On the Origin and Evolution of the Mosquito Male-determining Factor Nix.

The mosquito family Culicidae is divided into 2 subfamilies named the Culicinae and Anophelinae. Nix, the dominant male-determining factor, has only been found in the culicines Aedes aegypti and Aedes albopictus, 2 important arboviral vectors that belong to the subgenus Stegomyia. Here we performed sex-specific whole-genome sequencing and RNAseq of divergent mosquito species and explored additional male-inclusive datasets to investigate the distribution of Nix. Except for the Culex genus, Nix homologs were found in all species surveyed from the Culicinae subfamily, including 12 additional species from 3 highly divergent tribes comprising 4 genera, suggesting Nix originated at least 133 to 165 million years ago (MYA). Heterologous expression of 1 of 3 divergent Nix open reading frames (ORFs) in Ae. aegypti resulted in partial masculinization of genetic females as evidenced by morphology and doublesex splicing. Phylogenetic analysis suggests Nix is related to femaleless (fle), a recently described intermediate sex-determining factor found exclusively in anopheline mosquitoes. Nix from all species has a conserved structure, including 3 RNA-recognition motifs (RRMs), as does fle. However, Nix has evolved at a much faster rate than fle. The RRM3 of both Nix and fle are distantly related to the single RRM of a widely distributed and conserved splicing factor transformer-2 (tra2). The RRM3-based phylogenetic analysis suggests this domain in Nix and fle may have evolved from tra2 or a tra2-related gene in a common ancestor of mosquitoes. Our results provide insights into the evolution of sex determination in mosquitoes and will inform broad applications of mosquito-control strategies based on manipulating sex ratios toward nonbiting males.

Aedes aegypti D7 long salivary proteins modulate blood feeding and parasite infection.

Mosquito saliva facilitates blood meal acquisition through pharmacologically active compounds that prevent host hemostasis and immune responses. Here, we generated two knockout (KO) mosquito lines by CRISPR/Cas9 to functionally characterize D7L1 and D7L2, two abundantly expressed salivary proteins from the yellow fever mosquito vector Aedes aegypti. The D7s bind and scavenge biogenic amines and eicosanoids involved in hemostasis at the bite site. The absence of D7 proteins in the salivary glands of KO mosquitoes was confirmed by mass spectrometry, enzyme-linked immunosorbent assay, and fluorescence microscopy of the salivary glands with specific antibodies. D7-KO mosquitoes had longer probing times than parental wildtypes. The differences in probing time were abolished when mutant mice resistant to inflammatory insults were used. These results confirmed the role of D7 proteins as leukotriene scavengers in vivo. We also investigated the role of D7 salivary proteins in Plasmodium gallinaceum infection and transmission. Both KO lines had significantly fewer oocysts per midgut. We hypothesize that the absence of D7 proteins in the midgut of KO mosquitoes might be responsible for creating a harsh environment for the parasite. The information generated by this work highlights the biological functionality of salivary gene products in blood feeding and pathogen infection. IMPORTANCE During blood feeding, mosquitoes inject saliva into the host skin, preventing hemostasis and inflammatory responses. D7 proteins are among the most abundant components of the saliva of blood-feeding arthropods. Aedes aegypti, the vector of yellow fever and dengue, expresses two D7 long-form salivary proteins: D7L1 and D7L2. These proteins bind and counteract hemostatic agonists such as biogenic amines and leukotrienes. D7L1 and D7L2 knockout mosquitoes showed prolonged probing times and carried significantly less Plasmodium gallinaceum oocysts per midgut than wild-type mosquitoes. We hypothesize that reingested D7s play a vital role in the midgut microenvironment with important consequences for pathogen infection and transmission.

Marker-assisted mapping enables forward genetic analysis in Aedes aegypti, an arboviral vector with vast recombination deserts.

