Manipulating M factor alters mosquito sex: Female mosquitoes feed on blood and in so doing transmit pathogens to millions annually. Although the molecular mechanism for determining sex in many animals is known, the specific factors in mosquitoes have been elusive. This is because sex determination in insects involves a section of the genome that is highly repetitive. Hall et al. now identify a male-determining factor (M factor) in Aedes aegypti. Manipulation of the M factor produced sex-change phenotypes. Knocking out the gene Nix resulted in feminized males, and ectopic expression gave masculinized females. These findings should help to advance strategies for converting female mosquitoes into nonbiting males. Science , this issue p. 1268

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.

Lumpy skin disease virus (LSDV) is a vector-transmitted poxvirus that causes disease in cattle. Vector species involved in LSDV transmission and their ability to acquire and transmit the virus are poorly characterized. Using a highly representative bovine experimental model of lumpy skin disease, we fed four model vector species (Aedes aegypti, Culex quinquefasciatus, Stomoxys calcitrans, and Culicoides nubeculosus) on LSDV-inoculated cattle in order to examine their acquisition and retention of LSDV. Subclinical disease was a more common outcome than clinical disease in the inoculated cattle. Importantly, the probability of vectors acquiring LSDV from a subclinical animal (0.006) was very low compared with that from a clinical animal (0.23), meaning an insect feeding on a subclinical animal was 97% less likely to acquire LSDV than one feeding on a clinical animal. All four potential vector species studied acquired LSDV from the host at a similar rate, but Aedes aegypti and Stomoxys calcitrans retained the virus for a longer time, up to 8 days. There was no evidence of virus replication in the vector, consistent with mechanical rather than biological transmission. The parameters obtained in this study were combined with data from studies of LSDV transmission and vector life history parameters to determine the basic reproduction number of LSDV in cattle mediated by each of the model species. This reproduction number was highest for Stomoxys calcitrans (19.1), followed by C. nubeculosus (7.1) and Ae. aegypti (2.4), indicating that these three species are potentially efficient transmitters of LSDV; this information can be used to inform LSD control programs. IMPORTANCE Lumpy skin disease virus (LSDV) causes a severe systemic disease characterized by cutaneous nodules in cattle. LSDV is a rapidly emerging pathogen, having spread since 2012 into Europe and Russia and across Asia. The vector-borne nature of LSDV transmission is believed to have promoted this rapid geographic spread of the virus; however, a lack of quantitative evidence about LSDV transmission has hampered effective control of the disease during the current epidemic. Our research shows subclinical cattle play little part in virus transmission relative to clinical cattle and reveals a low probability of virus acquisition by insects at the preclinical stage. We have also calculated the reproductive number of different insect species, therefore identifying efficient transmitters of LSDV. This information is of utmost importance, as it will help to define epidemiological control measures during LSDV epidemics and of particular consequence in resource-poor regions where LSD vaccination may be less than adequate.

Morphogens are secreted signaling molecules that form concentration gradients and control cell fate in developing tissues. During development, it is essential that morphogen range is strictly regulated in order for correct cell type specification to occur. One of the best characterized morphogens is Drosophila Decapentaplegic (Dpp), a BMP signaling molecule that patterns the dorsal ectoderm of the embryo by activating the Mad and Medea (Med) transcription factors. We demonstrate that there is a spatial and temporal expansion of the expression patterns of Dpp target genes in SUMO pathway mutant embryos. We identify Med as the primary SUMOylation target in the Dpp pathway, and show that failure to SUMOylate Med leads to the increased Dpp signaling range observed in the SUMO pathway mutant embryos. Med is SUMO modified in the nucleus, and we provide evidence that SUMOylation triggers Med nuclear export. Hence, Med SUMOylation provides a mechanism by which nuclei can continue to monitor the presence of extracellular Dpp signal to activate target gene expression for an appropriate duration. Overall, our results identify an unusual strategy for regulating morphogen range that, rather than impacting on the morphogen itself, targets an intracellular transducer.

The preservation of a homomorphic sex-determining chromosome in some organisms without transformation into a heteromorphic sex chromosome is a long-standing enigma in evolutionary biology. A dominant sex-determining locus (or M-locus) in an undifferentiated homomorphic chromosome confers the male phenotype in the yellow fever mosquito Aedes aegypti. Genetic evidence suggests that the M-locus is in a nonrecombining region. However, the molecular nature of the M-locus has not been characterized. Using a recently developed approach based on Illumina sequencing of male and female genomic DNA, we identified a novel gene, myo-sex, that is present almost exclusively in the male genome but can sporadically be found in the female genome due to recombination. For simplicity, we define sequences that are primarily found in the male genome as male-biased. Fluorescence in situ hybridization (FISH) on A. aegypti chromosomes demonstrated that the myo-sex probe localized to region 1q21, the established location of the M-locus. Myo-sex is a duplicated myosin heavy chain gene that is highly expressed in the pupa and adult male. Myo-sex shares 83% nucleotide identity and 97% amino acid identity with its closest autosomal paralog, consistent with ancient duplication followed by strong purifying selection. Compared with males, myo-sex is expressed at very low levels in the females that acquired it, indicating that myo-sex may be sexually antagonistic. This study establishes a framework to discover male-biased sequences within a homomorphic sex-determining chromosome and offers new insights into the evolutionary forces that have impeded the expansion of the nonrecombining M-locus in A. aegypti.