An Insight Into the microRNA Profile of the Ectoparasitic Mite Varroa destructor (Acari: Varroidae), the Primary Vector of Honey Bee Deformed Wing Virus

Abstract

The remarkably adaptive mite Varroa destructor is the most important honey bee ectoparasite. Varroa mites are competent vectors of deformed wing virus (DWV), and the Varroa-virus complex is a major determinant of annual honey bee colony mortality and collapse. MicroRNAs (miRNAs) are 22-24 nucleotide non-coding RNAs produced by all plants and animals and some viruses that influence biological processes through post-transcriptional regulation of gene expression. Knowledge of miRNAs and their function in mite biology remains limited. Here we constructed small RNA libraries from male and female V. destructor using Illumina's small RNA-Seq platform. A total of 101,913,208 and 91,904,732 small RNA reads (>18 nucleotides) from male and female mites were analyzed using the miRDeep2 algorithm. A conservative approach predicted 306 miRNAs, 18 of which were upregulated and 13 downregulated in female V. destructor compared with males. Quantitative real-time PCR validated the expression of selected differentially-expressed female Varroa miRNAs. This dataset provides a list of potential miRNA targets involved in regulating vital Varroa biological processes and paves the way for developing strategies to target Varroa and their viruses.

Keywords: Varroa destructor; deformed wing virus; honey bee (Apis mellifera L.); microRNAs; small RNA-seq.

Figure 1

Small RNA sequence length distribution of microRNAs in male and female Varroa destructor (VD) mites. MicroRNAs are twenty-four (24) nucleotides in length.

Figure 2

(A) Basic stem-loop structure of a predicted microRNA. miRDeep2 was used to identify potential miRNA precursors based on nucleotide length, star sequence, stem-loop folding, and homology to the Varroa reference genome. Shown are the predicted stem-loop structures (yellow), star (violet), and mature sequences (red). (B) Annotation of predicted Varroa destructor microRNAs. 306 microRNAs were predicted in Varroa samples. 50 were categorized as high-confidence and 80 as low-confidence miRNAs based on standard criteria.

Figure 3

(A) In silico differential expression analysis of predicted microRNAs in female Varroa destructor relative to males. EdgeR was used for differential expression analysis. 13 predicted microRNAs were downregulated, 18 upregulated, and 60 were unaffected. (B) Differential expression of individual microRNAs in Varroa females relative to males. miRNAs with a log2 fold-change expression > |1| and FDR ≤ 0.1 were considered significantly differentially expressed with respect to male miRNAs (refer to Table 1 ).

Figure 4

(A) MicroRNA candidates predicted in this study, conserved in other arthropods, and validated in the available literature play significant roles in replication, harboring, and inhibition of viral pathogens. (B) qRT-PCR expression of potential microRNAs found in the present study in DWV-B-infected male and female Varroa mites. Succinate dehydrogenase (SDHA) was used as the housekeeping gene for normalization. Expression of these microRNAs is comparatively higher in female than male Varroa. Statistical significance for qRT-PCR-based differential expression was determined using the two-tailed Student’s t-test, where * denotes p < 0.05.

Figure 5

Summary of Varroa-derived female (A) and male (B) small RNA reads matching various small RNA categories.

Figure 6

(A) A network built exclusively from Varroa proteins targeted by predicted upregulated/downregulated miRNAs. (B) The total number of predicted target transcripts significantly upregulated and downregulated in female Varroa miRNAs relative to male miRNAs.

Figure 7

qPCR validation of selected mature miRNAs (high confidence) in female Varroa differentially expressed relative to male Varroa. Statistical significance for qRT-PCR-based differential expression was determined by the 2-tailed Student’s t-test, where * denotes p<0.05. miRNA expression of female Varroa was normalized to that of male Varroa (indicated as 1 on y-axis). Succinate dehydrogenase (SDHA) was used as a housekeeping control.

Figure 8

Gene ontology (GO)-derived biological processes related to genes targeted by upregulated/downregulated miRNAs in female V. destructor.