Introducing desirable transgenes into insect populations using Y-linked meiotic drive - a theoretical assessment

The use of genetic drive mechanisms to replace native mosquito genotypes with individuals bearing antipathogen transgenes is a potential strategy for repressing insect transmission of human diseases such as malaria and dengue. Antipathogen transgenes have been developed and tested, but efficient gene drive mechanisms are lacking. Here we theoretically assess the feasibility of introducing antipathogen genes into wild Aedes aegypti populations by using a naturally occurring meiotic drive system. We consider the release of males having both a Y-linked meiotic drive gene and an X-linked drive-insensitive response allele to which an antipathogen gene is linked. We use mathematical models and computer simulations to determine how the post-introduction dynamics of the antipathogen gene are affected by specific genetic characteristics of the system. The results show that when the natural population is uniformly sensitive to the meiotic drive gene, the antipathogen gene may be driven close to fixation if the fitness costs of the drive gene, the insensitive response allele, and the antipathogen gene are low. However, when the natural population has a small proportion of an X-linked insensitive response allele or an autosomal gene that strongly reduces the effect of the drive gene, the antipathogen gene does not spread if it has an associated fitness cost. Our modeling results provide a theoretical foundation for further experimental tests.     

Inverse Medea as a novel gene drive system for local population replacement: a theoretical analysis

One strategy to control mosquito-borne diseases, such as malaria and dengue fever, on a regional scale is to use gene drive systems to spread disease-refractory genes into wild mosquito populations. The development of a synthetic Medea element that has been shown to drive population replacement in laboratory Drosophila populations has provided encouragement for this strategy but has also been greeted with caution over the concern that transgenes may spread into countries without their consent. Here, we propose a novel gene drive system, inverse Medea, which is strong enough to bring about local population replacement but is unable to establish itself beyond an isolated release site. The system consists of 2 genetic components--a zygotic toxin and maternal antidote--which render heterozygous offspring of wild-type mothers unviable. Through population genetic analysis, we show that inverse Medea will only spread when it represents a majority of the alleles in a population. The element is best located on an autosome and will spread to fixation provided any associated fitness costs are dominant and to very high frequency otherwise. We suggest molecular tools that could be used to build the inverse Medea system and discuss its utility for a confined release of transgenic mosquitoes.     

Selection, Generalized Transmission and the Evolution of Modifier Genes. I. the Reduction Principle

Modifier gene models are used to explore the evolution of features of organisms, such as the genetic system, that are not directly involved in the determination of fitness. Recent work has shown that a general "reduction principle" holds in models of selectively neutral modifiers of recombination, mutation, and migration. Here we present a framework for models of modifier genes that shows these reduction results to be part of a more general theory, for which recombination and mutation are special cases. The deterministic forces that affect the genetic composition of a population can be partitioned into two categories: selection and transmission. Selection includes differential viabilities, fertilities, and mating success. Imperfect transmission occurs as a result of such phenomena as recombination, mutation and migration, meiosis, gene conversion, and meiotic drive. Selectively neutral modifier genes affect transmission, and a neutral modifier gene can evolve only by generating association with selected genes whose transmission it affects. We show that, in randomly mating populations at equilibrium, imperfect transmission of selected genes allows a variance in their marginal fitnesses to be maintained. This variance in the marginal fitnesses of selected genes is what drives the evolution of neutral modifier genes. Populations with a variance in marginal fitnesses at equilibrium are always subject to invasion by modifier genes that bring about perfect transmission of the selected genes. It is also found, within certain constraints, that for modifier genes producing what we call "linear variation" in the transmission processes, a new modifier allele can invade a population at equilibrium if it reduces the level of imperfect transmission acting on the selected genes, and will be expelled if it increases the level of imperfect transmission. Moreover, the strength of the induced selection on the modifier gene is shown to range up to the order of the departure of the genetic system from perfect transmission.     

