Silencing of the Hsf gene, the transcriptional regulator of A. gambiae male accessory glands, inhibits the formation of the mating plug in mated females and disrupts their monogamous behaviour

Discovering the molecular factors that shape the mating behaviour and the fertility of the mosquito Anopheles gambiae, the principal vector of human malaria, is regarded as critical to better understand its reproductive success as well as for identifying new leads for malaria control measures. In A. gambiae mating induces complex behavioural and physiological changes in the females, including refractoriness to subsequent mating and induction of egg-laying. In other insects including Drosophila a group of proteins named Accessory gland proteins (Acps), produced by males and transferred with sperm to the female reproductive tract, have been implicated in this post-mating response. Although Acps represent a set of promising candidates for unravelling the mating physiology, their role in inducing behavioural changes in mated A. gambiae females remains largely unknown. In this work, we demonstrate that a down-regulation of a large fraction of Acp genes via silencing of the Acp regulating transcription factor Hsf, abolishes the formation of mating plug in mated females and fails to induce refractoriness of mated female to subsequent inseminations. A significant fraction of females mated to Hsf silenced males (66%) failed to receive the mating plug though seminal fluid had been transferred as documented by the presence of spermatozoa in the female sperm storage organ. Furthermore, nearly all females (95%) mated to HSF-silenced males were re-inseminated when exposed to males carrying EGPF marked sperm. Our findings provide evidence showing that Acp genes regulated by the transcription factor HSF play a key role in the function of the male accessory glands

Rapid evolution of female-biased genes among four species of Anopheles malaria mosquitoes.

Understanding how phenotypic differences between males and females arise from the sex-biased expression of nearly identical genomes can reveal important insights into the biology and evolution of a species. Among Anopheles mosquito species, these phenotypic differences include vectorial capacity, as it is only females that blood feed and thus transmit human malaria. Here, we use RNA-seq data from multiple tissues of four vector species spanning the Anopheles phylogeny to explore the genomic and evolutionary properties of sex-biased genes. We find that, in these mosquitoes, in contrast to what has been found in many other organisms, female-biased genes are more rapidly evolving in sequence, expression, and genic turnover than male-biased genes. Our results suggest that this atypical pattern may be due to the combination of sex-specific life history challenges encountered by females, such as blood feeding. Furthermore, female propensity to mate only once in nature in male swarms likely diminishes sexual selection of post-reproductive traits related to sperm competition among males. We also develop a comparative framework to systematically explore tissue- and sex-specific splicing to document its conservation throughout the genus and identify a set of candidate genes for future functional analyses of sex-specific isoform usage. Finally, our data reveal that the deficit of male-biased genes on the X Chromosomes in Anopheles is a conserved feature in this genus and can be directly attributed to chromosome-wide transcriptional regulation that de-masculinizes the X in male reproductive tissues

Gene-drive suppression of mosquito populations in large cages as a bridge between lab and field.

CRISPR-based gene-drives targeting the gene doublesex in the malaria vector Anopheles gambiae effectively suppressed the reproductive capability of mosquito populations reared in small laboratory cages. To bridge the gap between laboratory and the field, this gene-drive technology must be challenged with vector ecology.Here we report the suppressive activity of the gene-drive in age-structured An. gambiae populations in large indoor cages that permit complex feeding and reproductive behaviours.The gene-drive element spreads rapidly through the populations, fully supresses the population within one year and without selecting for resistance to the gene drive. Approximate Bayesian computation allowed retrospective inference of life-history parameters from the large cages and a more accurate prediction of gene-drive behaviour under more ecologically-relevant settings.Generating data to bridge laboratory and field studies for invasive technologies is challenging. Our study represents a paradigm for the stepwise and sound development of vector control tools based on gene-drive

Stimulating Anopheles gambiae swarms in the laboratory: application for behavioural and fitness studies

Male Anopheles mosquitoes that swarm rely in part on features of the environment including visual stimuli to locate swarms. Swarming is believed to be the primary behaviour during which mating occurs in the field, but is not a common behaviour in the laboratory. Features that stimulate male Anopheles gambiae G3 strain swarming were created in novel large indoor cages

Large-cage assessment of a transgenic sex-ratio distortion strain on populations of an African malaria vector

