Micro RNAs are indispensable post-transcriptional regulators of gene expression involving various biological pathways, including cell growth, apoptosis, metamorphosis, development, and vector competence (Kloosterman and Plasterk 2006). However, little is known about the expression of miRNAs in parasitic mites such as V. destructor. Therefore, identifying and understanding the expression profiles of miRNAs in male and female Varroa will expand our fundamental knowledge of mite miRNAs and provide insight into the development and vector competence of Varroa. A conservative in silico approach identified 306 microRNAs, among which 80 were of low confidence and 50 of high confidence. Among these high confidence microRNAs 18 were up- and 13 were down regulated in female compared to male Varroa. Our qRT-PCR assay confirmed the up-regulation of Varroa miRNAs such as vde-miR-87-3p, vde-bantam-3p, vde-miR-375-3p, and vde-miR-34-5p, which have known roles in DENV replication, CHIKV infection, and immune response during DENV-2 infection in mosquitoes (Hussain et al. 2013; Liu et al., 2013; Yan et al., 2014; Maharaj et al., 2015; Lucas et al., 2015; Sempere et al., 2003). This small RNA dataset provides a new resource to characterize the function of miRNAs in mite biology. Other Varroa microRNAs predicted have also shown roles in development, pesticide resistance, metamorphosis, immune response, and reproduction (Fig. 4A, Table 2) in other arthropods. The new and novel microRNAs predicted in this study remain to be characterized using miRNA inhibitory assays.
Several of the predicted miRNAs identified in this study are also conserved in D. melanogaster (i.e. the assessed species along with V. destructor in this study) such as let-7-5p, bantam-3p, miR-8-3p, miR-34-5p, miR-263a-5p, miR-87-3p, miR-252-5p, miR-12-5p, miR-375-3p, miR-9a-3p, miR-306-5p, miR-133-3p, miR-6-3p, miR-276-a-3p, miR-1002-5p, miR-304-5p, and are known as having conserved roles in other arthropods as well. The bantam-3p targets the proapoptotic gene hid and is involved in several cellular processes such as cell proliferation, apoptosis, development, and the circadian clock (Brennecke et al., 2003; Kadener et al., 2009). In Aedes aegypti, bantam-3p is significantly up-regulated during pupal developmental, and the highest expression has been reported at the mid-pupal period (Bryant et al.,2010). In another study, it was most abundantly expressed in both pupal and adult male and female mosquitoes, indicating its functional importance (Feng et al., 2018) and its role in Varroa development.
Another conserved miRNA miR-8-3p is significantly up-regulated in Aedes aegypti during pupation and has the highest expression at the mid-pupal period. The miR-8-3p is also significantly up-regulated in the fat body. Bryant et al., (2010) showed the up-regulation of miR-8-3p in the fat body of a blood-fed female mosquito and suggested a potential regulatory role in Ae. aegypti reproduction. Contrary to Ae. aegypti, miR-8-3p is abundantly expressed in Anopheles stephensi developmental stages (Feng et al., 2018) and found to be equally expressed in uninfected or infected Aedes albopictus saliva upon infection by CHIKV (Maharaj et al., 2015). In Ae. aegypti, miR-8 has been validated to target SWIM (secreted wingless-interacting molecule), thereby regulating reproductive events (Lucas et al.,2015). Additionally, miR-8 shows cell-type specificity and is expressed in S2 cells (a cell line derived from Drosophila melanogaster embryos). Its temporal expression is less restricted in Drosophila, and has been observed across all developmental stages occasionally with significant variation in expression (Jin et al., 2012). On the basis of above-mentioned studies, the conserved nature of miR-8-3p also indicates its possible role in Varroa development, reproduction, and virus infection and requires further investigation. Another miRNA predicted in this study that is common to Drosophila is miR-34-5p, which is more pronounced in female midguts in Anopheles gambiae (Winter et al., 2007). In contrast, it is down-regulated in Drosophila during the metamorphosis period (pupa to adult stage transition) (Sempere et al., 2003). Interestingly, in Anopheles gambiae, miR-34-5p expression was found down-regulated in the midgut upon Plasmodium falciparum infection (Dennison et al., 2015). The miR-34-5p is suggested to contribute to anti-pathogen and immune responses during DENV-2 infection in Ae. albopictus (Liu et al., 2015). Conserved nature of this microRNA indicates its possible role towards development regulation in Varroa but requires further studies. Our in silico data showed upregulation, and our qRT-PCR data has shown its upregulation by ~ 5000 fold in DWV-B infected varroa females suggesting its possible role in anti-DWV-B and immune response during DWV-B infection, or it might have contribution towards DWV-B survival inside Varroa which requires further work to dissect it. In our insilico study, miR-87-3p was found upregulated in female Varroa. Previous studies have suggested its role in development in Drosophila (Sempere et al. 2003), and anti-viral, immune responses during DENV-2 infection in Ae. albopictus (Liu et al., 2015). Our qRT-PCR analysis has also demonstrated its high expression of ~ 300 folds in DWV-B infected Varroa females suggesting its possible role either in anti-DWV-B immune response or in harboring DWV-B subject to further investigation. Another microRNA surfaced in our data is miR-375-3p. In Ae. aegypti, miR-375-3p is expressed in the blood-fed mosquitoes. miR-375 was found as the key to dengue virus (DENV) replication, enhancing DENV-2 infection in an Ae.aegypti cell line (Hussain et al., 2013). Ae. aegypti miR-375 