Search

Only female insects transmit diseases such as malaria, dengue and Zika; therefore, control methods that bias the sex ratio of insect offspring have long been sought. Genetic elements such as sex-chromosome drives can distort sex ratios to produce unisex populations that eventually collapse, but the underlying molecular mechanisms are unknown. We report a male-biased sex-distorter gene drive (SDGD) in the human malaria vector Anopheles gambiae. We induced super-Mendelian inheritance of the X-chromosome-shredding I-PpoI nuclease by coupling this to a CRISPR-based gene drive inserted into a conserved sequence of the doublesex (dsx) gene. In modeling of invasion dynamics, SDGD was predicted to have a quicker impact on female mosquito populations than previously developed gene drives targeting female fertility. The SDGD at the dsx locus led to a male-only population from a 2.5% starting allelic frequency in 10–14 generations, with population collapse and no selection for resistance. Our results support the use of SDGD for malaria vector control., A sex-distorter gene drive causes population collapse in the malaria mosquito.

We evaluated the associations between marine recreational fishing, stress, seafood consumption, and sleep quality in a cross-sectional questionnaire-based survey of a convenience sample of 244 fishers recruited in 2019 in Spain. Fishers' stress levels were moderate, with a mean stress index score of 36.4 units on a scale from 14 (very low stress) to 70 (very high). Their average emotional condition was positive, with a mean index of negative affect of 7.8 units on a scale from 5 (very low negative affect) to 25 (very high). Seafood intake was low, with a mean index of seafood in diets of 38.0 units on a scale from 20 (very low seafood consumption) to 160 (very high). Fishers' perceived quality of night sleep was good because the mean index of sleep problems was 39.5 units on a scale from 21 (very low sleep problems) to 107 (very high). Each hour of self-reported monthly fishing activity was associated with 0.016 units of lower stress score. Thus, the most engaged fishers reported up to 15.4% lower stress score than less avid fishers. Since recreational fishing is a highly accessible outdoor activity for people in older age groups, it is possible that public health could be improved by access to sustainably managed recreational fisheries. Fishing engagement was positively associated with seafood intake. Each hour of fishing per month was associated with one-unit higher seafood consumption. The higher seafood consumption observed among avid recreational fishers compared with less avid fishers might have health implications.

CRISPR-based homing gene drives can be designed to disrupt essential genes whilst biasing their own inheritance, leading to suppression of mosquito populations in the laboratory. This class of gene drives relies on CRISPR-Cas9 cleavage of a target sequence and copying (‘homing’) therein of the gene drive element from the homologous chromosome. However, target site mutations that are resistant to cleavage yet maintain the function of the essential gene are expected to be strongly selected for. Targeting functionally constrained regions where mutations are not easily tolerated should lower the probability of resistance. Evolutionary conservation at the sequence level is often a reliable indicator of functional constraint, though the actual level of underlying constraint between one conserved sequence and another can vary widely. Here we generated a novel adult lethal gene drive (ALGD) in the malaria vector Anopheles gambiae, targeting an ultra-conserved target site in a haplosufficient essential gene (AGAP029113) required during mosquito development, which fulfils many of the criteria for the target of a population suppression gene drive. We then designed a selection regime to experimentally assess the likelihood of generation and subsequent selection of gene drive resistant mutations at its target site. We simulated, in a caged population, a scenario where the gene drive was approaching fixation, where selection for resistance is expected to be strongest. Continuous sampling of the target locus revealed that a single, restorative, in-frame nucleotide substitution was selected. Our findings show that ultra-conservation alone need not be predictive of a site that is refractory to target site resistance. Our strategy to evaluate resistance in vivo could help to validate candidate gene drive targets for their resilience to resistance and help to improve predictions of the invasion dynamics of gene drives in field populations., Author summary Gene drives have the potential to be applied as a novel control strategy of disease-transmitting mosquitoes, by spreading genetic traits that suppress or modify the target population. Many gene drive elements work by recognising and cutting a specific target sequence in the mosquito genome and copying themselves into that target sequence allowing the gene drive to increase in frequency in the population. Like other mosquito control interventions, efficacy will greatly depend on minimising the development of resistance to the gene drive mechanism—most likely via a change in the target sequence that prevents further cutting. One strategy to reduce resistance is to target sequences that are highly conserved, which implies that changes cannot easily be tolerated. We developed a strategy that simulates high selection pressure, under which resistance is most likely to emerge, and therefore provides a stringent test of its propensity to arise. Unlike previous results with another gene drive, we recovered a resistant allele within a few generations of gene drive exposure and at high frequency. Our results show that conserved sequences can vary hugely in ability to tolerate mutations and highlights the need to functionally validate future candidate gene drive target sites for their robustness to resistance.

