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Due to limitations in conventional disease vector control strategies including the rise of insecticide resistance in natural populations of mosquitoes, genetic control strategies using CRISPR gene drive systems have been under serious consideration. The identification of CRISPR target sites in mosquito populations is a key aspect for developing efficient genetic vector control strategies. While genome-wide Cas9 target sites have been explored in mosquitoes, a precise evaluation of target sites focused on coding sequence (CDS) is lacking. Additionally, target site polymorphisms have not been characterized for other nucleases such as Cas12a, which require a different DNA recognition site (PAM) and would expand the accessibility of mosquito genomes for genetic engineering. We undertook a comprehensive analysis of potential target sites for both Cas9 and Cas12a nucleases within the genomes of natural populations of Anopheles gambiae and Aedes aegypti from multiple continents. We demonstrate that using two nucleases increases the number of targets per gene. Also, we identified differences in nucleotide diversity between North American and African Aedes populations, impacting the abundance of good target sites with a minimal degree of polymorphisms that can affect the binding of gRNA. Lastly, we screened for gRNAs targeting sex-determination genes that could be widely applicable for developing field genetic control strategies. Overall, this work highlights the utility of employing both Cas9 and Cas12a nucleases and underscores the importance of designing universal genetic strategies adaptable to diverse mosquito populations., (© 2024. The Author(s).)

Virome studies among metazoans have revealed the ubiquity of RNA viruses in animals, contributing to a fundamental rethinking of the relationships between organisms and their microbiota. Mosquito viromes, often scrutinized due to their public health relevance, may also provide insight into broadly applicable concepts, such as a "core virome," a set of viruses consistently associated with a host species or population that may fundamentally impact its basic biology. A subset of mosquito-associated viruses (MAVs) could comprise such a core, and MAVs can be categorized as (i) arboviruses, which alternate between mosquito and vertebrate hosts, (ii) insect-specific viruses, which cannot replicate in vertebrate cells, and (iii) viruses with unknown specificity. MAVs have been widely characterized in the disease vector Aedes aegypti, and the occurrence of a core virome in this species has been proposed but remains unclear. Using a wild population previously surveyed for MAVs and a common laboratory strain, we investigated viromes in reproductive tissue via metagenomic RNA sequencing. Virome composition varied across samples, but four groups comprised >97% of virus sequences: a novel partiti-like virus (Partitiviridae), a toti-like virus (Totiviridae), unclassified Riboviria, and four orthomyxo-like viruses (Orthormyxoviridae). Whole or partial genomes for the partiti-like virus, toti-like virus, and one orthomyxo-like virus were assembled and analysed phylogenetically. Multigenerational maintenance of these MAVs was confirmed by RT-PCR, indicating vertical transmission as a mechanism for persistence. This study provides fundamental information regarding MAV ecology and variability in A. aegypti and the potential for vertically maintained core viromes at the population level., (© 2022 John Wiley & Sons Ltd.)

Dengue viruses (DENVs) cause the greatest public health burden globally among the arthropod-borne viruses. DENV transmission risk has also expanded from tropical to subtropical regions due to the increasing range of its principal mosquito vector, Aedes aegypti . Focal outbreaks of dengue fever (dengue) in the state of Florida (FL) in the USA have increased since 2009. However, little is known about the competence of Ae. aegypti populations across different regions of FL to transmit DENVs. To understand the effects of DENV genotype and serotype variations on vector susceptibility and transmission potential in FL, we orally infected a colony of Ae. aegypti (Orlando/ORL) with low passage or laboratory DENV-1 through -4. Low passage DENVs were more infectious to and had higher transmission potential by ORL mosquitoes. We used these same DENVs to examine natural Ae. aegypti populations to determine whether spatial distributions correlated with differential vector competence. Vector competence across all DENV serotypes was greater for mosquitoes from areas with the highest dengue incidence in south FL compared to north FL. Vector competence for low passage DENVs was significantly higher, revealing that transmission risk is influenced by virus/vector combinations. These data support a targeted mosquito-plus-pathogen screening approach to more accurately estimate DENV transmission risk.

The incidence of locally acquired dengue infections increased during the last decade in the United States, compelling a sustained research effort concerning the dengue mosquito vector, Aedes aegypti , and its microbiome, which has been shown to influence virus transmission success. We examined the "metavirome" of four populations of Aedes aegypti mosquitoes collected in 2016 to 2017 in Manatee County, FL. Unexpectedly, we discovered that dengue virus serotype 4 (DENV4) was circulating in these mosquito populations, representing the first documented case of such a phenomenon in the absence of a local DENV4 human case in this county over a 2-year period. We confirmed that all of the mosquito populations carried the same DENV4 strain, assembled its full genome, validated infection orthogonally by reverse transcriptase PCR, traced the virus origin, estimated the time period of its introduction to the Caribbean region, and explored the viral genetic signatures and mosquito-specific virome associations that potentially mediated DENV4 persistence in mosquitoes. We discuss the significance of prolonged maintenance of the DENV4 infections in A. aegypti that occurred in the absence of a DENV4 human index case in Manatee County with respect to the inability of current surveillance paradigms to detect mosquito vector infections prior to a potential local outbreak. IMPORTANCE Since 1999, dengue outbreaks in the continental United States involving local transmission have occurred only episodically and only in Florida and Texas. In Florida, these episodes appear to be coincident with increased introductions of dengue virus into the region through human travel and migration from countries where the disease is endemic. To date, the U.S. public health response to dengue outbreaks has been largely reactive, and implementation of comprehensive arbovirus surveillance in advance of predictable transmission seasons, which would enable proactive preventative efforts, remains unsupported. The significance of our finding is that it is the first documented report of DENV4 transmission to and maintenance within a local mosquito vector population in the continental United States in the absence of a human case during two consecutive years. Our data suggest that molecular surveillance of mosquito populations in high-risk, high-tourism areas of the United States may enable proactive, targeted vector control before potential arbovirus outbreaks.