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.

Partial masculinization of Aedes aegypti females by conditional expression of Nix.

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.

Improved reference genome of the arboviral vector Aedes albopictus.

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.

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.

Characterization of a female germline and early zygote promoter from the transcription factor bZip1 in the dengue mosquito Aedes aegypti.

BACKGROUND: The wide distribution of Aedes aegypti, the main vector of dengue and yellow fever viruses, currently puts three billion people in the world at risk of infection with these viruses. Continuous transmission of these and other viruses despite aggressive efforts to prevent this emphasizes the need to develop new control strategies. Proposals to control disease transmission based on vector engineering, including both population suppression and population replacement, rely on the development of transgenes under the control of regulatory elements able to drive molecules in a specific tissue, time and strength. METHODS: Here we report the characterization of a promoter active in both the female germline and early zygote, derived from the transcription factor bZip1 in the mosquito Ae. aegypti, using transposon-based methods and RT-qPCR. RESULTS: We generated seven transgenic lines carrying AabZip1-reporter constructs and observed expression in both the ovary and early embryo. RT-qPCR analysis was performed to evaluate transcript expression patterns for each line, confirming that transgenic expression from the AabZip1 promoter largely recapitulated the endogenous expression pattern, albeit the strength of maternal expression appeared to be strongly influenced by chromosomal position. CONCLUSIONS: This study provides a new regulatory sequence that can be useful for generating transgenic lines that can become a tool in vector control strategies.

Nix is a male-determining factor in the Asian tiger mosquito Aedes albopictus.

The initial signal that governs sex determination is highly variable among insects. A homolog of Nix, the male-determining factor in Aedes aegypti, was previously found in the Asian tiger mosquito Ae. albopictus. Here we show that the Ae. albopictus Nix (AalNix) is more complex in gene structure and splice isoforms than its Ae. aegypti homolog (AaeNix). AalNix shows a similar transcription profile compared to AaeNix. CRISPR/Cas9-mediated knockouts of AalNix in vivo and in the Ae. albopictus C6/36 cells lead to a shift of dsx and fru splicing towards the female isoforms. G0 knockout males showed feminization and deformities including feminized antennae, absence or partial absence of gonocoxites, gonostyli, testes and accessory glands, and the formation of ovaries. Despite ~70 MY of divergence, Nix functions as a conserved male-determining factor in the two most important arboviral vectors, namely Ae. aegypti and Ae. albopictus.

Pure early zygotic genes in the Asian malaria mosquito Anopheles stephensi.

BACKGROUND: The Asian malaria mosquito, Anopheles stephensi, is a major urban malaria vector in the Middle East and on the Indian subcontinent. Early zygotic transcription, which marks the maternal-to-zygotic transition, has not been systematically studied in An. stephensi or any other Anopheles mosquitoes. Improved understanding of early embryonic gene expression in An. stephensi will facilitate genetic and evolutionary studies and help with the development of novel control strategies for this important disease vector. RESULTS: We obtained RNA-seq data in biological triplicates from four early An. stephensi embryonic time points. Using these data, we identified 70 and 153 pure early zygotic genes (pEZGs) under stringent and relaxed conditions, respectively. We show that these pEZGs are enriched in functional groups related to DNA-binding transcription regulators, cell cycle modulators, proteases, transport, and cellular metabolism. On average these pEZGs are shorter and have less introns than other An. stephensi genes. Some of the pEZGs may arise de novo while others have clear non-pEZG paralogs. There is no or very limited overlap between An. stephensi pEZGs and Drosophila melanogaster or Aedes aegypti pEZGs. Interestingly, the upstream region of An. stephensi pEZGs lack significant enrichment of a previously reported TAGteam/VBRGGTA motif found in the regulatory region of pEZGs in D. melanogaster and Ae. aegypti. However, a GT-rich motif was found in An. stephensi pEZGs instead. CONCLUSIONS: We have identified a number of pEZGs whose predicted functions and structures are consistent with their collective roles in the degradation of maternally deposited components, activation of the zygotic genome, cell division, and metabolism. The pEZGs appear to rapidly turn over within the Dipteran order and even within the Culicidae family. These pEZGs, and the shared regulatory motif, could provide the promoter or regulatory sequences to drive gene expression in the syncytial or early cellular blastoderm, a period when the developing embryo is accessible to genetic manipulation. In addition, these molecular resources may be used to achieve sex separation of mosquitoes for sterile insect technique.

Complete Dosage Compensation in Anopheles stephensi and the Evolution of Sex-Biased Genes in Mosquitoes.

