During blood feeding hematophagous arthropods inject into their hosts a cocktail of salivary proteins whose main role is to counteract vertebrate responses to tissue injury, namely hemostasis, inflammation and immunity. However, animal body fluids, including saliva of the few blood sucking arthropods analyzed so far, also carry miRNAs. We recently analyzed the salivary miRNA repertoire of the malaria vector Anopheles coluzzii and found an enrichment of specific miRNAs in saliva as compared to salivary glands. Interestingly, eleven of the most abundant An. coluzzii saliva miRNAs mimicked human endogenous miRNAs targeting host genes involved in immune and inflammatory responses. Overall, these findings raise the intriguing hypothesis that specific miRNAs, perhaps incorporated within exosomes, may be selectively directed toward the secretory pathway and saliva and, injected into vertebrates, may contribute to host manipulation, with potential implications for vector-host interaction and pathogen transmission. To follow-up these initial observations, we plan to characterize salivary exosome-like vesicles and verify whether vector miRNAs can be detected into the host after mosquito blood feeding. Moreover, since anopheline mosquitoes are vectors of Plasmodium parasites, we intend to investigate whether infection by the murine malaria parasite P. berghei modulates Anopheles salivary gland miRNA composition. Finally, we aim to extend our initial observations from Anopheles to Aedes mosquitoes, that are vectors of arboviruses of large relevance for human health. To this end we will perform a small RNA-Seq analysis on both salivary glands and saliva of Aedes aegypti mosquitoes infected and non-infected with the chikungunya virus. We expect the research work proposed here to shed some light on the possible role of mosquito salivary miRNAs in the intricate molecular interactions taking place between mosquitoes, the pathogens they transmit and their vertebrate hosts.
While both the role and the mechanism of action of miRNAs acting on endogenous genes is pretty well established, the possible contribution of miRNAs and other small non-coding RNAs (sncRNAs) in cross-species and even cross-kingdom communication is much less known/understood and still subject of debate. Besides the rather well know case of viral-encoded miRNAs [24] several additional examples coming from bacteria, protozoa, parasitic nematodes, plants and even human erythrocytes have emerged in the last few years [25, 26, 29-32]. Extracellular miRNAs are present in all animal body fluids analyzed so far and are extremely stable, being complexed to proteins or included within exosomal microvesicles. Pretty convincing experimental proofs of exosome involvement in cell-cell communication are available [18, 19], even though their role is still controversial [16]. Recently, our group and others reported the presence of miRNAs in the saliva of Anopheles [11] and Aedes [12] mosquitoes as well as of an Ixodes tick [13], all vectors of diseases of great relevance to human health. In Anopheles mosquitoes and ticks, where both saliva and salivary glands were analyzed, saliva was found enriched in specific miRNAs, which likely implies the existence of mechanisms conveying selected miRNAs in saliva. The observation that blood feeding arthropods as diverse as mosquitoes and ticks share a common set of miRNAs that are abundant in saliva and able to target host immune and inflammatory genes [11] represents a novel intriguing finding. In fact, considering their role of ectoparasites, the complex biochemical/morphological/behavioral adaptations required from the blood feeding style of life, the convergent evolutionary nature of hematophagy as well as parasite-host coevolution, it appears highly unlikely that this common set of miRNAs appeared in the saliva of these blood feeding arthropods just by serendipity. The emerging, biologically fascinating, hypothesis is that miRNAs from the saliva of blood feeding arthropods, perhaps enclosed within exosomal microvesicles, are injected into the vertebrate hosts during blood feeding and, along with salivary proteins, may contribute to host manipulation by post-transcriptional regulation of host gene expression. The observation that a subset of miRNAs present in the saliva of mosquitoes and ticks is also found in exosomal-like microvesicles secreted by parasitic nematodes that establish long-term relationships with their vertebrate hosts [11] raises the question whether this may be an even more general strategy in parasite-host interactions. The research proposed here is aimed at (i) setting up conditions for purifying exosomes from the saliva of An. coluzzii, a prerequisite for their molecular and biochemical characterization; (ii) confirming the asymmetric distribution of miRNAs between saliva and salivary glands in Aedes mosquitoes; (iii) verifying if and how mosquito salivary glands infection by different pathogens as Plasmodium parasites or an arbovirus affects the miRNA composition of saliva and/or salivary glands. In our opinion, also considering the rather poor and sometime controversial knowledge in the field, this research proposal has several innovative aspects with high potential to represent a substantial step forward in the understanding of the complex molecular interactions taking place between vectors and their vertebrate host, with possible implications for pathogen transmission.
[29] Mayoral JG et al. Proc Natl Acad Sci U S A. 2014;111(52): 18721-6.
[30] Quintana JF et al. Parasit Vectors. 2015;8: 58.
[31] Shahid S et al. Nature. 2018;553(7686): 82-5.
[32] Walzer KA, Chi JT. RNA Biol. 2017;14(4): 442-9.