targets Cactus, and REL1 genes, and the injection of a miRNA mimic into mosquitoes led to fold-changes in immune gene transcripts, suggesting that aae-miR-375 enhanced DENV-2 infection (Hussain et al., 2013). It enhances Dengue virus serotype-2 infection in Ae. aegypti. Our qRT-PCR data has also shown its ~ 20,000 fold upregulation in DWV-B infected Varroa suggesting its possible role in DWV-B infection. Interestingly, the expression of miR-263a-5p in uninfected and CHIKV-infected Ae. aegypti saliva was up-regulated considerably (Maharaj et al., 2015). The miR263a-5p is constitutively expressed across many developmental stages in several mosquito species (Hu et al., 2015). Another conserved dme-miR-252-5p was induced more than three-fold after DENV-2 infection in an Ae. albopictus C6/36 cell line and inhibited DENV replication by suppressing the expression of the DENV envelope protein. It has been validated to target DENV-2 envelope gene in Ae. albopictus, thereby regulating expression of DENV-2 E protein (Yan et al., 2014). In An. gambiae, dme-miR-12-5p is shown in the thorax of males and females, predominantly in midguts, and in their heads considerably constitutively expressed (Winter et al., 2007). The dme-miR-12-5p targets DNA replication licensing factor (MCM6) and monocarboxylate transporter (MCT1) genes, as validated in Ae. aegypti, by which it affects Wolbachia density in host cells (Osei-Amo et al., 2012). The dme-mir-279-3p is expressed evenly and ubiquitously throughout the An. gambiae body (Winter et al., 2007). Another conserved miRNA predicted in this study is dme-miR-278-3p, and it is highly and abundantly expressed miRNA in the reported research (Feng et al., 2018). It was up-regulated in the susceptible Culex pipiens pallens strain upon pyrethroid exposure; a widely and indiscriminately used insecticide. The miR-278-3p targets CYP6AG11 in Culex pipiens pallens to regulate Pyrethroid resistance (Lei et al., 2015). The pyrethroid tau-fluvalinate (Apistan®) is among the first synthetic varroacides registered in the United States, and Varroa resistant to pyrethroid is a big problem in controlling this mite. Varroa resistant to pyrethroid is associated to point mutations in the gene voltage-gated sodium channel gene at position 925 (Millan-Leiva et al., 2021). Further dissection of mechanism of miR-278-3p and pyrethroid resistance along with voltage-gated sodium channel gene might provide some cue towards negating pyrethroid resistance in Varroa.
The predicted miRNAs in the Varroa small RNA-seq provide a list of conserved miRNA targets involved in regulating key biological processes. As discussed earlier, conserved Drosophila homolog microRNAs predicted in the Varroa (Fig. 4A, Table 2), including vde-miR-87-3p, vde-bantam-3p, vde-miR-375-3p and, vde-miR-34-5p. These miRNAs have been shown to play a role in inhibiting or replicating dengue (DENV) and chikungunya virus (CHIKV) in mosquito species (Sempere et al., 2003, Maharaj et al., 2015, Hussain et al., 2013). The expressions of these miRNAs were significantly up-regulated (100 − 20,000 fold) in DWV-B infected Varroa female mites (Fig. 4B). These results indicate a functional role of these miRNAs in virus load and warrant a detailed study to characterize these miRNAs in Varroa-borne virus transmission studies.
Some of the predicted mature microRNAs such as nDS_019211455.1_10989, nDS_019211455.1_7097, nDS_019211459.1_37116 and, nDS_019211459.1_24860 were female specific, detected only in the Varroa females but not in males, and 2 of the predicted miRNAs, nDS_019211455.1_10989 and nDS_019211455.1_16072 were male-specific, detected in males but not in females. These male and female-specific microRNAs in Varroa need to be dissected on the basis of their functional roles. A previous study in a nematode Ascaris suum has predicted the role of gender specific miRNAs as a set of elongation factors, heat shock proteins, and growth factors essential for the development of the organism (Xu et al., 2012). Moreover, sperm proteins and sperm cell motility proteins were found as the targets of the male-specific miRNAs while ovarian message proteins were found as targets of female-specific miRNAs (Xu et al., 2012).
In this study, the size distributions of miRNAs in the Varroa male and female data sets established two peaks, with a predominant peak correlated to miRNAs of 24 nucleotides in size, consistent with genome-wide identification of miRNA study of the Varroa (Fonseca et al., 2021). These miRNAs are major contributors to the total small RNAs from the Varroa males and females. Moreover, it has been suggested that miRNA might be actively involved in the vector competence of the Varroa-borne viruses.
Using publicly available Varroa destructor genome data, we annotated and evaluated possible targets and infer functions of those female Varroa putative miRNAs which showed in silico up-regulation or down-regulation relative to male. The most conserved miRNAs are involved in several biological, cellular, and development processes.
Main KEGG pathways predicted by STRING web analysis were oxidative phosphorylation, endocytosis, protein processing in the endoplasmic reticulum (ER), and other metabolic pathways. In the predicted KEGG pathways, 14 proteins out of 810 have been annotated for metabolic pathways, 12 out of 80 for oxidative phosphorylation, 4 out of 116 for endocytosis, and 2 out of 109 for protein processing in the endoplasmic reticulum (Table 3). Not surprisingly, this data suggests the significance of these biological processes. Proteins involved in endocytosis and protein processing in ER could provide significant clues towards viral infection (Wang et al., 2013; Rosche et al., 2021) process in Varroa.