Gene drive systems for insect disease vectors

The elegant mechanisms by which naturally occurring selfish genetic elements, such as transposable elements, meiotic drive genes, homing endonuclease genes and Wolbachia, spread at the expense of their hosts provide some of the most fascinating and remarkable subjects in evolutionary genetics. These elements also have enormous untapped potential to be used in the control of some of the world's most devastating diseases. Effective gene drive systems for spreading genes that can block the transmission of insect-borne pathogens are much needed. Here we explore the potential of natural gene drive systems and discuss the artificial constructs that could be envisaged for this purpose.     

nanos-Driven expression of piggyBac transposase induces mobilization of a synthetic autonomous transposon in the malaria vector mosquito,Anopheles stephensi

Transposons are a class of selfish DNA elements that can mobilize within a genome. If mobilization is accompanied by an increase in copy number (replicative transposition), the transposon may sweep through a population until it is fixed in all of its interbreeding members. This introgression has been proposed as the basis for drive systems to move genes with desirable phenotypes into target species. One such application would be to use them to move a gene conferring resistance to malaria parasites throughout a population of vector mosquitos. We assessed the feasibility of using the piggyBac transposon as a gene-drive mechanism to distribute anti-malarial transgenes in populations of the malaria vector, Anopheles stephensi. We designed synthetic gene constructs that express the piggyBac transposase in the female germline using the control DNA of the An. stephensi nanos orthologous gene linked to marker genes to monitor inheritance. Two remobilization events were observed with a frequency of one every 23 generations, a rate far below what would be useful to drive anti-pathogen transgenes into wild mosquito populations. We discuss the possibility of optimizing this system and the impetus to do so.     

Sperm competition suppresses gene drive among experimentally evolving populations of house mice

Drive genes are genetic elements that manipulate the 50% ratio of Mendelian inheritance in their own favour, allowing them to rapidly propagate through populations. The action of drive genes is often hidden, making detection and identification inherently difficult. Yet drive genes can have profound evolutionary consequences for the populations that harbour them: most known drivers are detrimental to organismal gamete development, reproduction and survival. In this study, we identified the presence of a well‐known drive gene called t haplotype post hoc in eight replicate selection lines of house mice that had been evolving under enforced monandry or polyandry for 20 generations. Previous work on these selection lines reported an increase in sperm competitive ability in males evolving under polyandry. Here, we show that this evolutionary response can be partly attributed to gene drive. We demonstrate that drive‐carrying males are substantially compromised in their sperm competitive ability. As a consequence, we found that t frequencies declined significantly in the polyandrous lines while remaining at stable, high levels in the monandrous lines. For the first time in a vertebrate, we thus provide direct experimental evidence that the mating system of a species can have important repercussions on the spread of drive genes over evolutionary relevant timescales. Moreover, our work highlights how the covert action of drive genes can have major, potentially unintended impact on our study systems.     

Origin,evolution, and population genetics of the selfish Segregation Distorter gene duplication in European and African populations of Drosophila melanogaster

Meiotic drive elements are a special class of evolutionarily “selfish genes” that subvert Mendelian segregation to gain preferential transmission at the expense of homologous loci. Many drive elements appear to be maintained in populations as stable polymorphisms, their equilibrium frequencies determined by the balance between drive (increasing frequency) and selection (decreasing frequency). Here we show that a classic, seemingly balanced, drive system is instead characterized by frequent evolutionary turnover giving rise to dynamic, rather than stable, equilibrium frequencies. The autosomal Segregation Distorter (SD) system of the fruit fly Drosophila melanogaster is a selfish coadapted meiotic drive gene complex in which the major driver corresponds to a partial duplication of the gene Ran‐GTPase activating protein (RanGAP). SD chromosomes segregate at similar, low frequencies of 1–5% in natural populations worldwide, consistent with a balanced polymorphism. Surprisingly, our population genetic analyses reveal evidence for parallel, independent selective sweeps of different SD chromosomes in populations on different continents. These findings suggest that, rather than persisting at a single stable equilibrium, SD chromosomes turn over frequently within populations.     

Making gene drive biodegradable

Gene drive systems have long been sought to modify mosquito populations and thus combat malaria and dengue. Powerful gene drive systems have been developed in laboratory experiments, but may never be used in practice unless they can be shown to be acceptable through rigorous field-based testing. Such testing is complicated by the anticipated difficulty in removing gene drive transgenes from nature. Here, we consider the inclusion of self-elimination mechanisms into the design of homing-based gene drive transgenes. This approach not only caused the excision of the gene drive transgene, but also generates a transgene-free allele resistant to further action by the gene drive. Strikingly, our models suggest that this mechanism, acting at a modest rate (10%) as part of a single-component system, would be sufficient to cause the rapid reversion of even the most robust homing-based gene drive transgenes, without the need for further remediation. Modelling also suggests that unlike gene drive transgenes themselves, self-eliminating transgene approaches are expected to tolerate substantial rates of failure. Thus, self-elimination technology may permit rigorous field-based testing of gene drives by establishing strict time limits on the existence of gene drive transgenes in nature, rendering them essentially biodegradable.This article is part of the theme issue ‘Novel control strategies for mosquito-borne diseases''.     

