Aedes aegypti acts as a vector for several arboviral diseases that impose a major socio-economic burden. Moreover, the absence of a vaccine against these diseases and drug resistance in mosquitoes necessitates the development of new control strategies for vector-borne diseases. ABC transporters that play a vital role in immunity and other cellular processes in different organisms may act as non-canonical immune molecules against arboviruses, however, their role in mosquito immunity remains unexplored. This study comprehensively analyzed various genetic features of putative ABC transporters and classified them into A-H subfamilies based on their evolutionary relationships. Existing RNA-sequencing data analysis indicated higher expression of cytosolic ABC transporter genes (E & F Subfamily) throughout the mosquito development, while members of other subfamilies exhibited tissue and time-specific expression. Furthermore, comparative gene expression analysis from the microarray dataset of mosquito infected with dengue, yellow fever and West Nile viruses revealed 31 commonly expressed ABC transporters suggesting a potentially conserved transcriptomic signature of arboviral infection. Among these, only a few transporters of ABCA, ABCC and ABCF subfamily were upregulated, while most were downregulated. This indicates the possible involvement of ABC transporters in mosquito immunity.

Background: The mosquito midgut is crucial for digestion and immune interactions. It produces several immune factors that protect the organ from invading pathogens and can limit their propagation. Studies on mosquito midgut transcriptome following pathogen exposure have revealed the presence of non-canonical immune genes, such as ABC transporters, whose function in insect immunity remains unexplored. Therefore, this study focuses on identifying and characterising the immune role of ABC transporters in the midgut of Aedes aegypti, a primary arboviral vector. Methods: To identify the midgut-expressed ABC transporters, the mosquitoes were challenged with a mixture of gram-negative (Escherichia coli) and gram-positive (Micrococcus luteus) bacteria, and the expression of all ABC transporters was analysed with PCR using gene-specific primers. Furthermore, the transcriptional alterations of midgut ABC transporters were explored at different time points upon a thoracic nano-injection (systemic challenge) or infectious blood meal (local challenge) of the bacterial mixture through quantitative real-time PCR (qPCR), and one gene was selected for RNAi-mediated gene silencing and its role assessment in midgut immune responses. Results: The expression of all 48 microbial-induced midgut-expressing Ae. aegypti ABC transporter genes upon systemic or local bacterial challenges was analyzed. Based on the transcriptomic data and potential immune expression similar to the well-known immune gene defensin, AaeABCG3 was selected for RNAi-mediated gene silencing and characterization. The AaeABCG3 gene silencing exhibited a significant reduction of midgut bacterial load through the induction of nitric oxide synthase (NOS) in sugar-fed and systemic bacterial-challenged mosquitoes. In contrast, midgut bacterial load was significantly regulated by induction of defensin A and cecropin G in the late hours of local bacterial challenges in AaeABCG3-silenced mosquitoes. Conclusions: The silencing of AaeABCG3 modulated the mosquito midgut immune response and disturbed the midgut microbiota homeostasis. The systemic immune responses of AaeABCG3-silenced mosquitoes were influenced by the JAK-STAT pathway with no induction of Toll and IMD immune pathways. Interestingly, Toll and IMD immune pathways actively participated in the late hours of local bacterial challenges, suggesting that the route of infection influences these immune responses; however, the molecular mechanism behind these phenomena still needs to be explored. Overall, this work provides significant insight into the importance of ABC transporters in mosquito immunity. Graphical

One third of all emerging infectious diseases are vector-borne, with the vector’s ecology and physiology playing key roles in determining whether viruses can access new vertebrate host species and spread globally. Innate immunity is a known barrier to virus replication in mosquito vectors that influences arboviral vector tropism. We here generated novel CRISPR-Cas9-mediated knockouts of the NF-κB family transcription factor Rel2 in Aedes aegypti -derived Aag2 cells and tested the impact on the replication of a diverse range of arboviruses in the Flaviviridae and Togaviridae families and the class Bunyaviricetes . We found that NF-κB-mediated innate immunity has broad antiviral activity against the Ae. aegypti -borne orthoflaviviruses dengue virus (DENV), yellow fever virus (YFV) and Zika virus (ZIKV) in mosquito cells. In contrast, little impact of NF-κB-loss-of-function was observed for the alphavirus chikungunya virus (CHIKV) or phlebovirus Rift Valley fever virus (RVFV), indicating specificity in the antiviral effects of NF-κB-mediated immunity. By comparing orthoflaviviruses with different transmission routes (mosquito-borne, tick-borne, no known vector), we demonstrated that NF-κB-mediated immunity exerts its antiviral effects both early and late in the viral replication cycle, and that NF-κB-mediated immunity is not the only molecular barrier influencing the ability of orthoflaviviruses to replicate in Ae. aegypti cells. Overall, our work demonstrates the importance of mosquito NF-κB-mediated innate immunity in suppressing arbovirus replication, and shows that the barriers for arboviruses to adapt to new vector species are multifactorial and virus-specific. Our findings increase our understanding of the molecular barriers influencing arboviral emergence, and could inform the development of refractory mosquitoes incapable of transmitting human pathogens. Author Summary: Mosquito-borne viruses are causing an increasing global disease burden due to urbanisation, globalisation, changing land use and the climate change-driven invasion of mosquitoes into new locations. Few vaccines and no medicines for mosquito-borne viral infections are licensed for human use. Historically, the most effective way to reduce human disease has been to kill mosquitoes using insecticides, which also harms beneficial insects and is leading to resistance. Newer technologies with lower ecological impacts are being developed, including genetically modified mosquitoes with a reduced ability to transmit viruses. Like humans, mosquitoes have an immune system that protects against viral infections, and strengthening this immune system through genetic modification has shown promise for reducing virus transmission. We here expand our understanding of the ways in which mosquito immunity could be harnessed by demonstrating that a gene called “Rel2” helps the mosquito fight mosquito-borne virus infection. Unlike previously studied mosquito immune responses, Rel2 has a very broad (but not universal) protective capacity against viruses. Our work also indicates that Rel2 works in concert with other arms of the mosquito immune system, highlighting a need for further research to fully realise the potential of genetically modifying mosquito immune responses for the prevention of human disease.