The Asian tiger mosquito Aedes albopictus represents a serious threat for public health worldwide. Due to both its invasive aptitude and vector competence, it is extending to temperate regions the risk of transmission of important human arboviruses. Indeed, several cases of autochtonous transmission of dengue, chikungunya and Zika viruses have been recorded in Europe (e.g., Italy, France, Croatia, etc.) in the last fifteen years. The virus life cycle in the mosquito starts with the blood meal on an infected host. The ingested virus first invades the midgut epithelium and replicates, then disseminates through the hemolymph, and finally infects the salivary glands; from here it is transmitted to a new host with mosquito saliva during the next blood meal. Our understanding of the Ae. albopictus innate immune pathways activated during CHIKV infection is still limited, and recent evidence also highlighted that arboviral infection may affect mosquito physiology modulating, for instance, host-seeking, blood feeding and egg maturation. This project builds on a RNA-seq analysis of CHIKV-infected Ae. albopictus that we recently completed that planned to clarify the molecular interactions between the tiger mosquito and CHIKV. First, mosquito genes previously identified as modulated by CHIKV and potentially involved in key aspects of immune response, olfaction/chemoperception, feeding, egg maturation or transmission, will be screened by a reverse genetic approach based on dsRNA-mediated gene silencing. The most promising candidates will be targeted by the CRISPR-Cas9 methodology, which is currently applied to other mosquitoes but not yet established in Ae. albopictus, making its implementation in the tiger mosquito a key innovative aspect of the project. Finally, a small RNA-seq analysis of mosquito midguts soon after CHIKV invasion is expected to provide novel information on organ-specific modulation of miRNA expression and activation of antiviral siRNA and piRNA pathways.
The main aim of this project is to clarify some basic aspects of the molecular interactions taking place between the tiger mosquito Ae. albopictus and CHIKV; however, on the other side, it also includes the implementation of molecular tools with potential practical applications for vector control strategies. In the last few years, an increasing number of datasets describing changes in mosquito gene expression during virus lifecycle have been published and/or made available. However, while an adequate and also growing number of transcriptomic studies investigated several biological aspects of the relationships between mosquitoes and flavivirus (Dengue, Zika, etc.), only very few reports so far described the application of molecular technologies to the study of alphavirus (such as CHIKV) and their vectors.
We have recently completed an extensive RNA-seq analysis on CHIKV-infected Ae. albopictus. The study focused on the molecular interactions occurring between the two organisms at two time-points, 1 dpf and 5 dpf, to describe the virus passage across the midgut and the dissemination stage in the mosquito hemolymph, respectively. At day 1, a specific focus was addressed on dissected midguts, to get deeper inside on the specific gene regulation during the tissue invasion by the virus. At day 5, both midgut and carcasses (whole body without the midgut) were analysed to gain a complete picture of the virus effect on the mosquito transcriptional profile during the dissemination stage. This analysis allowed for the identification of genes differentially expressed (DE) between control and infected samples.
The experimental workflow designed for this project represents a robust follow up of the transcriptomic study mentioned above and will guide the exploration of the transcriptional regulation in key organs and in crucial stages of the virus lifecycle in the mosquito from a functional point of view. In fact, using a reverse genetic approach (throughout transient dsRNA-mediated RNAi), this project is expected to shed light on Ae. albopictus immune responses during early infection (1 dpi) and CHIKV dissemination (5 dpi). This will allow not only to identify genes playing crucial roles in antiviral immune response and CHIKV transmission, but also genes involved in manipulation of mosquito behaviors, such as host-seeking (changes in olfaction/chemoperception), probing and feeding efficiency (changes in saliva production and composition) and egg development[1]. A special attention will be paid to genes belonging to the ubiquitination/SUMOylation pathways, which have been recently shown to have a role in the mosquito defense against arbovirus[2]. Moreover, it will be extremely interesting the evaluation of direct and/or indirect effects of CHIKV infection on Ae. albopictus behavioral aspects such as olfaction or oviposition, which might also help designing novel strategies to control mosquito vectors[3,4]. Indeed, functional analyses of novel olfactory factors involved in enhancing/reducing transmission rates might help the development of novel repellent/attractants able to reduce the burden of mosquito feeding on the human host and therefore the transmission of arboviruses.
The implementation of the CRISPR-Cas9 technology in Ae. albopictus is probably the most ambitious/innovative aspect of this project. The adaptation of the CRISPR-Cas9 technology to the tiger mosquito is expected to provide a novel powerful molecular tool for studies on this invasive species and to pave the way toward the development of novel control strategies.
Finally, the focus on small non-coding RNAs during CHIKV invasion of the mosquito midgut will contribute a more complete picture of Ae. albopictus-CHIKV molecular interactions. This is certainly going to extend the available information on Ae. albopictus miRNAs and to provide novel insights on the involvement of the siRNA and piRNA pathways in the antiviral response against CHIKV. All the raw sequencing data as well as the transcriptomic data assembled and analysed, including DE results, will be provided to the scientific community through the upload in open-access public databases.
Bibliography
1. Murdock, C. C. et al. Curr. Opin. Insect Sci. 20, 28-33 (2017).
2. Stokes, S. et al. PLoS Pathog. 16, 1-25 (2020).
3. Lopez, S. B. G. et al. Sci. Rep. 9, 1-13 (2019).
4. Achee, N. L. et al. PLoS Negl. Trop. Dis. 13, e0006822 (2019).