Medusa: A Novel Gene Drive System for Confined Suppression of Insect Populations

Gene drive systems provide novel opportunities for insect population suppression by driving genes that confer a fitness cost into pest or disease vector populations; however regulatory issues arise when genes are capable of spreading across international borders. Gene drive systems displaying threshold properties provide a solution since they can be confined to local populations and eliminated through dilution with wild-types. We propose a novel, threshold-dependent gene drive system, Medusa, capable of inducing a local and reversible population crash. Medusa consists of four components - two on the X chromosome, and two on the Y chromosome. A maternally-expressed, X-linked toxin and a zygotically-expressed, Y-linked antidote results in suppression of the female population and selection for the presence of the transgene-bearing Y because only male offspring of Medusa-bearing females are protected from the effects of the toxin. At the same time, the combination of a zygotically-expressed, Y-linked toxin and a zygotically-expressed, X-linked antidote selects for the transgene-bearing X in the presence of the transgene-bearing Y. Together these chromosomes create a balanced lethal system that spreads while selecting against females when present above a certain threshold frequency. Simple population dynamic models show that an all-male release of Medusa males, carried out over six generations, is expected to induce a population crash within 12 generations for modest release sizes on the order of the wild population size. Re-invasion of non-transgenic insects into a suppressed population can result in a population rebound; however this can be prevented through regular releases of modest numbers of Medusa males. Finally, we outline how Medusa could be engineered with currently available molecular tools.     

The toxin and antidote puzzle: new ways to control insect pest populations through manipulating inheritance

Insects carry out essential ecological functions, such as pollination, but also cause extensive damage to agricultural crops, and transmit human diseases such as malaria and dengue fever. Advances in insect transgenesis are making it increasingly feasible to engineer genes conferring desirable phenotypes, and gene drive systems are required to spread these genes into wild populations. Medea provides one solution, being able to spread into a population from very low initial frequencies through the action of a maternally-expressed toxin linked to a zygotically-expressed antidote. Several other toxin-antidote combinations are imaginable that distort the offspring ratio in favor of a desired transgene, or drive the population towards an all-male crash. We explore two such systems--Semele, which is capable of spreading a desired transgene into an isolated population in a confined manner; and Merea, which is capable of inducing a local population crash when located on the Z chromosome of a Lepidopteron pest.     

The effect of gene drive on containment of transgenic mosquitoes

Mosquito-borne diseases such as malaria and dengue fever continue to be a major health problem through much of the world. Several new potential approaches to disease control utilize gene drive to spread anti-pathogen genes into the mosquito population. Prior to a release, these projects will require trials in outdoor cages from which transgenic mosquitoes may escape, albeit in small numbers. Most genes introduced in small numbers are very likely to be lost from the environment; however, gene drive mechanisms enhance the invasiveness of introduced genes. Consequently, introduced transgenes may be more likely to persist than ordinary genes following an accidental release. Here, we develop stochastic models to analyze the loss probabilities for several gene drive mechanisms, including homing endonuclease genes, transposable elements, Medea elements, the intracellular bacterium Wolbachia, engineered underdominance genes, and meiotic drive. We find that Medea and Wolbachia present the best compromise between invasiveness and containment for the six gene drive systems currently being considered for the control of mosquito-borne disease.     

Engineered underdominance allows efficient and economical introgression of traits into pest populations

A novel form of underdominance is suggested as a mechanism that is able to drive desired genes into pest populations through the release of transgenic individuals over one or more generations. Such a mechanism is urgently needed by those working to reduce the impact of malaria by releasing strains of Anopheles, the vector of the disease, that are not susceptible to malaria parasites. We use simple population genetics models to quantify the benefits conferred when heterozygous genotypes, arising from matings between introduced and wild individuals, are not viable. In a randomly mating population, underdominant systems accelerate introgression of desired alleles and allow the release of individuals to be discontinued once the frequency of transgenic alleles attains a threshold. A set of two constructs, which together are selectively neutral but lethal when one is carried without the other, are found to produce dynamics that are characteristic of underdominant systems. When these constructs are carried on non-homologous chromosomes, then the ratio of released to natural born individuals need only be greater than 3:100 for introgression to occur. Furthermore, the threshold for the gene frequencies over which the introduced genes are expected to become fixed upon discontinuing the release of transgenic individuals is surprisingly low. The location of the threshold suggests that the introduced genes are expected to spread in space, at least locally. For the first time, the prospect of a practical drive mechanism for the genetic manipulation of pest populations is raised.     

