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.

Y chromosomes control essential male functions in many species, including sex determination and fertility. However, because of obstacles posed by repeat-rich heterochromatin, knowledge of Y chromosome sequences is limited to a handful of model organisms, constraining our understanding of Y biology across the tree of life. Here, we leverage long single-molecule sequencing to determine the content and structure of the nonrecombining Y chromosome of the primary African malaria mosquito, Anopheles gambiae. We find that the An. gambiae Y consists almost entirely of a few massively amplified, tandemly arrayed repeats, some of which can recombine with similar repeats on the X chromosome. Sex-specific genome resequencing in a recent species radiation, the An. gambiae complex, revealed rapid sequence turnover within An. gambiae and among species. Exploiting 52 sex-specific An. gambiae RNA-Seq datasets representing all developmental stages, we identified a small repertoire of Y-linked genes that lack X gametologs and are not Y-linked in any other species except An. gambiae, with the notable exception of YG2, a candidate male-determining gene. YG2 is the only gene conserved and exclusive to the Y in all species examined, yet sequence similarity to YG2 is not detectable in the genome of a more distant mosquito relative, suggesting rapid evolution of Y chromosome genes in this highly dynamic genus of malaria vectors. The extensive characterization of the An. gambiae Y provides a long-awaited foundation for studying male mosquito biology, and will inform novel mosquito control strategies based on the manipulation of Y chromosomes.

BackgroundY chromosomes are responsible for the initiation of male development, male fertility, and other male-related functions in diverse species. However, Y genes are rarely characterized outside a few model species due to the arduous nature of studying the repeat-rich Y.ResultsThe chromosome quotient (CQ) is a novel approach to systematically discover Y chromosome genes. In the CQ method, genomic DNA from males and females is sequenced independently and aligned to candidate reference sequences. The female to male ratio of the number of alignments to a reference sequence, a parameter called the chromosome quotient (CQ), is used to determine whether the sequence is Y-linked. Using the CQ method, we successfully identified known Y sequences from Homo sapiens and Drosophila melanogaster. The CQ method facilitated the discovery of Y chromosome sequences from the malaria mosquitoes Anopheles stephensi and An. gambiae. Comparisons to transcriptome sequence data with blastn led to the discovery of six Anopheles Y genes, three from each species. All six genes are expressed in the early embryo. Two of the three An. stephensi Y genes were recently acquired from the autosomes or the X. Although An. stephensi and An. gambiae belong to the same subgenus, we found no evidence of Y genes shared between the species.ConclusionsThe CQ method can reliably identify Y chromosome sequences using the ratio of alignments from male and female sequence data. The CQ method is widely applicable to species with fragmented genome assemblies produced from next-generation sequencing data. Analysis of the six Y genes characterized in this study indicates rapid Y chromosome evolution between An. stephensi and An. gambiae. The Anopheles Y genes discovered by the CQ method provide unique markers for population and phylogenetic analysis, and opportunities for novel mosquito control measures through the manipulation of sexual dimorphism and fertility.

Paracentric chromosomal inversions are major architects of organismal evolution and have been associated with adaptations relevant to malaria transmission in anopheline mosquitoes. The processes responsible for their origin and maintenance, still poorly understood, can be illuminated by analysis of inversion breakpoint sequences. Here, we report the breakpoint structure of chromosomal inversion 2La from the principal malaria vector Anopheles gambiae and its relatives in the A. gambiae complex. The distal and proximal breakpoints of the standard (2L؉ a ) arrangement contain gene duplications: full-length genes and their truncated copies at opposite ends. Intact genes without pseudogene copies in the alternative arrangement (2La) imply that 2L؉ a is derived and was viable despite damage to genes, because duplication preserved gene function. A unique origin for the interspecific 2La inversion was challenged previously by indirect genetic evidence, but breakpoint sequences determined from members of the A. gambiae complex strongly suggest their descent from a single event. The derived position of 2L؉ a , long considered ancestral in this medically important group, has significant implications for the phylogenetic history and the evolution of vectorial capacity in the A. gambiae complex. genome evolution ͉ transposable elements ͉ malaria vectors ͉ inversion monophyly ͉ sibling species