Complete dosage compensation refers to hyperexpression of the entire X or Z chromosome in organisms with heterogametic sex chromosomes (XY male or ZW female) in order to compensate for having only one copy of the X or Z chromosome. Recent analyses suggest that complete dosage compensation, as in Drosophila melanogaster, may not be the norm. There has been no systematic study focusing on dosage compensation in mosquitoes. However, analysis of dosage compensation in Anopheles mosquitoes provides opportunities for evolutionary insights, as the X chromosome of Anopheles and that of its Dipteran relative, D. melanogaster formed independently from the same ancestral chromosome. Furthermore, Culicinae mosquitoes, including the Aedes genus, have homomorphic sex-determining chromosomes, negating the need for dosage compensation. Thus, Culicinae genes provide a rare phylogenetic context to investigate dosage compensation in Anopheles mosquitoes. Here, we performed RNA-seq analysis of male and female samples of the Asian malaria mosquito Anopheles stephensi and the yellow fever mosquito Aedes aegypti. Autosomal and X-linked genes in An. stephensi showed very similar levels of expression in both males and females, indicating complete dosage compensation. The uniformity of average expression levels of autosomal and X-linked genes remained when An. stephensi gene expression was normalized by that of their Ae. aegypti orthologs, strengthening the finding of complete dosage compensation in Anopheles. In addition, we comparatively analyzed the differentially expressed genes between adult males and adult females in both species, investigated sex-biased gene chromosomal distribution patterns in An. stephensi and provided three examples where gene duplications may have enabled the acquisition of sex-specific expression during mosquito evolution.

SEX DETERMINATION. A male-determining factor in the mosquito Aedes aegypti.

Sex determination in the mosquito Aedes aegypti is governed by a dominant male-determining factor (M factor) located within a Y chromosome-like region called the M locus. Here, we show that an M-locus gene, Nix, functions as an M factor in A. aegypti. Nix exhibits persistent M linkage and early embryonic expression, two characteristics required of an M factor. Nix knockout with clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 resulted in largely feminized genetic males and the production of female isoforms of two key regulators of sexual differentiation: doublesex and fruitless. Ectopic expression of Nix resulted in genetic females with nearly complete male genitalia. Thus, Nix is both required and sufficient to initiate male development. This study provides a foundation for mosquito control strategies that convert female mosquitoes into harmless males.

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. Promoters from two of these candidates, vitellogenin receptor and nanos, were used in independent transgenic cassettes for the expression of artificial microRNAs against suspected mosquito maternal-effect genes, discontinuous actin hexagon and myd88. We show these promoters have early germline-specific expression and demonstrate 73% and 42% knockdown of myd88 and discontinuous actin hexagon mRNA in ovaries 48 hr after blood meal, respectively. Additionally, we demonstrate maternal-specific delivery of mRNA and protein to progeny embryos. We discuss the application of this system of maternal delivery of mRNA/miRNA/protein in research on mosquito reproduction and embryonic development, and for the development of a gene drive system based on maternal-effect dominant embryonic arrest.

Identification of early zygotic genes in the yellow fever mosquito Aedes aegypti and discovery of a motif involved in early zygotic genome activation.

During early embryogenesis the zygotic genome is transcriptionally silent and all mRNAs present are of maternal origin. The maternal-zygotic transition marks the time over which embryogenesis changes its dependence from maternal RNAs to zygotically transcribed RNAs. Here we present the first systematic investigation of early zygotic genes (EZGs) in a mosquito species and focus on genes involved in the onset of transcription during 2-4 hr. We used transcriptome sequencing to identify the "pure" (without maternal expression) EZGs by analyzing transcripts from four embryonic time ranges of 0-2, 2-4, 4-8, and 8-12 hr, which includes the time of cellular blastoderm formation and up to the start of gastrulation. Blast of 16,789 annotated transcripts vs. the transcriptome reads revealed evidence for 63 (P<0.001) and 143 (P<0.05) nonmaternally derived transcripts having a significant increase in expression at 2-4 hr. One third of the 63 EZG transcripts do not have predicted introns compared to 10% of all Ae. aegypti genes. We have confirmed by RT-PCR that zygotic transcription starts as early as 2-3 hours. A degenerate motif VBRGGTA was found to be overrepresented in the upstream sequences of the identified EZGs using a motif identification software called SCOPE. We find evidence for homology between this motif and the TAGteam motif found in Drosophila that has been implicated in EZG activation. A 38 bp sequence in the proximal upstream sequence of a kinesin light chain EZG (KLC2.1) contains two copies of the mosquito motif. This sequence was shown to support EZG transcription by luciferase reporter assays performed on injected early embryos, and confers early zygotic activity to a heterologous promoter from a divergent mosquito species. The results of these studies are consistent with the model of early zygotic genome activation via transcriptional activators, similar to what has been found recently in Drosophila.

Evolutionary analysis of the kinesin light chain genes in the yellow fever mosquito Aedes aegypti: gene duplication as a source for novel early zygotic genes.