Aedes aegypti is a major vector of arboviruses that cause dengue, chikungunya, yellow fever, and Zika. Although recent success in reverse genetics has facilitated rapid progress in basic and applied research, integration of forward genetics with modern technologies remains challenging in this important species, as up to 47% of its chromosome is refractory to genetic mapping due to extremely low rate of recombination. Here, we report the development of a marker-assisted mapping strategy to readily screen for and genotype only the rare but informative recombinants, drastically increasing both the resolution and signal-to-noise ratio. Using marker-assisted mapping, we mapped a transgene that was inserted in a >100-Mb recombination desert and a sex-linked spontaneous red-eye (re) mutation just outside the region. We subsequently determined, by CRISPR/Cas9-mediated knockout, that cardinal is the causal gene of re, which is the first forward genetic identification of a causal gene in Ae. aegypti. The identification of the causal gene of the sex-linked re mutation provides the molecular foundation for using gene editing to develop versatile and stable genetic sexing methods. To facilitate genome-wide forward genetics in Ae. aegypti, we generated and compiled a number of lines with markers throughout the genome. Thus, by overcoming the challenges presented by the vast recombination deserts and the scarcity of markers, we have shown that effective forward genetic analysis is increasingly feasible in this important arboviral vector species.

Aedes aegypti sialokinin facilitates mosquito blood feeding and modulates host immunity and vascular biology.

Saliva from mosquitoes contains vasodilators that antagonize vasoconstrictors produced at the bite site. Sialokinin is a vasodilator present in the saliva of Aedes aegypti. Here, we investigate its function and describe its mechanism of action during blood feeding. Sialokinin induces nitric oxide release similar to substance P. Sialokinin-KO mosquitoes produce lower blood perfusion than parental mosquitoes at the bite site during probing and have significantly longer probing times, which result in lower blood feeding success. In contrast, there is no difference in feeding between KO and parental mosquitoes when using artificial membrane feeders or mice that are treated with a substance P receptor antagonist, confirming that sialokinin interferes with host hemostasis via NK1R signaling. While sialokinin-KO saliva does not affect virus infection in vitro, it stimulates macrophages and inhibits leukocyte recruitment in vivo. This work highlights the biological functionality of salivary proteins in blood feeding.

Nix alone is sufficient to convert female Aedes aegypti into fertile males and myo-sex is needed for male flight.

A dominant male-determining locus (M-locus) establishes the male sex (M/m) in the yellow fever mosquito, Aedes aegyptiNix, a gene in the M-locus, was shown to be a male-determining factor (M factor) as somatic knockout of Nix led to feminized males (M/m) while transient expression of Nix resulted in partially masculinized females (m/m), with male reproductive organs but retained female antennae. It was not clear whether any of the other 29 genes in the 1.3-Mb M-locus are also needed for complete sex-conversion. Here, we report the generation of multiple transgenic lines that express Nix under the control of its own promoter. Genetic and molecular analyses of these lines provided insights unattainable from previous transient experiments. We show that the Nix transgene alone, in the absence of the M-locus, was sufficient to convert females into males with all male-specific sexually dimorphic features and male-like gene expression. The converted m/m males are flightless, unable to perform the nuptial flight required for mating. However, they were able to father sex-converted progeny when presented with cold-anesthetized wild-type females. We show that myo-sex, a myosin heavy-chain gene also in the M-locus, was required for male flight as knockout of myo-sex rendered wild-type males flightless. We also show that Nix-mediated female-to-male conversion was 100% penetrant and stable over many generations. Therefore, Nix has great potential for developing mosquito control strategies to reduce vector populations by female-to-male sex conversion, or to aid in a sterile insect technique that requires releasing only non-biting males.

A mosquito juvenile hormone binding protein (mJHBP) regulates the activation of innate immune defenses and hemocyte development.

