Novel mosquito genetic control tools, such as CRISPR-based gene drives, hold great promise in reducing the global burden of vector-borne diseases. As these technologies advance through the research and development pipeline, there is a growing need for modeling frameworks incorporating increasing levels of entomological and epidemiological detail in order to address questions regarding logistics and biosafety. Epidemiological predictions are becoming increasingly relevant to the development of target product profiles and the design of field trials and interventions, while entomological surveillance is becoming increasingly important to regulation and biosafety. We present MGDrivE 3 (Mosquito Gene Drive Explorer 3), a new version of a previously-developed framework, MGDrivE 2, that investigates the spatial population dynamics of mosquito genetic control systems and their epidemiological implications. The new framework incorporates three major developments: i) a decoupled sampling algorithm allowing the vector portion of the MGDrivE framework to be paired with a more detailed epidemiological framework, ii) a version of the Imperial College London malaria transmission model, which incorporates age structure, various forms of immunity, and human and vector interventions, and iii) a surveillance module that tracks mosquitoes captured by traps throughout the simulation. Example MGDrivE 3 simulations are presented demonstrating the application of the framework to a CRISPR-based homing gene drive linked to dual disease-refractory genes and their potential to interrupt local malaria transmission. Simulations are also presented demonstrating surveillance of such a system by a network of mosquito traps. MGDrivE 3 is freely available as an open-source R package on CRAN (https://cran.r-project.org/package=MGDrivE2) (version 2.1.0), and extensive examples and vignettes are provided. We intend the software to aid in understanding of human health impacts and biosafety of mosquito genetic control tools, and continue to iterate per feedback from the genetic control community.

The mosquito, Culex tarsalis, is a key vector in the western United States due to its role in transmission of zoonotic arboviruses that affect human health. Extensive research has been conducted on Cx. tarsalis ecology, feeding behavior, vector competence, autogeny, diapause, genetics, and insecticide resistance. Population genetic analyses in the western U.S. have identified at least three genetic clusters that are geographically distinct. However, in-depth genetic studies have been hindered by the lack of a reference genome. In this study, we present the first whole-genome assembly of this mosquito species (CtarK1) based on PacBio HiFi reads from high-molecular-weight DNA extracted from a single male. The CtarK1 assembly is 790 Mb with an N50 of 58 kb, which is 27% larger than Culex quinquefasciatus (578 Mb). This difference appears to be mostly composed of transposable elements. To annotate CtarK1, we used a previously assembled Cx. tarsalis transcriptome and approximately 17,456 protein genes from Cx. quinquefasciatus (N = 17,456). Genome completeness was assessed using the Benchmarking Universal Single-Copy Orthologs (BUSCO) tool, which identified 84.8% of the 2799 Dipteran BUSCO genes. Using a Bayesian phylogeny based on mitochondrial genomes, we place Cx. tarsalis in the context of other mosquito species and estimate the divergence between Cx. tarsalis and Cx. quinquefasciatus to be between 15.8 and 22.2 million years ago (MYA). Important next steps from this work include characterizing the genetic basis of diapause and sex determination in Culex mosquitoes.

Local vector control and public health agencies in California use the California Mosquito-Borne Virus Surveillance and Response Plan to monitor and evaluate West Nile virus (WNV) activity and guide responses to reduce the burden of WNV disease. All available data from environmental surveillance, such as the abundance and WNV infection rates in Culex tarsalis and the Culex pipiens complex mosquitoes, the numbers of dead birds, seroconversions in sentinel chickens, and ambient air temperatures, are fed into a formula to estimate the risk level and associated risk of human infections. In many other areas of the US, the vector index, based only on vector mosquito abundance and infection rates, is used by vector control programs to estimate the risk of human WNV transmission. We built models to determine the association between risk level and the number of reported symptomatic human disease cases with onset in the following three weeks to identify the essential components of the risk level and to compare California's risk estimates to vector index. Risk level calculations based on Cx. tarsalis and Cx. pipiens complex levels were significantly associated with increased human risk, particularly when accounting for vector control area and population, and were better predictors than using vector index. Including all potential environmental components created an effective tool to estimate the risk of WNV transmission to humans in California.

Mosquito-borne diseases, such as dengue and malaria, pose significant global health burdens. Unfortunately, current control methods based on insecticides and environmental maintenance have fallen short of eliminating the disease burden. Scalable, deployable, genetic-based solutions are sought to reduce the transmission risk of these diseases. Pathogen-blocking Wolbachia bacteria, or genome engineering-based mosquito control strategies including gene drives have been developed to address these problems, both requiring the release of modified mosquitoes into the environment. Here, we review the latest developments, notable similarities, and critical distinctions between these promising technologies and discuss their future applications for mosquito-borne disease control.

Innovative tools are essential for advancing malaria control and depend on an understanding of molecular mechanisms governing transmission of malaria parasites by Anopheles mosquitoes. CRISPR/Cas9-based gene disruption is a powerful method to uncover underlying biology of vector-pathogen interactions and can itself form the basis of mosquito control strategies. However, embryo injection methods used to genetically manipulate mosquitoes (especially Anopheles) are difficult and inefficient, particularly for non-specialist laboratories. Here, we adapted the ReMOT Control (Receptor-mediated Ovary Transduction of Cargo) technique to deliver Cas9 ribonucleoprotein complex to adult mosquito ovaries, generating targeted and heritable mutations in the malaria vector Anopheles stephensi without injecting embryos. In Anopheles, ReMOT Control gene editing was as efficient as standard embryo injections. The application of ReMOT Control to Anopheles opens the power of CRISPR/Cas9 methods to malaria laboratories that lack the equipment or expertise to perform embryo injections and establishes the flexibility of ReMOT Control for diverse mosquito species.

Although mosquitoes are major transmission vectors for pathogenic arboviruses, viral infection has little impact on mosquito health. This immunity is caused in part by mosquito RNA interference (RNAi) pathways that generate antiviral small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). RNAi also maintains genome integrity by potently repressing mosquito transposon activity in the germline and soma. However, viral and transposon small RNA regulatory pathways have not been systematically examined together in mosquitoes. Therefore, we developed an integrated mosquito small RNA genomics (MSRG) resource that analyzes the transposon and virus small RNA profiles in mosquito cell cultures and somatic and gonadal tissues across four medically important mosquito species. Our resource captures both somatic and gonadal small RNA expression profiles within mosquito cell cultures, and we report the evolutionary dynamics of a novel Mosquito-Conserved piRNA Cluster Locus (MCpiRCL) made up of satellite DNA repeats. In the larger culicine mosquito genomes we detected highly regular periodicity in piRNA biogenesis patterns coinciding with the expansion of Piwi pathway genes. Finally, our resource enables detection of cross talk between piRNA and siRNA populations in mosquito cells during a response to virus infection. The MSRG resource will aid efforts to dissect and combat the capacity of mosquitoes to tolerate and spread arboviruses.

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