BACKGROUND: The maternal zygotic transition marks the time at which transcription from the zygotic genome is initiated and a subset of maternal RNAs are progressively degraded in the developing embryo. A number of early zygotic genes have been identified in Drosophila melanogaster and comparisons to sequenced mosquito genomes suggest that some of these early zygotic genes such as bottleneck are fast-evolving or subject to turnover in dipteran insects. One objective of this study is to identify early zygotic genes from the yellow fever mosquito Aedes aegypti to study their evolution. We are also interested in obtaining early zygotic promoters that will direct transgene expression in the early embryo as part of a Medea gene drive system. RESULTS: Two novel early zygotic kinesin light chain genes we call AaKLC2.1 and AaKLC2.2 were identified by transcriptome sequencing of Aedes aegypti embryos at various time points. These two genes have 98% nucleotide and amino acid identity in their coding regions and show transcription confined to the early zygotic stage according to gene-specific RT-PCR analysis. These AaKLC2 genes have a paralogous gene (AaKLC1) in Ae. aegypti. Phylogenetic inference shows that an ortholog to the AaKLC2 genes is only found in the sequenced genome of Culex quinquefasciatus. In contrast, AaKLC1 gene orthologs are found in all three sequenced mosquito species including Anopheles gambiae. There is only one KLC gene in D. melanogaster and other sequenced holometabolous insects that appears to be similar to AaKLC1. Unlike AaKLC2, AaKLC1 is expressed in all life stages and tissues tested, which is consistent with the expression pattern of the An. gambiae and D. melanogaster KLC genes. Phylogenetic inference also suggests that AaKLC2 genes and their likely C. quinquefasciatus ortholog are fast-evolving genes relative to the highly conserved AaKLC1-like paralogs. Embryonic injection of a luciferase reporter under the control of a 1 kb fragment upstream of the AaKLC2.1 start codon shows promoter activity at least as early as 3 hours in the developing Ae. aegypti embryo. The AaKLC2.1 promoter activity reached ~1600 fold over the negative control at 5 hr after egg deposition. CONCLUSIONS: Transcriptome profiling by use of high throughput sequencing technologies has proven to be a valuable method for the identification and discovery of early and transient zygotic genes. The evolutionary investigation of the KLC gene family reveals that duplication is a source for the evolution of new genes that play a role in the dynamic process of early embryonic development. AaKLC2.1 may provide a promoter for early zygotic-specific transgene expression, which is a key component of the Medea gene drive system.

Evolution and horizontal transfer of a DD37E DNA transposon in mosquitoes.

ITmD37E, a unique class II transposable element (TE) with an ancient origin, appears to have been involved in multiple horizontal transfers in mosquitoes as ITmD37E sequences from 10 mosquito species of five genera share high nucleotide (nt) identities. For example, ITmD37E sequences from Aedes aegypti and Anopheles gambiae, which have an estimated common ancestor of 145-200 million years ago, display 92% nt identity. The comparison of ITmD37E and host mosquito phylogenies shows a lack of congruence. The wide distribution of conserved ITmD37Es in mosquitoes and the presence of intact copies suggest that this element may have been recently active.

The Juan non-LTR retrotransposon in mosquitoes: genomic impact, vertical transmission and indications of recent and widespread activity.

BACKGROUND: In contrast to DNA-mediated transposable elements (TEs), retrotransposons, particularly non-long terminal repeat retrotransposons (non-LTRs), are generally considered to have a much lower propensity towards horizontal transfer. Detailed studies on site-specific non-LTR families have demonstrated strict vertical transmission. More studies are needed with non-site-specific non-LTR families to determine whether strict vertical transmission is a phenomenon related to site specificity or a more general characteristic of all non-LTRs. Juan is a Jockey clade non-LTR retrotransposon first discovered in mosquitoes that is widely distributed in the mosquito family Culicidae. Being a non-site specific non-LTR, Juan offers an opportunity to further investigate the hypothesis that non-LTRs are genomic elements that are primarily vertically transmitted. RESULTS: Systematic analysis of the ~1.3 Gbp Aedes aegypti (Ae. aegypti) genome sequence suggests that Juan-A is the only Juan-type non-LTR in Aedes aegypti. Juan-A is highly reiterated and comprises approximately 3% of the genome. Using minimum cutoffs of 90% length and 70% nucleotide (nt) identity, 663 copies were found by BLAST using the published Juan-A sequence as the query. All 663 copies are at least 95% identical to Juan-A, while 378 of these copies are 99% identical to Juan-A, indicating that the Juan-A family has been transposing recently in evolutionary history. Using the 0.34 Kb 5' UTR as the query, over 2000 copies were identified that may contain internal promoters, leading to questions on the genomic impact of Juan-A. Juan sequences were obtained by PCR, library screening, and database searches for 18 mosquito species of six genera including Aedes, Ochlerotatus, Psorophora, Culex, Deinocerites, and Wyeomyia. Comparison of host and Juan phylogenies shows overall congruence with few exceptions. CONCLUSION: Juan-A is a major genomic component in Ae. aegypti and it has been retrotransposing recently in evolutionary history. There are also indications that Juan has been recently active in a wide range of mosquito species. Furthermore, our research demonstrates that a Jockey clade non-LTR without target site-specificity has been sustained by vertical transmission in the mosquito family. These results strengthen the argument that non-LTRs tend to be genomic elements capable of persistence by vertical descent over a long evolutionary time.