Insects rely on the innate immune system for defense against pathogens, some aspects of which are under hormonal control. Here we provide direct experimental evidence showing that the juvenile hormone-binding protein (mJHBP) of Aedes aegypti is required for the regulation of innate immune responses and the development of mosquito blood cells (hemocytes). Using an mJHBP-deficient mosquito line generated by means of CRISPR-Cas9 gene editing technology we uncovered a mutant phenotype characterized by immunosuppression at the humoral and cellular levels, which profoundly affected susceptibility to bacterial infection. Bacteria-challenged mosquitoes exhibited significantly higher levels of septicemia and mortality relative to the wild type (WT) strain, delayed expression of antimicrobial peptides (AMPs), severe developmental dysregulation of embryonic and larval hemocytes (reduction in the total number of hemocytes) and increased differentiation of the granulocyte lineage. Interestingly, injection of recombinant wild type mJHBP protein into adult females three-days before infection was sufficient to restore normal immune function. Similarly, injection of mJHBP into fourth-instar larvae fully restored normal larval/pupal hemocyte populations in emerging adults. More importantly, the recovery of normal immuno-activation and hemocyte development requires the capability of mJHBP to bind JH III. These results strongly suggest that JH III functions in mosquito immunity and hemocyte development in a manner that is perhaps independent of canonical JH signaling, given the lack of developmental and reproductive abnormalities. Because of the prominent role of hemocytes as regulators of mosquito immunity, this novel discovery may have broader implications for the understanding of vector endocrinology, hemocyte development, vector competence and disease transmission.

Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9.

BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology has rapidly emerged as a very effective tool for gene editing. Although great advances on gene editing in the medical entomology field have arisen, no attempts of gene editing have been reported in sand flies, the vectors of Leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we described a detailed protocol for sand fly embryo microinjection taking into consideration the sand fly life cycle, and manipulation and oviposition requirements of this non-model organism. Following our microinjection protocol, a hatching rate of injected embryos of 11.90%-14.22% was achieved, a rate consistent with other non-model organism dipterans such as mosquitoes. Essential factors for the adaptation of CRISPR/Cas9 technology to the sand fly field were addressed including the selection of a target gene and the design and production of sgRNA. An in vitro cleavage assay was optimized to test the activity of each sgRNA and a protocol for Streptococcus pyogenes Cas9 (spCas9) protein expression and purification was described. Relevant considerations for a successful gene editing in the sand fly such as specifics of embryology and double-stranded break DNA repair mechanisms were discussed. CONCLUSION AND SIGNIFICANCE: The step-by-step methodology reported in this article will be of significant use for setting up a sand fly embryo microinjection station for the incorporation of CRISPR/Cas9 technology in the sand fly field. Gene editing strategies used in mosquitoes and other model insects have been adapted to work with sand flies, providing the tools and relevant information for adapting gene editing techniques to the vectors of Leishmaniasis. Gene editing in sand flies will provide essential information on the biology of these vectors of medical and veterinary relevance and will rise a better understanding of vector-parasite-host interactions.

Methods for TALEN Evaluation, Use, and Mutation Detection in the Mosquito Aedes aegypti.

The generation and study of transgenic Aedes aegypti mosquitoes provides an essential tool for elucidating the complex molecular biology of this important vector. Within the field, genetic manipulation has surpassed the proof of principle stage and is now utilized in both applied and theoretical vector control strategies. The application of new instruments, technologies and techniques allows ever more controlled experiments to be conducted. In this text we describe microinjection of Ae. aegypti embryos in the context of evaluating and performing genomic editing with transcription activator-like effector nucleases (TALENs).

Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti.

Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that reprogrammable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24-90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2-3%, similar to traditional transposon- or ΦC31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems.

Understanding the DNA damage response in order to achieve desired gene editing outcomes in mosquitoes.

