Mosquitoes have evolved divergent sex-determining chromosomes, and they employ diverse primary signals for sex determination. As only females feed on vertebrate blood, manipulating genes involved in sex determination can facilitate genetic control measures for mosquito-borne infectious diseases, such as dengue and malaria. We highlight new advances in mosquito sex determination, describe innovative applications, and discuss relevant evolutionary insights and future directions.

Genetic biocontrol is an increasingly important way to suppress insect pest populations and to mitigate their economic and health impact. One key advantage is that it is species-specific, as it relies on the mating of released males with wild females to either suppress or modify populations. The latter is through rendering females incompetent at disease transmission. Sex separation is critical to ensure the efficiency of these control programs, and it is essential in the case of vector control to avoid releasing females that can transmit pathogens. Modern genetic methods provide the opportunity to target or manipulate components of the sex determination systems to facilitate genetic biocontrol with new means to effectively accomplish sex-specific selection, lethality, or sterility. For example, sex-specific splicing elements in genes in the sex determination pathway are used to produce sex-specific markers. Sex-linked recessive lethal alleles are used to differentially eliminate the transgene-marked sex chromosome from males to produce nontransgenic males. Knocking out or knocking down sex-specific isoforms of genes in the sex determination pathway is employed to confer female-specific lethality or sterility. Sex determination pathways and sex chromosomes are also targeted for gene drives that suppress pest populations by introducing extreme sex ratio biases. Here, we review these and other recent advances in genetic technologies for pest control that have benefited from knowledge of sex determination systems in Diptera.

Chromosomal inversions play a crucial role in evolution and have been found to regulate epidemiologically significant traits in malaria mosquitoes. However, they have not been characterized in Aedes aegypti, the primary vector of arboviruses, due to the poor structure of its polytene chromosomes. The Hi-C proximity ligation approach was used to identify chromosomal inversions in 25 strains of A. aegypti obtained from its worldwide distribution and in one strain of Aedes mascarensis. The study identified 21 multimegabase polymorphic inversions ranging in size from 5 to 55 Mbp. Inversions were more abundant in African than in non-African strains, 15 versus 3 inversions, with the highest number observed in West Africa. All inversions were grouped into two geographic clusters of African or non-African origin, suggesting their association with A. aegypti subspecies. Inversions were unevenly distributed along chromosomal arms, with the highest number found in the 1q and 3p arms homologous to the inversion-rich 2R chromosomal arm in the malaria vector Anopheles gambiae. Direct comparison of inversions between A. aegypti and An. gambiae revealed significant overlap in their genomic locations. This finding may explain the parallel evolution of the two species under similar environmental conditions. Some of the inversions colocalized with chemoreceptor genes and quantitative trait loci associated with pathogen infection, suggesting their potential role in host preference and disease transmission. Our study revealed the large pool of structural variations in the A. aegypti genome and provides the foundation for future studies of their impact on the biology of this important arboviral vector.

Genetic biocontrol methods are species-specific ways to suppress or modify pest insect populations to mitigate their economic or health impact. Successful genetic biocontrol often requires mass releases of only males of the target species. Reliable and cost-effective sex separation is a major bottleneck to the implementation of genetic biocontrol of many important species of agricultural and medical importance. Conditional selection is critical to resolving this major challenge. A diverse array of tools, such as the temperature-sensitive systems, the Gal4/UAS, QF/QUAS, and Tet-on/off bipartite systems, and the photoactivatable systems, have been established in various insect species. In this review, we focus on how various means of conditional expression have been used to achieve sex separation. We also describe other means of selection and counterselection to achieve sex separation without conditional gene expression. By providing examples across many species and discussing the strengths and weaknesses of each method, we hope to facilitate the design and application of conditional systems to improve genetic biocontrol of insect pests.

Diverse genetic strategies are being pursued to control mosquito-borne infectious diseases. These strategies often rely on the release of nonbiting males to either reduce the target mosquito population or render them resistant to pathogens. Male-only releases are important as any contaminating females can bite and potentially transmit pathogens. Despite significant efforts, it remains a major bottleneck to reliably and efficiently separate males from females, especially when nontransgenic males are preferred. In the yellow fever mosquito Aedes aegypti, sex is determined by a pair of homomorphic sex chromosomes, with the dominant male-determining locus (the M locus) and its counterpart (the m locus) embedded in an M-bearing and an m-bearing chromosome 1, respectively. We utilized both naturally occurring and engineered sex-linked recessive lethal alleles (RLAs) to create sex separation strains for Ae. aegypti on the basis of differential elimination of marked sex chromosomes (DeMark). DeMark strains are self-sustaining and produce nontransgenic males that are readily separated from individuals carrying RLA- and transgene-marked m chromosomes. For example, the marked m chromosome in the heterozygous mother in some strains was only inherited by her female progeny due to RLA-mediated incompatibility with the M-bearing chromosome in the father, producing nontransgenic males and transgenic females, generation after generation. We further explore strategies to conditionally eliminate females that contain marked sex chromosomes. We also discuss DeMark designs that are applicable for efficient sex separation in organisms with well-differentiated X and Y chromosomes, such as the Anopheles mosquitoes.

