Transposon and Transgene Tribulations in Mosquitoes: A Perspective of piRNA Proportions.

Mosquitoes, like , are dipterans, the order of "true flies" characterized by a single set of two wings. 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.

Alpha-mannosidase-2 modulates arbovirus infection in a pathogen- and Wolbachia-specific manner in Aedes aegypti mosquitoes.

Multiple Wolbachia strains can block pathogen infection, replication and/or transmission in Aedes aegypti mosquitoes under both laboratory and field conditions. However, Wolbachia effects on pathogens can be highly variable across systems and the factors governing this variability are not well understood. It is increasingly clear that the mosquito host is not a passive player in which Wolbachia governs pathogen transmission phenotypes; rather, the genetics of the host can significantly modulate Wolbachia-mediated pathogen blocking.

The Development and Expansion of in vivo Germline Editing Technologies in Arthropods: Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) and Beyond.

In the past 20 years, sequencing technologies have led to easy access to genomic data from nonmodel organisms in all biological realms. Insect genetic manipulation, however, continues to be a challenge due to various factors, including technical and cost-related issues. Traditional techniques such as microinjection of gene-editing vectors into early stage embryos have been used for arthropod transgenesis and the discovery of Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR-Cas) technologies allowed for targeted mutagenesis and the creation of knockouts or knock-ins in arthropods.

Leaning Into the Bite: The piRNA Pathway as an Exemplar for the Genetic Engineering Need in Mosquitoes.

The piRNA pathway is a specialized small RNA interference that in mosquitoes is mechanistically distant from analogous biology in the model. Current genetic engineering methods, such as targeted genome manipulation, have a high potential to tease out the functional complexity of this intricate molecular pathway. However, progress in utilizing these methods in arthropod vectors has been geared mostly toward the development of new vector control strategies rather than to study cellular functions.

Cas9-Mediated Gene-Editing in the Malaria Mosquito by ReMOT Control.

Innovative tools are essential for advancing malaria control and depend on an understanding of molecular mechanisms governing transmission of malaria parasites by 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 ) are difficult and inefficient, particularly for non-specialist laboratories.

Heat shock protein 70 (Hsp70) mediates Zika virus entry, replication, and egress from host cells.

Zika virus (ZIKV) is a historically neglected mosquito-borne flavivirus that has caused recent epidemics in the western hemisphere. ZIKV has been associated with severe symptoms including infant microcephaly and Guillain-Barré syndrome, stimulating interest in understanding factors governing ZIKV infection. Heat shock protein 70 (Hsp70) has been shown to be an infection factor for multiple viruses, leading us to investigate the role of Hsp70 in the ZIKV infection process.

Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod ovaries for heritable germline gene editing.

Cas9-mediated gene editing is a powerful tool for addressing research questions in arthropods. Current approaches rely upon delivering Cas9 ribonucleoprotein (RNP) complex by embryonic microinjection, which is challenging, is limited to a small number of species, and is inefficient even in optimized taxa. Here we develop a technology termed Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) to deliver Cas9 RNP to the arthropod germline by injection into adult female mosquitoes.

Gene Drive for Mosquito Control: Where Did It Come from and Where Are We Headed?

Mosquito-borne pathogens place an enormous burden on human health. The existing toolkit is insufficient to support ongoing vector-control efforts towards meeting disease elimination and eradication goals. The perspective that genetic approaches can potentially add a significant set of tools toward mosquito control is not new, but the recent improvements in site-specific gene editing with CRISPR/Cas9 systems have enhanced our ability to both study mosquito biology using reverse genetics and produce genetics-based tools.

nanos-Driven expression of piggyBac transposase induces mobilization of a synthetic autonomous transposon in the malaria vector mosquito, Anopheles stephensi.

Transposons are a class of selfish DNA elements that can mobilize within a genome. If mobilization is accompanied by an increase in copy number (replicative transposition), the transposon may sweep through a population until it is fixed in all of its interbreeding members. This introgression has been proposed as the basis for drive systems to move genes with desirable phenotypes into target species.

Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi.

Genetic engineering technologies can be used both to create transgenic mosquitoes carrying antipathogen effector genes targeting human malaria parasites and to generate gene-drive systems capable of introgressing the genes throughout wild vector populations. We developed a highly effective autonomous Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)-mediated gene-drive system in the Asian malaria vector Anopheles stephensi, adapted from the mutagenic chain reaction (MCR). This specific system results in progeny of males and females derived from transgenic males exhibiting a high frequency of germ-line gene conversion consistent with homology-directed repair (HDR).

Maternal germline-specific genes in the Asian malaria mosquito Anopheles stephensi: characterization and application for disease control.

Anopheles stephensi is a principal vector of urban malaria on the Indian subcontinent and an emerging model for molecular and genetic studies of mosquito biology. To enhance our understanding of female mosquito reproduction, and to develop new tools for basic research and for genetic strategies to control mosquito-borne infectious diseases, we identified 79 genes that displayed previtellogenic germline-specific expression based on RNA-Seq data generated from 11 life stage-specific and sex-specific samples. Analysis of this gene set provided insights into the biology and evolution of female reproduction.