Mosquitoes are high-impact disease vectors with the capacity to transmit pathogenic agents that cause diseases such as malaria, yellow fever, chikungunya, and dengue. Continued growth in knowledge of genetic, molecular, and physiological pathways in mosquitoes allows for the development of novel control methods and for the continued optimization of existing ones. The emergence of site-specific nucleases as genomic engineering tools promises to expedite research of crucial biological pathways in these disease vectors. The utilization of these nucleases in a more precise and efficient manner is dependent upon knowledge and manipulation of the DNA repair pathways utilized by the mosquito. While progress has been made in deciphering DNA repair pathways in some model systems, research into the nature of the hierarchy of mosquito DNA repair pathways, as well as in mechanistic differences that may exist, is needed. In this review, we will describe progress in the use of site-specific nucleases in mosquitoes, along with the hierarchy of DNA repair in the context of mosquito chromosomal organization and structure, and how this knowledge may be manipulated to achieve precise chromosomal engineering in mosquitoes.

Targeted genome editing in Aedes aegypti using TALENs.

The Culicine mosquito, Aedes aegypti, is both a major vector of arthropod-borne viruses (arboviruses) and a genetic model organism for arbovirus transmission. TALE nucleases (TALENs), a group of artificial enzymes capable of generating site-specific DNA lesions, consist of a non-specific FokI endonuclease cleavage domain fused to an engineered DNA binding domain specific to a target site. While TALENs have become an important tool for targeted gene disruption in a variety of organisms, application to the mosquito genome is a new approach. We recently described the use of TALENs to perform heritable genetic disruptions in A. aegypti. Here, we provide detailed methods that will allow other research laboratories to capitalize on the potential of this technology for understanding mosquito gene function. We describe target site selection, transient embryo-based assays to rapidly assess TALEN activity, embryonic microinjection and downstream screening steps to identify target site mutations.

Germline excision of transgenes in Aedes aegypti by homing endonucleases.

Aedes (Ae.) aegypti is the primary vector for dengue viruses (serotypes1-4) and chikungunya virus. Homing endonucleases (HEs) are ancient selfish elements that catalyze double-stranded DNA breaks (DSB) in a highly specific manner. In this report, we show that the HEs Y2-I-AniI, I-CreI and I-SceI are all capable of catalyzing the excision of genomic segments from the Ae. aegypti genome in a heritable manner. Y2-I-AniI demonstrated the highest efficiency at two independent genomic targets, with 20-40% of Y2-I-AniI-treated individuals producing offspring that had lost the target transgene. HE-induced DSBs were found to be repaired via the single-strand annealing (SSA) and non-homologous end-joining (NHEJ) pathways in a manner dependent on the availability of direct repeat sequences in the transgene. These results support the development of HE-based gene editing and gene drive strategies in Ae. aegypti, and confirm the utility of HEs in the manipulation and modification of transgenes in this important vector.

TALEN-based gene disruption in the dengue vector Aedes aegypti.

In addition to its role as the primary vector for dengue viruses, Aedes aegypti has a long history as a genetic model organism for other bloodfeeding mosquitoes, due to its ease of colonization, maintenance and reproductive productivity. Though its genome has been sequenced, functional characterization of many Ae. aegypti genes, pathways and behaviors has been slow. TALE nucleases (TALENs) have been used with great success in a number of organisms to generate site-specific DNA lesions. We evaluated the ability of a TALEN pair to target the Ae. aegypti kmo gene, whose protein product is essential in the production of eye pigmentation. Following injection into pre-blastoderm embryos, 20-40% of fertile survivors produced kmo alleles that failed to complement an existing kh(w) mutation. Most of these individuals produced more than 20% white-eyed progeny, with some producing up to 75%. Mutant alleles were associated with lesions of 1-7 bp specifically at the selected target site. White-eyed individuals could also be recovered following a blind intercross of G1 progeny, yielding several new white-eyed strains in the genetic background of the sequenced Liverpool strain. We conclude that TALENs are highly active in the Ae. aegypti germline, and have the potential to transform how reverse genetic experiments are performed in this important disease vector.