Chromosomal inversions play a fundamental role in evolution and have been shown to regulate epidemiologically important traits in malaria mosquitoes. However, they have never been characterized in Aedes aegypti, the major vector of arboviruses, because of the poor structure of its polytene chromosomes. In this study, we applied a Hi-C proximity ligation approach to identify chromosomal inversions in 25 strains of Ae. aegypti, acquired from its worldwide distribution, as well as in one strain of Ae. mascarensis. The study identified 21 multi-megabase inversions with uneven distributions along the three chromosomes. All chromosomal inversions, including one specific for Ae. mascarensis, were polymorphic. Nevertheless, geographic origin separated the strains into two clusters carrying African and non-African inversions suggesting their potential association with Ae. aegypti subspecies. Some of the inversions colocalized with chemoreceptor genes and quantitative trait loci associated with pathogen infection, implicating the potential role of inversions in host choice and disease transmission.

Background Understanding genome organization and evolution is important for species involved in transmission of human diseases, such as mosquitoes. Anophelinae and Culicinae subfamilies of mosquitoes show striking differences in genome sizes, sex chromosome arrangements, behavior, and ability to transmit pathogens. However, the genomic basis of these differences is not fully understood. Methods In this study, we used a combination of advanced genome technologies such as Oxford Nanopore Technology sequencing, Hi-C scaffolding, Bionano, and cytogenetic mapping to develop an improved chromosome-scale genome assembly for the West Nile vector Culex quinquefasciatus . Results We then used this assembly to annotate odorant receptors, odorant binding proteins, and transposable elements. A genomic region containing male-specific sequences on chromosome 1 and a polymorphic inversion on chromosome 3 were identified in the Cx. quinquefasciatus genome. In addition, the genome of Cx. quinquefasciatus was compared with the genomes of other mosquitoes such as malaria vectors An. coluzzi and An. albimanus , and the vector of arboviruses Ae. aegypti . Our work confirms significant expansion of the two chemosensory gene families in Cx. quinquefasciatus , as well as a significant increase and relocation of the transposable elements in both Cx. quinquefasciatus and Ae. aegypti relative to the Anophelines. Phylogenetic analysis clarifies the divergence time between the mosquito species. Our study provides new insights into chromosomal evolution in mosquitoes and finds that the X chromosome of Anophelinae and the sex-determining chromosome 1 of Culicinae have a significantly higher rate of evolution than autosomes. Conclusion The improved Cx. quinquefasciatus genome assembly uncovered new details of mosquito genome evolution and has the potential to speed up the development of novel vector control strategies.

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.

Insect Molecular Biology (IMB) publishes high-quality research related to the molecular biology of insects. We are a society journal, overseen by the Royal Entomological Society, which publishes IMB as part of its mission to enrich the world with insect science. Many of the IMB editors are also members of the Royal Entomological Society, working to support its mission. When you publish in or review for IMB, you are also supporting this mission. IMB seeks to publish research on a variety of subjects in molecular biology using a range of model systems. Common topics are the molecular genetics of development, insecticide resistance, behaviour, neurobiology, sociality, reproduction, immunity, genomics, ecology, evolution and symbiont interactions. Focal insects include the models Drosophila and Tribolium, as well as a range of less-studied, but economically important insects. Importantly, IMB also considers manuscripts that focus on non-insect arthropods, such as ticks and spiders. We say a reluctant good-bye and a hearty thank you to Dr. Mark Paine, who served in the Editor-in-Chief role for 6 years. We have a vibrant, diverse and high-profile global editorial board to ensure publication of high-quality original research and review articles. We are still keen to recruit new Associate Editors to the board, so please look out for recruitment opportunities coming up. IMB is a strong supporter of transparency, scientific rigour and reproducibility, and we will shortly be requiring authors to deposit their data at the revision stage and make this available to editors and reviewers. IMB welcomes both original research and review papers. Original research papers can be in long or short format. Please see the information under 'Instructions for Authors' to understand the distinction between the two. Review papers should be timely and should comprehensively review the focal field. For either submission type, the Editors-in-Chief and the Review Editor are happy to correspond regarding pre-submission inquiries. IMB also welcomes proposals for special issues. Special issues are an excellent way to make a larger impact with your work. Wiley has support services in place to help Guest Editors through every stage of the process. We particularly encourage submissions or special issue proposals that address the grand challenges in entomology (Luke et al., 2023). Methodological techniques came out strongly as a challenge as did conservation and anthropogenic impacts. Works addressing these areas and in scope for IMB are welcome.

