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Background/objectives: Culex species mosquitoes are globally distributed and transmit several pathogens that impact animal and public health, including West Nile virus, Usutu virus, and Plasmodium relictum. Despite their relevance, Culex species are less widely studied than Aedes and Anopheles mosquitoes. To expand the genetic tools used to study Culex mosquitoes, we previously developed an optimized plasmid for transient Cas9 and single-guide RNA (sgRNA) expression in Culex quinquefasciatus cells to generate gene knockouts. Here, we established a monoclonal cell line that consistently expresses Cas9 and can be used for screens to determine gene function or antiviral activity.

Methods: We used this system to perform the successful gene editing of seven genes and subsequent testing for potential antiviral effects, using a simple single-guide RNA (sgRNA) transfection and subsequent virus infection.

Results: We were able to show antiviral effects for the Cx. quinquefasciatus genes dicer-2, argonaute-2b, vago, piwi5, piwi6a, and cullin4a. In comparison to the RNAi-mediated gene silencing of dicer-2, argonaute-2b, and piwi5, our Cas9/sgRNA approach showed an enhanced ability to detect antiviral effects.

Conclusions: We propose that this cell line offers a new tool for studying gene function in Cx. quinquefasciatus mosquitoes that avoids the use of RNAi. This short study also serves as a proof-of-concept for future gene knock-ins in these cells. Our cell line expands the molecular resources available for vector competence research and will support the design of future research strategies to reduce the transmission of mosquito-borne diseases.

Oxidative stress-mediated biomolecular damage is a characteristic feature of ionizing radiation (IR) injury, leading to genomic instability and chronic health implications. Specifically, a dose- and linear energy transfer (LET)-dependent persistent increase in oxidative DNA damage has been reported in many tissues and biofluids months after IR exposure. Contrary to low-LET photon radiation, high-LET IR exposure is known to cause significantly higher accumulations of DNA damage, even at sublethal doses, compared to low-LET IR. High-LET IR is prevalent in the deep space environment (i.e., beyond Earth's magnetosphere), and its exposure could potentially impair astronauts' health. Therefore, the development of biomarkers to assess and monitor the levels of oxidative DNA damage can aid in the early detection of health risks and would also allow timely intervention. Among the recognized biomarkers of oxidative DNA damage, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodG) has emerged as a promising candidate, indicative of chronic oxidative stress. It has been reported to exhibit differing levels following equivalent doses of low- and high-LET IR. This review discusses 8-OxodG as a potential biomarker of high-LET radiation-induced chronic stress, with special emphasis on its potential sources, formation, repair mechanisms, and detection methods. Furthermore, this review addresses the pathobiological implications of high-LET IR exposure and its association with 8-OxodG. Understanding the association between high-LET IR exposure-induced chronic oxidative stress, systemic levels of 8-OxodG, and their potential health risks can provide a framework for developing a comprehensive health monitoring biomarker system to safeguard the well-being of astronauts during space missions and optimize long-term health outcomes.

Mosquitoes, like Drosophila, are dipterans, the order of "true flies" characterized by a single set of two wings. Drosophila are prime model organisms for biomedical research, while mosquito researchers struggle to establish robust molecular biology in these that are arguably the most dangerous vectors of human pathogens. Both insects utilize the RNA interference (RNAi) pathway to generate small RNAs to silence transposons and viruses, yet details are emerging that several RNAi features are unique to each insect family, such as how culicine mosquitoes have evolved extreme genomic feature differences connected to their unique RNAi features. A major technical difference in the molecular genetic studies of these insects is that generating stable transgenic animals are routine in Drosophila but still variable in stability in mosquitoes, despite genomic DNA-editing advances. By comparing and contrasting the differences in the RNAi pathways of Drosophila and mosquitoes, in this review we propose a hypothesis that transgene DNAs are possibly more intensely targeted by mosquito RNAi pathways and chromatin regulatory pathways than in Drosophila. We review the latest findings on mosquito RNAi pathways, which are still much less well understood than in Drosophila, and we speculate that deeper study into how mosquitoes modulate transposons and viruses with Piwi-interacting RNAs (piRNAs) will yield clues to improving transgene DNA expression stability in transgenic mosquitoes.