CRISPR-based gene drives offer promising means to reduce the burden of pests and vector-borne diseases. These techniques consist of releasing genetically modified organisms carrying CRISPR-Cas nucleases designed to bias their inheritance and rapidly propagate desired modifications. Gene drives can be intended to reduce reproductive capacity of harmful insects or spread anti-pathogen effectors through wild populations, even when these confer fitness disadvantages. Technologies capable of halting the spread of gene drives may prove highly valuable in controlling, counteracting, and even reverting their effect on individual organisms as well as entire populations. Here we show engineering and testing of a genetic approach, based on the germline expression of a phage-derived anti-CRISPR protein (AcrIIA4), able to inactivate CRISPR-based gene drives and restore their inheritance to Mendelian rates in the malaria vector Anopheles gambiae. Modeling predictions and cage testing show that a single release of male mosquitoes carrying the AcrIIA4 protein can block the spread of a highly effective suppressive gene drive preventing population collapse of caged malaria mosquitoes., Technologies that can halt the spread of gene drives would be highly useful in controlling or reverting their effect. Here the authors use the anti-CRISPR protein AcrIIA4 to inactivate drives in A. gambiae.

Homing-based gene drives use a germline source of nuclease to copy themselves at specific target sites in a genome and bias their inheritance. Such gene drives can be designed to spread and deliberately suppress populations of malaria mosquitoes by impairing female fertility. However, strong unintended fitness costs of the drive and a propensity to generate resistant mutations can limit a gene drive’s potential to spread. Alternative germline regulatory sequences in the drive element confer improved fecundity of carrier individuals and reduced propensity for target site resistance. This is explained by reduced rates of end-joining repair of DNA breaks from parentally deposited nuclease in the embryo, which can produce heritable mutations that reduce gene drive penetrance. We tracked the generation and selection of resistant mutations over the course of a gene drive invasion of a population. Improved gene drives show faster invasion dynamics, increased suppressive effect and later onset of target site resistance. Our results show that regulation of nuclease expression is as important as the choice of target site when developing a robust homing-based gene drive for population suppression., Author summary Gene drives are selfish genetic elements that are able to drastically bias their own inheritance. They can rapidly invade populations, even starting from a very low frequency. Recent advances have allowed the engineering of gene drives deliberately designed to spread genetic traits of choice into populations of malaria-transmitting mosquito species–for example traits that impair a mosquito’s ability to reproduce or its ability to transmit parasites. The class of gene drive in question uses a very precise cutting and copying mechanism, termed ‘homing’, that allows it to increase its numbers in the cells that go on to form sperm or eggs, thereby increasing the chances that a copy of the gene drive is transmitted to offspring. However, while this type of gene drive can rapidly invade a mosquito population, mosquitoes can also eventually become resistant to the gene drive in some cases. Here we show that restricting the cutting activity of the gene drive to the germline tissue is crucial to maintaining its potency and we illustrate how failure to restrict this activity can lead to the generation of mutations that can make mosquitoes resistant to the gene drive.

Only female insects transmit diseases such as malaria, dengue and Zika; therefore, control methods that bias the sex ratio of insect offspring have long been sought. Genetic elements such as sex-chromosome drives can distort sex ratios to produce unisex populations that eventually collapse, but the underlying molecular mechanisms are unknown. We report a male-biased sex-distorter gene drive (SDGD) in the human malaria vector Anopheles gambiae. We induced super-Mendelian inheritance of the X-chromosome-shredding I-PpoI nuclease by coupling this to a CRISPR-based gene drive inserted into a conserved sequence of the doublesex (dsx) gene. In modeling of invasion dynamics, SDGD was predicted to have a quicker impact on female mosquito populations than previously developed gene drives targeting female fertility. The SDGD at the dsx locus led to a male-only population from a 2.5% starting allelic frequency in 10-14 generations, with population collapse and no selection for resistance. Our results support the use of SDGD for malaria vector control.

