Thesis title: Unraveling the possible role of salivary miRNAs in mosquito-host-pathogen interactions
Mosquitoes are vectors of diseases of great relevance in public health. This is the case of malaria, which is transmitted by anopheline species, as well as of several arboviral diseases transmitted by culicine mosquitoes (yellow fever, dengue, West Nile fever, Zika virus fever, chikungunya, Japanese encephalitis). According to estimates of the WHO billions people worldwide are at risk of mosquito-borne diseases, which may have caused over half million deaths in 2019. The control of these diseases is challenging and, so far, no vaccines or drugs are available against most of them. Mosquitoes acquire and transmit pathogens during their blood meals. Typically, pathogens are injected into the host skin along with mosquito saliva, a complex cocktail of bioactive compounds with anti-hemostatic activity (platelet inhibitors, vasodilators, anticoagulants) that help the mosquito during blood-feeding. However, mosquito saliva is also known to affect host immune responses with important implications for pathogen transmission.
In the last decades transcriptomic and proteomic studies allowed to significantly increase our knowledge of blood feeding arthropod saliva. These studies mainly focused on the functional analysis of salivary proteins; however, blood feeding arthropod saliva, as all animal body fluids analyzed so far, also carries non-coding RNAs (ncRNA). MicroRNAs (miRNAs) are certainly the most well known class of small ncRNAs. They are molecules of ∼22 nt in length with a relevant role in post-transcriptional gene regulation, found in all animal cell types, with a tissue-specific expression patterns, and involved in the regulation of every aspect of cell life (growth and differentiation, apoptosis, development and immunity). MiRNAs circulating in animal body fluids have been the subject of several studies focused on the identification of possible novel biomarkers of human diseases. However, there is a poor understanding and an ongoing debate on the possible role of extracellular miRNAs (circulating either in complex with proteins or within exosomal microvesicles) in cell-cell communication. So far, the only information available on miRNAs from blood feeding arthropod saliva is limited to an Ixodes tick and to Aedes mosquitoes. The presence of miRNAs in mosquito saliva raises the hypothesis that salivary miRNAs may represent additional players in vector-pathogen-host interactions. My PhD research work has been mainly focused on trying to shed some light on miRNAs from mosquito saliva.
Since no information was available on salivary miRNAs in Anopheles mosquito species, I initially focused my attention on the important African malaria vector Anopheles coluzzii.We used Illumina small RNA-Seq to identify miRNAs expressed in saliva and salivary glands of An. coluzzii. Interestingly, this analysis revealed an asymmetric distribution of miRNAs between salivary glands and saliva, indicating that saliva miRNA content does not simply mirror the salivary gland content, and suggesting that some specific mechanism may selectively transport a distinct subset of miRNAs to Anopheles saliva. This is not too surprising by itself but had not been shown before for any blood-feeding insect. Intriguingly, eleven of the most abundant miRNAs in An. coluzzii saliva were identical to human miRNAs known to target genes involved in immune and inflammatory responses. To confirm and further validate this observation in an evolutionarily distant mosquito, we also analyzed miRNAs from saliva and salivary glands of Aedes aegypti infected or not by the chikungunya virus (note that anophelines and culicines separated ~120-150
million years ago). Noteworthy, a similar asymmetric miRNAs distribution also emerged in this species, and a wide overlap between the most abundant salivary miRNAs was found in Anopheles and Aedes, with several mimicking human miRNAs as seen before for An. coluzzii. These observations support the idea that miRNAs in mosquito saliva play a biological function and may contribute to host manipulation, raising an additional interesting question on the possible influence on pathogen infection/transmission. To start addressing this difficult question I also analyzed both An. gambiae salivary glands after Plasmodium berghei infection and Ae. aegypti saliva and salivary glands after infection with the chikungunya virus. Surprising no significant difference was found between Plasmodium-infected and -uninfected salivary glands, whereas some miRNAs were found differentially expressed between chikungunya-infected and -uninfected Ae. aegypti saliva/salivary glands. Further studies will be certainly needed to experimentally prove the hypothesis that miRNAs from mosquito saliva contribute to host manipulation with possible implications for pathogen transmission. This is certainly going to be challenging considering the difficulty of working at the mosquito-host-pathogen interface. Nevertheless, I believe that our observations contribute to a better understanding of the many functions of mosquito saliva and represent an useful starting point for future investigations on the role of mosquito miRNAs in pathogens transmission.