Background Novel transgenic mosquito control methods require progressively more realistic evaluation. The goal of this study was to determine the effect of a transgene that causes a male-bias sex ratio on Anopheles gambiae target populations in large insectary cages. Methods Life history characteristics of Anopheles gambiae wild type and Ag(PMB)1 (aka gfp124L-2) transgenic mosquitoes, whose progeny are 95% male, were measured in order to parameterize predictive population models. Ag(PMB)1 males were then introduced at two ratios into large insectary cages containing target wild type populations with stable age distributions and densities. The predicted proportion of females and those observed in the large cages were compared. A related model was then used to predict effects of male releases on wild mosquitoes in a west African village. Results The frequency of transgenic mosquitoes in target populations reached an average of 0.44 ± 0.02 and 0.56 ± 0.02 after 6 weeks in the 1:1 and in the 3:1 release ratio treatments (transgenic male:wild male) respectively. Transgenic males caused sex-ratio distortion of 73% and 80% males in the 1:1 and 3:1 treatments, respectively. The number of eggs laid in the transgenic treatments declined as the experiment progressed, with a steeper decline in the 3:1 than in the 1:1 releases. The results of the experiment are partially consistent with predictions of the model; effect size and variability did not conform to the model in two out of three trials, effect size was over-estimated by the model and variability was greater than anticipated, possibly because of sampling effects in restocking. The model estimating the effects of hypothetical releases on the mosquito population of a West African village demonstrated that releases could significantly reduce the number of females in the wild population. The interval of releases is not expected to have a strong effect. Conclusions The biological data produced to parameterize the model, the model itself, and the results of the experiments are components of a system to evaluate and predict the performance of transgenic mosquitoes. Together these suggest that the Ag(PMB)1 strain has the potential to be useful for reversible population suppression while this novel field develops.</p

Data from: Detecting the population dynamics of an autosomal sex-ratio distorter transgene in malaria vector mosquitoes

1. The development of genetically modified mosquitoes and their subsequent field release offers innovative and cost-effective approaches to reduce mosquito-borne diseases, such as malaria. A sex-distorting autosomal transgene has been developed recently in G3 mosquitoes, a lab strain of the malaria vector Anopheles gambiae s.l. The transgene expresses an endonuclease called I-PpoI during spermatogenesis, which selectively cleaves the X chromosome to result in ~95% male progeny. Following the World Health Organization Guidance Framework for the testing of genetically modified mosquitoes, we assessed the dynamics of this transgene in large cages using a joint experimental-modelling approach. 2. We performed a four-month experiment in indoor large cages to study the population genetics of the transgene. The cages were set up to mimic a simple tropical environment with a diurnal light-cycle, constant temperature, and constant humidity. We allowed the generations to overlap to engender a stable age structure in the populations. We constructed a model to mimic the experiments, and used the experimental data to infer the key model parameters. 3. We identified two fitness costs associated to the transgene. First, transgenic adult males have reduced fertility and, second, their female progeny have reduced pupal survival rates. Our results demonstrate that the transgene is likely to disappear in less than three years under our confined conditions. Model predictions suggest this will be true over a wide range of background population sizes and transgene introduction rates. 4. Synthesis and applications: Our semi-field indoor cage experiments are in line with WHO guidance recommendations in regards to the development and testing of self-limiting technologies. Since the transgenic strain (Ag(PMB)1) has been considered for genetic vector control of malaria, our results are fundamentally important for determining expectations on the persistence of the transgene post-release. Our results provide a demonstration of the self-limiting nature of the transgene, and indicate that longevity will be further reduced by fitness costs that were not previously identified. Finally, our study has showcased an alternative and effective method for characterising the phenotypic expression of a transgene in an insect pest population.1. The development of genetically modified mosquitoes and their subsequent field release offers innovative and cost-effective approaches to reduce mosquito-borne diseases, such as malaria. A sex-distorting autosomal transgene has been developed recently in G3 mosquitoes, a lab strain of the malaria vector Anopheles gambiae s.l. The transgene expresses an endonuclease called I-PpoI during spermatogenesis, which selectively cleaves the X chromosome to result in ~95% male progeny. Following the World Health Organization Guidance Framework for the testing of genetically modified mosquitoes, we assessed the dynamics of this transgene in large cages using a joint experimental-modelling approach. 2. We performed a four-month experiment in indoor large cages to study the population genetics of the transgene. The cages were set up to mimic a simple tropical environment with a diurnal light-cycle, constant temperature, and constant humidity. We allowed the generations to overlap to engender a stable age structure in the populations. We constructed a model to mimic the experiments, and used the experimental data to infer the key model parameters. 3. We identified two fitness costs associated to the transgene. First, transgenic adult males have reduced fertility and, second, their female progeny have reduced pupal survival rates. Our results demonstrate that the transgene is likely to disappear in less than three years under our confined conditions. Model predictions suggest this will be true over a wide range of background population sizes and transgene introduction rates. 4. Synthesis and applications: Our semi-field indoor cage experiments are in line with WHO guidance recommendations in regards to the development and testing of self-limiting technologies. Since the transgenic strain (Ag(PMB)1) has been considered for genetic vector control of malaria, our results are fundamentally important for determining expectations on the persistence of the transgene post-release. Our results provide a demonstration of the self-limiting nature of the transgene, and indicate that longevity will be further reduced by fitness costs that were not previously identified. Finally, our study has showcased an alternative and effective method for characterising the phenotypic expression of a transgene in an insect pest population