Chikungunya virus (CHIKV) and Mayaro virus (MAYV) are closely related alphaviruses that cause acute febrile illness accompanied by an incapacitating polyarthralgia that can persist for years following initial infection. In conjunction with sporadic outbreaks throughout the sub-tropical regions of the Americas, increased global travel to CHIKV- and MAYV-endemic areas has resulted in imported cases of MAYV, as well as imported cases and autochthonous transmission of CHIKV, within the United States and Europe. With increasing prevalence of CHIKV worldwide and MAYV throughout the Americas within the last decade, a heavy focus has been placed on control and prevention programs. To date, the most effective means of controlling the spread of these viruses is through mosquito control programs. However, current programs have limitations in their effectiveness; therefore, novel approaches are necessary to control the spread of these crippling pathogens and lessen their disease burden. We have previously identified and characterized an anti-CHIKV single-domain antibody (sdAb) that potently neutralizes several alphaviruses including Ross River virus and Mayaro virus. Given the close antigenic relationship between MAYV and CHIKV, we formulated a single defense strategy to combat both emerging arboviruses: we generated transgenic Aedes aegypti mosquitoes that express two camelid-derived anti-CHIKV sdAbs. Following an infectious bloodmeal, we observed significant reduction in CHIKV and MAYV replication and transmission potential in sdAb-expressing transgenic compared to wild-type mosquitoes; thus, this strategy provides a novel approach to controlling and preventing outbreaks of these pathogens that reduce quality of life throughout the tropical regions of the world.

Aedes albopictus is one of the most invasive insect species in the world and an effective vector for many important arboviruses. We reported previously that Ae . albopictus Nix ( AalNix ) is the male-determining factor of this species. However, whether AalNix alone is sufficient to initiate male development is unknown. Transgenic lines that express each of the three AalNix isoforms from the native promoter were obtained using piggyBac transformation. We verified the stable expression of AalNix isoforms in the transgenic lines and confirm that one isoform, AalNix3&4 , is sufficient to convert females into fertile males (pseudo-males) that are indistinguishable from wild-type males. We also established a stable sex-converted female mosquito strain, AalNix3&4 -♂4-pseudo-male. The pseudo-male mosquitoes can fly and mate normally with wild-type female, although their mating competitiveness is lower than wild-type. This work further clarifies the role of AalNix in the sex determination pathway and will facilitate the development of Ae . albopictus control strategies that rely on male-only releases such as SIT and sex-ratio distortion.

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.

Insects have evolved highly diverse genetic sex-determination mechanisms and a relatively balanced male to female sex ratio is generally expected. However, selection may shift the optimal sex ratio while meiotic drive and endosymbiont manipulation can result in sex ratio distortion (SRD). Recent advances in sex chromosome genomics and CRISPR/Cas9-mediated genome editing brought significant insights into the molecular regulators of sex determination in an increasing number of insects and provided new ways to engineer SRD. We review these advances and discuss both naturally occurring and engineered SRD in the context of the Anthropocene. We emphasize SRD-mediated biological control of insects to help improve One Health, sustain agriculture, and conserve endangered species.

Mosquitoes are vectors for a number of infectious diseases. Only females feed on blood to provision for their embryos and, in doing so, transmit pathogens to the associated vertebrate hosts. Therefore, sex is an important phenotype in the context of genetic control programs, both for sex separation in the rearing facilities to avoid releasing biting females and for ways to distort the sex ratio towards nonbiting males. We review recent progress in the fundamental knowledge of sex determination and sex chromosomes in mosquitoes and discuss new methods to achieve sex separation and sex ratio distortion to help control mosquito-borne infectious diseases. We conclude by suggesting a few critical areas for future research.