Why mitochondrial genes are most often found in nuclei

A very small fraction of the proteins required for the propagation and function of mitochondria are coded by their genomes, while nuclear genes code the vast majority. We studied the migration of genes between the two genomes when transfer mechanisms mediate this exchange. We could calculate the influence of differential mutation rates, as well as that of biased transfer rates, on the partitioning of genes between the two genomes. We observe no significant difference in partitioning for haploid and diploid cell populations, but the effective size of cell populations is important. For infinitely large effective populations, higher mutation rates in mitochondria than in nuclear genomes are required to drive mitochondrial genes to the nuclear genome. In the more realistic case of finite populations, gene transfer favoring the nucleus and/or higher mutation rates in the mitochondrion will drive mitochondrial genes to the nucleus. We summarize experimental data that identify a gene transfer process mediated by vacuoles that favors the accumulation of mitochondrial genes in the nuclei of modern cells. Finally, we compare the behavior of mitochondrial genes for which transfer to the nucleus is neutral or influenced by purifying selection.     

First Steps towards Underdominant Genetic Transformation of Insect Populations

The idea of introducing genetic modifications into wild populations of insects to stop them from spreading diseases is more than 40 years old. Synthetic disease refractory genes have been successfully generated for mosquito vectors of dengue fever and human malaria. Equally important is the development of population transformation systems to drive and maintain disease refractory genes at high frequency in populations. We demonstrate an underdominant population transformation system in Drosophila melanogaster that has the property of being both spatially self-limiting and reversible to the original genetic state. Both population transformation and its reversal can be largely achieved within as few as 5 generations. The described genetic construct {Ud} is composed of two genes; (1) a UAS-RpL14.dsRNA targeting RNAi to a haploinsufficient gene RpL14 and (2) an RNAi insensitive RpL14 rescue. In this proof-of-principle system the UAS-RpL14.dsRNA knock-down gene is placed under the control of an Actin5c-GAL4 driver located on a different chromosome to the {Ud} insert. This configuration would not be effective in wild populations without incorporating the Actin5c-GAL4 driver as part of the {Ud} construct (or replacing the UAS promoter with an appropriate direct promoter). It is however anticipated that the approach that underlies this underdominant system could potentially be applied to a number of species.     

Evolving disease resistance genes

Defenses against most specialized plant pathogens are often initiated by a plant disease resistance gene. Plant genomes encode several classes of genes that can function as resistance genes. Many of the mechanisms that drive the molecular evolution of these genes are now becoming clear. The processes that contribute to the diversity of R genes include tandem and segmental gene duplications, recombination, unequal crossing-over, point mutations, and diversifying selection. Diversity within populations is maintained by balancing selection. Analyses of whole-genome sequences have and will continue to provide new insight into the dynamics of resistance gene evolution.     

Empirical evidence for son-killing X chromosomes and the operation of SA-zygotic drive

Background

Diploid organisms have two copies of all genes, but only one is carried by each haploid gamete and diploid offspring. This causes a fundamental genetic conflict over transmission rate between alternative alleles. Single genes, or gene clusters, only rarely code for the complex phenotypes needed to give them a transmission advantage (drive phenotype). However, all genes on a male''s X and Y chromosomes co-segregate, allowing different sex-linked genes to code for different parts of the drive phenotype. Correspondingly, the well-characterized phenomenon of male gametic drive, occurring during haploid gametogenesis, is especially common on sex chromosomes. The new theory of sexually antagonistic zygotic drive of the sex chromosomes (SA-zygotic drive) extends the logic of gametic drive into the diploid phase of the lifecycle, whenever there is competition among siblings or harmful sib-sib mating. The X and Y are predicted to gain a transmission advantage by harming offspring of the sex that does not carry them.

Results

Here we analyzed a mutant X-chromosome in Drosophila simulans that produced an excess of daughters when transmitted from males. We developed a series of tests to differentiate between gametic and SA-zygotic drive, and provide multiple lines of evidence that SA-zygotic drive is responsible for the sex ratio bias. Driving sires produce about 50% more surviving daughters than sons.

Conclusion

Sex-ratio distortion due to genetic conflict has evolved via gametic drive and maternally transmitted endosymbionts. Our data indicate that sex chromosomes can also drive by harming the non-carrier sex of offspring.     

