| Popis: |
The mosquito Aedes aegypti is currently one of the leading avenues for human disease throughout the world. The mosquito is responsible for the spread of severe human pathogens such as dengue and Zika viruses as well as several other arboviruses. Dengue alone is responsible for an estimated 300,000 cases per year with two billion of the world’s population at risk. Currently, there are no suitable medical interventions for the disease, therefore, the burden is placed on mosquito population control. This has primarily been done by pesticide spraying, sterile insect technique, and the release of Wolbachia-infected mosquitoes. The later involves the release of mosquitoes infected with the endosymbiotic bacterium Wolbachia, which has been shown to restrict the replication of a number of arboviruses. This has proved effective in many cases, however, the mechanisms in which Wolbachia restricts viruses is still largely unknown. Therefore, the future of how we defend against vector borne diseases is still unclear, meaning it is far too early to forego conventional research into the understanding of the spread of diseases by the mosquito.One avenue of research that has already proved fruitful in vector research, and also in many other models of disease, has been through the exploration of non-coding RNAs (ncRNAs). ncRNAs span a range of different RNAs of varying lengths most of which are involved in various facets of gene regulation. Some of the most common and understood types are microRNAs (miRNAs). These are canonically derived from non-coding primary transcripts and cleaved by stepwise processes leading to the final products of short 21-22 nucleotide RNAs. miRNAs are carried by Dicer and Ago proteins and direct a complex of proteins to degrade targeted mRNAs or repress translation. The other major process for ncRNA routinely used in molecular biology is the short interfering RNA (siRNA) pathway under the general RNA interference (RNAi) pathway. This process involves the use of exogenous dsRNA to target host genes and inhibit translation by a similar but slightly different process as miRNAs. These two processes alone have provided a great depth of knowledge and provided many avenues to further research across many fields.A more recently discovered related role for ncRNAs is mediating RNA activation. This differs from RNAi by targeting promoter regions in the genome upstream of targeted genes and results in the induction of gene expression. While there are a number of reports describing these processes, mainly in mammalian models, to date no evidence has been shown to describe this in insects. Here, we described how exogenous dsRNA can be used to upregulate targeted gene expression in Ae. aegypti, as an insect system. We aimed to initially describe this through the targeting of a reporter gene construct and moved to show that we could induce the expression of mosquito immune genes. We also showed that a number of the binding interactions acted in a conserved manner with those described in mammals. RNA activation presents a viable option for modulating gene expression for molecular studies both in vivo and in vitro.Through previous exploration of the effects that Wolbachia has on the host, it was identified that a miRNA, aae-miR-2940-5p, was drastically induced in Wolbachia-infected Ae. aegypti mosquitoes. Some further studies characterized the necessary upregulation of this miRNA facilitating colonization and maintenance of infection in the host. To further understand the role this miRNA plays in the host, we looked to describe the expression profile of this miRNA during the blood feeding, reproductive and developmental stages of the mosquito. We identified an increase in the miRNA’s expression during the late stages of egg maturation after blood feeding and then showed that depleting the miRNA during this phase delayed egg maturation. We also found that this resulted in reduced hatching rates while maintaining the number of eggs that were laid. We also found that the miRNA increased in the ovary after mating and that early larval stages had higher levels of the miRNA present.With the importance of aae-miR-2940-5p being demonstrated not only in Wolbachia infection but also during development, we looked to expand identification of the number of potential target genes that may be modulated by the miRNA. We employed two techniques through indirect and direct interactions to identify different subsets of genes that were affected by the miRNA. Interestingly, we noted that the miRNA induced an IMD response through identification of upregulated Defensin and C-type lectin genes associated with the pathway. We also discovered that some genes associated with lipid processing and storage were also modified by the presence of the miRNA suggesting it may also contribute to the maintenance of Wolbachia through these avenues. Together, these results highlight the importance of aae-miR-2940-5p in not only Wolbachia infection, but also host reproductive pathways and present an alternative way to investigate the role the targeted genes play in the mosquito. |