Chromosomes are hierarchically folded within cell nuclei into territories, domains and subdomains, but the functional importance and evolutionary dynamics of these hierarchies are poorly defined. Here, we comprehensively profile genome organizations of five Anopheles mosquito species and show how different levels of chromatin architecture influence each other. Patterns observed on Hi-C maps are associated with known cytological structures, epigenetic profiles, and gene expression levels. Evolutionary analysis reveals conservation of chromatin architecture within synteny blocks for tens of millions of years and enrichment of synteny breakpoints in regions with increased genomic insulation. However, in-depth analysis shows a confounding effect of gene density on both insulation and distribution of synteny breakpoints, suggesting limited causal relationship between breakpoints and regions with increased genomic insulation. At the level of individual loci, we identify specific, extremely long-ranged looping interactions, conserved for ~100 million years. We demonstrate that the mechanisms underlying these looping contacts differ from previously described Polycomb-dependent interactions and clustering of active chromatin.

BACKGROUND:Aedes aegypti, the main vector of dengue, yellow fever, and other arboviruses thrives in tropical and subtropical areas around the globe putting half of the world's population at risk. Despite aggressive efforts to control the transmission of those viruses, an unacceptable number of cases occur every year, emphasizing the need to develop new control strategies. Proposals for vector control focused on population suppression could offer a feasible alternative method to reduce disease transmission. The induction of extreme male-biased sex ratios has been hypothesized to be able to suppress or collapse a population, with previous experiments showing that stable expression of the male determining factor Nix in A. aegypti is sufficient to convert females into fertile males.METHODOLOGY/PRINCIPAL FINDINGS:Here, we report on the conditional expression of Nix in transgenic A. aegypti under the control of the tetracycline-dependent (Tet-off) system, with the goal of establishing repressible sex distortion. A masculinization phenotype was observed in three of the seven transgenic lines with females exhibiting male-like long maxillary palps and most importantly, the masculinized females were unable to blood feed. Doxycycline treatment of the transgenic lines only partially restored the normal phenotype from the masculinized transgenic lines, while RT-qPCR analysis of early embryos or adults showed no correlation between the level of masculinization and ectopic Nix expression.CONCLUSIONS/SIGNIFICANCE:While the conditional expression of Nix produced intersex phenotypes, the level of expression was insufficient to program full conversion. Modifications that increase both the level of activation (no tet) and the level of repression (with tet) will be necessary, as such this study represents one step forward in the development of genetic strategies to control vector-borne diseases via sex ratio distortion.

Background The Asian tiger mosquito Aedes albopictus is globally expanding and has become the main vector for human arboviruses in Europe. With limited antiviral drugs and vaccines available, vector control is the primary approach to prevent mosquito-borne diseases. A reliable and accurate DNA sequence of the Ae. albopictus genome is essential to develop new approaches that involve genetic manipulation of mosquitoes. Results We use long-read sequencing methods and modern scaffolding techniques (PacBio, 10X, and Hi-C) to produce AalbF2, a dramatically improved assembly of the Ae. albopictus genome. AalbF2 reveals widespread viral insertions, novel microRNAs and piRNA clusters, the sex-determining locus, and new immunity genes, and enables genome-wide studies of geographically diverse Ae. albopictus populations and analyses of the developmental and stage-dependent network of expression data. Additionally, we build the first physical map for this species with 75% of the assembled genome anchored to the chromosomes. Conclusion The AalbF2 genome assembly represents the most up-to-date collective knowledge of the Ae. albopictus genome. These resources represent a foundation to improve understanding of the adaptation potential and the epidemiological relevance of this species and foster the development of innovative control measures.

BACKGROUND:Anopheles coluzzii and Anopheles arabiensis belong to the Anopheles gambiae complex and are among the major malaria vectors in sub-Saharan Africa. However, chromosome-level reference genome assemblies are still lacking for these medically important mosquito species. FINDINGS:In this study, we produced de novo chromosome-level genome assemblies for A. coluzzii and A. arabiensis using the long-read Oxford Nanopore sequencing technology and the Hi-C scaffolding approach. We obtained 273.4 and 256.8 Mb of the total assemblies for A. coluzzii and A. arabiensis, respectively. Each assembly consists of 3 chromosome-scale scaffolds (X, 2, 3), complete mitochondrion, and unordered contigs identified as autosomal pericentromeric DNA, X pericentromeric DNA, and Y sequences. Comparison of these assemblies with the existing assemblies for these species demonstrated that we obtained improved reference-quality genomes. The new assemblies allowed us to identify genomic coordinates for the breakpoint regions of fixed and polymorphic chromosomal inversions in A. coluzzii and A. arabiensis. CONCLUSION:The new chromosome-level assemblies will facilitate functional and population genomic studies in A. coluzzii and A. arabiensis. The presented assembly pipeline will accelerate progress toward creating high-quality genome references for other disease vectors.