The impact of dissociation on transposon-mediated disease control strategies

Vector-borne diseases such as malaria and dengue fever continue to be a major health concern through much of the world. The emergence of chloroquine-resistant strains of malaria and insecticide-resistant mosquitoes emphasize the need for novel methods of disease control. Recently, there has been much interest in the use of transposable elements to drive resistance genes into vector populations as a means of disease control. One concern that must be addressed before a release is performed is the potential loss of linkage between a transposable element and a resistance gene. Transposable elements such as P and hobo have been shown to produce internal deletion derivatives at a significant rate, and there is concern that a similar process could lead to loss of the resistance gene from the drive system following a transgenic release. Additionally, transposable elements such as Himar1 have been shown to transpose significantly more frequently when free of exogenous DNA. Here, we show that any transposon-mediated gene drive strategy must have an exceptionally low rate of dissociation if it is to be effective. Additionally, the resistance gene must confer a large selective advantage to the vector to surmount the effects of a moderate dissociation rate and transpositional handicap.     

Modeling CRISPR gene drives for suppression of invasive rodents using a supervised machine learning framework

Invasive rodent populations pose a threat to biodiversity across the globe. When confronted with these invaders, native species that evolved independently are often defenseless. CRISPR gene drive systems could provide a solution to this problem by spreading transgenes among invaders that induce population collapse, and could be deployed even where traditional control methods are impractical or prohibitively expensive. Here, we develop a high-fidelity model of an island population of invasive rodents that includes three types of suppression gene drive systems. The individual-based model is spatially explicit, allows for overlapping generations and a fluctuating population size, and includes variables for drive fitness, efficiency, resistance allele formation rate, as well as a variety of ecological parameters. The computational burden of evaluating a model with such a high number of parameters presents a substantial barrier to a comprehensive understanding of its outcome space. We therefore accompany our population model with a meta-model that utilizes supervised machine learning to approximate the outcome space of the underlying model with a high degree of accuracy. This enables us to conduct an exhaustive inquiry of the population model, including variance-based sensitivity analyses using tens of millions of evaluations. Our results suggest that sufficiently capable gene drive systems have the potential to eliminate island populations of rodents under a wide range of demographic assumptions, though only if resistance can be kept to a minimal level. This study highlights the power of supervised machine learning to identify the key parameters and processes that determine the population dynamics of a complex evolutionary system.     

hAT element population genetics in Anopheles gambiae s.l. in Mozambique

Herves is a functional Class II transposable element in Anopheles gambiae belonging to the hAT superfamily of elements. Class II transposable elements are used as gene vectors in this species and are also being considered as genetic drive agents for spreading desirable genes through natural populations as part of an effort to control malaria transmission. In this study, Herves was investigated in populations of Anopheles gambiae s.s., Anopheles arabiensis and Anopheles merus in Mozambique over a period of 2 years. The copy number of Herves within these three species was approximately 5 copies per diploid genome and did not differ among species or between years. Based on the insertion-site occupancy-frequency distribution and existing models of transposable element dynamics, Herves appears to be transpositionally active currently or, at least recently, in all species tested. Ninety-five percent of the individuals within the populations of the three species tested contained intact elements with complete Herves transposase genes and this is consistent with the idea that these elements are currently active.     

Molecular population genetics of X-linked genes in Drosophila pseudoobscura

This article presents a nucleotide sequence analysis of 500 bp determined in each of five X-linked genes, runt, sisterlessA, period, esterase 5, and Heat-shock protein 83, in 40 Drosophila pseudoobscura strains collected from two populations. Estimates of the neutral migration parameter for the five loci show that gene flow among D. pseudoobscura populations is sufficient to homogenize inversion frequencies across the range of the species. Nucleotide diversity at each locus fails to reject a neutral model of molecular evolution. The sample of 40 chromosomes included six Sex-ratio inversions, a series of three nonoverlapping inversions that are associated with a strong meiotic drive phenotype. The selection driven by the Sex-ratio meiotic drive element has not fixed variation across the X chromosome of D. pseudoobscura because, while significant linkage disequilibrium was observed within the sisterlessA, period, and esterase 5 genes, we did not find evidence for nonrandom association among loci. The Sex-ratio chromosome was estimated to be 25,000 years old based on the decomposition of linkage disequilibrium between esterase 5 and Heat-shock protein 83 or 1 million years old based on the net divergence of esterase 5 between Standard and Sex-ratio chromosomes. Genetic diversity was depressed within esterase 5 within Sex-ratio chromosomes, while the four other genes failed to show a reduction in heterozygosity in the Sex-ratio background. The reduced heterogeneity in esterase 5 is due either to its location near one of the Sex-ratio inversion breakpoints or that it is closely linked to a gene or genes responsible for the Sex-ratio meiotic drive system.