CRISPR-Cas9 nuclease-based gene drives rely on inducing chromosomal breaks in the germline that are repaired in ways that lead to a biased inheritance of the drive. Gene drives designed to impair female fertility can suppress populations of the mosquito vector of malaria. However, strong unintended fitness costs, due to ectopic nuclease expression, and high levels of resistant mutations, limited the potential of the first generation of gene drives to spread.Here we show that changes to regulatory sequences in the drive element, designed to contain nuclease expression to the germline, confer improved fecundity over previous versions and generate drastically lower rates of target site resistance. We employed a genetic screen to show that this effect is explained by reduced rates of end-joining repair of DNA breaks at the target site caused by deposited nuclease in the embryo.Highlighting the impact of deposited Cas9, many of the mutations arising from this source of nuclease activity in the embryo are heritable, thereby having the potential to generate resistant target sites that reduce the penetrance of the gene drive.Finally, in cage invasion experiments these gene drives show improved invasion dynamics compared to first generation drives, resulting in greater than 90% suppression of the reproductive output and a delay in the emergence of target site resistance, even at a resistance-prone target sequence. We shed light on the dynamics of generation and selection of resistant alleles in a population by tracking, longitudinally, the frequency of resistant alleles in the face of an invading gene drive. Our results illustrate important considerations for future gene drive design and should expedite the development of gene drives robust to resistance.

Many animals steal food, termed kleptoparasitism, but attacks are frequently unsuccessful and may incur costs. Some species produce false alarm calls during kleptoparasitism, but the relative benefits of this versus physical attacks are not known. We investigated the kleptoparasitic behaviour of a population of individually recognizable and habituated fork-tailed drongos, Dicrurus adsimilis, which kleptoparasitize food using both false alarms and physical attacks. Drongos made false alarms more frequently when kleptoparasitizing small food items, probably because these can be more profitably stolen by false alarm than attack. False alarms were also more frequent when targets were larger species, which are likely to be more costly to attack since they more frequently defended food. Drongos were able to attack targets when a false alarm had failed but rarely did so, even though such attacks were no less successful than attacks when no false alarm had been made. It therefore appears that false alarms increase opportunities for kleptoparasitism because calls can be made when attacks are unprofitable. False alarm calls also appear to benefit drongos by increasing overall success in kleptoparasitism, because attacks could be made following failed false alarm calls. This is the first study to show the functional benefits of making false alarm calls, illustrating how they may be deployed strategically in kleptoparasitism when attacks would be less profitable or even unprofitable. More generally, false alarms may evolve where animals engage in physical conflict over resources because they can benefit individuals by decreasing the costs of competition.

Gene-drive systems that enable super-Mendelian inheritance of a transgene have the potential to modify insect populations over a timeframe of a few years [AU please provide a real estimate, this seems vague]. We describe CRISPR-Cas9 endonuclease constructs that function as gene-drive systems in Anopheles gambiae, the main vector for malaria [AU:OK?]. We identified three genes (AGAP005958, AGAP011377 and AGAP007280) that confer a recessive female sterility phenotype upon disruption, and inserted into each locus CRISPR-Cas9 gene-drive constructs designed to target and edit each gene [AU:OK?]. For each locus targeted we observed strong gene drive at the molecular level, with transmission rates to progeny of 91 to 99.6%. Population modelling and cage experiments indicate that a CRISPR-Cas9 construct targeting one of these loci, AGAP007280, meets the minimum requirement for a gene drive targeting female reproduction in an insect population. These findings could expedite the development of gene drives to control suppress mosquito populations to levels that do not support malaria transmission.

Trick and Treat Forked-tailed drongos are a particularly intelligent type of bird found in Africa. Drongos associate with many other bird and mammal species, which can learn to respond to drongo warning calls. Drongos are also exceptional mimics of the other species' alarm calls. Though the increased vigilance across these multi species associations is a benefit to all, drongos sometimes use these calls as ploys to scare associated species away from food, which the drongos then steal. However, without some approach to maintain the effectiveness of this deception, the drongos' ploy would soon be detected. Flower et al. (p. 513 ) now show that drongos are able to fool their target species longer by flexibly varying the type of call they give.