilRNAs and their target mRNAs were verified using a dual-luciferase reporter assay. In total, 974 A. apis milRNAs were identified. The first base of these milRNAs was biased toward U. The expression of six milRNAs was confirmed by stem-loop RT-PCR, and the sequences of milR-3245-y and milR-10285-y were validated using Sanger sequencing. These miRNAs grouped into four significant trends, with the target mRNAs of DEmilRNAs involving 42 GO terms and 120 KEGG pathways, such as the fungal-type cell wall and biosynthesis of secondary metabolites. Further investigation demonstrated that 299 DEmilRNAs (novel-m0011-3p, milR-10048-y, bantam-y, etc.) potentially targeted nine genes encoding secondary metabolite-associated enzymes, while 258 (milR-25-y, milR-14-y, milR-932-x, etc.) and 419 (milR-4561-y, milR-10125-y, let-7-x, etc.) DEmilRNAs putatively targeted virulence factor-encoded genes and nine genes involved in the MAPK signaling pathway, respectively. Additionally, the interaction between ADM-B and milR-6882-x, as well as between PKIA and milR-7009-x were verified. Together, these results not only offer a basis for clarifying the mechanisms underlying DEmilRNA-regulated pathogenesis of A. apis and a novel insight into the interaction between A. apis and honey bee larvae, but also provide candidate DEmilRNA-gene axis for further investigation.

Keywords: Apis mellifera; Ascosphaera apis; chalkbrood; infection mechanism; larva; milRNA; regulation.

Figure 1

Structural characteristics of A. apis milRNAs. (A) Length distribution of the milRNAs; (B) First nucleotide bias of the milRNAs of different lengths; (C) Base bias of the milRNAs at each nucleotide.

Figure 2

Molecular verification of A. apis milRNAs. (A) Agarose gel electrophoresis for the amplification products from stem–loop RT-PCR of six A. apis milRNAs and 5.8S rRNA; (B) Sanger sequencing of the amplified products from milR-3245-y and milR-10285-y.

Figure 3

Four significant profiles identified by STEM analysis. Colorful squares represent significant trends, while Black-and-White ones represent non-significant trends; the numbers located in the upper left of each square indicate the quantity of milRNAs within each trend, whereas those located in the lower left of each square indicate the p-value of each trend.

Figure 4

Top 20 GO terms significantly enriched by A. apis milRNA-targeted mRNAs. GO terms enriched by the target mRNAs of the A. apis milRNAs within profile2 (A), profile12 (B), profile16 (C), and profile17 (D).

Figure 5

Top 20 KEGG pathways significantly enriched by A. apis milRNA-targeted mRNAs. Pathways enriched by the target mRNAs of the A. apis milRNAs within profile2 (A), profile12 (B), profile16 (C), and profile17 (D).

Figure 6

Sub-regulatory network between the A. apis DEmilRNAs in the four significant trends and targets associated with virulence factors.

Figure 7

A. apis milRNAs target ADM-B and PKIA. (A,D) Relative expression level of milR-6882-x; (B) Relative expression level of ADM-B; (C) Dual-luciferase reporter assay of HEK293T cells co-transfected with milR-6882-x and pmirGLO vector containing target sequences of ADM-B; (D) Relative expression level of milR-7009-x; (E) Relative expression level of PKIA; (F) Dual-luciferase reporter assay of HEK293T cells co-transfected with milR-7009-x mimic and pmirGLO vector containing target sequences of PKIA.

Figure 8

A hypothetical working model of the DEmilRNA-mediated regulation in A. apis invading A. m. ligustica worker larvae.