Medea selfish genetic elements as tools for altering traits of wild populations: a theoretical analysis

One strategy for controlling transmission of insect-borne disease involves replacing the native insect population with transgenic animals unable to transmit disease. Population replacement requires a drive mechanism to ensure the rapid spread of linked transgenes, the presence of which may result in a fitness cost to carriers. Medea selfish genetic elements have the feature that when present in a female, only offspring that inherit the element survive, a behavior that can lead to spread. Here, we derive equations that describe the conditions under which Medea elements with a fitness cost will spread, and the equilibrium allele frequencies are achieved. Of particular importance, we show that whenever Medea spreads, the non-Medea genotype is driven out of the population, and we estimate the number of generations required to achieve this goal for Medea elements with different fitness costs and male-only introduction frequencies. Finally, we characterize two contexts in which Medea elements with fitness costs drive the non-Medea allele from the population: an autosomal element in which not all Medea-bearing progeny of a Medea-bearing mother survive, and an X-linked element in species in which X/Y individuals are male. Our results suggest that Medea elements can drive population replacement under a wide range of conditions.     

Modeling the Dynamics of a Non-Limited and a Self-Limited Gene Drive System in Structured Aedes aegypti Populations

Recently there have been significant advances in research on genetic strategies to control populations of disease-vectoring insects. Some of these strategies use the gene drive properties of selfish genetic elements to spread physically linked anti-pathogen genes into local vector populations. Because of the potential of these selfish elements to spread through populations, control approaches based on these strategies must be carefully evaluated to ensure a balance between the desirable spread of the refractoriness-conferring genetic cargo and the avoidance of potentially unwanted outcomes such as spread to non-target populations. There is also a need to develop better estimates of the economics of such releases. We present here an evaluation of two such strategies using a biologically realistic mathematical model that simulates the resident Aedes aegypti mosquito population of Iquitos, Peru. One strategy uses the selfish element Medea, a non-limited element that could permanently spread over a large geographic area; the other strategy relies on Killer-Rescue genetic constructs, and has been predicted to have limited spatial and temporal spread. We simulate various operational approaches for deploying these genetic strategies, and quantify the optimal number of released transgenic mosquitoes needed to achieve definitive spread of Medea-linked genes and/or high frequencies of Killer-Rescue-associated elements. We show that for both strategies the most efficient approach for achieving spread of anti-pathogen genes within three years is generally to release adults of both sexes in multiple releases over time. Even though females in these releases should not transmit disease, there could be public concern over such releases, making the less efficient male-only release more practical. This study provides guidelines for operational approaches to population replacement genetic strategies, as well as illustrates the use of detailed spatial models to assist in safe and efficient implementation of such novel genetic strategies.     

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.     

General principles of single-construct chromosomal gene drive

Gene drive systems are genetic elements capable of spreading into a population even if they confer a fitness cost to their host. We consider a class of drive systems consisting of a chromosomally located, linked cluster of genes, the presence of which renders specific classes of offspring arising from specific parental crosses unviable. Under permissive conditions, a number of these elements are capable of distorting the offspring ratio in their favor. We use a population genetic framework to derive conditions under which these elements spread to fixation in a population or induce a population crash. Many of these systems can be engineered using combinations of toxin and antidote genes, analogous to Medea, which consists of a maternal toxin and zygotic antidote. The majority of toxin-antidote drive systems require a critical frequency to be exceeded before they spread into a population. Of particular interest, a Z-linked Medea construct with a recessive antidote is expected to induce an all-male population crash for release frequencies above 50%. We suggest molecular tools that may be used to build these systems, and discuss their relevance to the control of a variety of insect pest species, including mosquito vectors of diseases such as malaria and dengue fever.     

Gene drive systems in mosquitoes: rules of the road

Population replacement strategies for controlling transmission of mosquito-borne diseases call for the introgression of antipathogen effector genes into vector populations. It is anticipated that these genes, if present at high enough frequencies, will impede transmission of the target pathogens and result in reduced human morbidity and mortality. Recent laboratory successes in the development of virus- and protozoan-resistant mosquito strains make urgent research of gene drive systems capable of moving effector genes into wild populations. A systematic approach to developing safe and effective gene drive systems that includes defining the requirements of the system, identifying naturally occurring or synthetic genetic mechanisms for gene spread upon which drive systems can be based and the successful adaptation of a mechanism to a drive system, should mitigate concerns about using genetically engineered mosquitoes for disease control.     

Semele: a killer-male, rescue-female system for suppression and replacement of insect disease vector populations

Two strategies to control mosquito-borne diseases, such as malaria and dengue fever, are reducing mosquito population sizes or replacing populations with disease-refractory varieties. We propose a genetic system, Semele, which may be used for both. Semele consists of two components: a toxin expressed in transgenic males that either kills or renders infertile wild-type female recipients and an antidote expressed in females that protects them from the effects of the toxin. An all-male release results in population suppression because wild-type females that mate with transgenic males produce no offspring. A release that includes transgenic females results in gene drive since females carrying the allele are favored at high population frequencies. We use simple population genetic models to explore the utility of the Semele system. We find that Semele can spread under a wide range of conditions, all of which require a high introduction frequency. This feature is desirable since transgenic insects released accidentally are unlikely to persist, transgenic insects released intentionally can be spatially confined, and the element can be removed from a population through sustained release of wild-type insects. We examine potential barriers to Semele gene drive and suggest molecular tools that could be used to build the Semele system.     

Engineering the genomes of wild insect populations: Challenges, and opportunities provided by synthetic Medea selfish genetic elements

Advances in insect transgenesis and our knowledge of insect physiology and genomics are making it possible to create transgenic populations of beneficial or pest insects that express novel traits. There are contexts in which we may want the transgenes responsible for these traits to spread so that all individuals within a wild population carry them, a process known as population replacement. Transgenes of interest are unlikely to confer an overall fitness benefit on those who carry them. Therefore, an essential component of any population replacement strategy is the presence of a drive mechanism that will ensure the spread of linked transgenes. We discuss contexts in which population replacement might be desirable and the requirements a drive system must satisfy to be both effective and safe. We then describe the creation of synthetic Medea elements, the first selfish genetic elements synthesized de novo, with the capability of driving population replacement, in this case in Drosophila. The strategy used to create DrosophilaMedea is applicable to a number of other insect species and the Medea system satisfies key requirements for scientific and social acceptance. Finally, we highlight several challenges to implementing population replacement in the wild.     

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.     

Double drives and private alleles for localised population genetic control

Synthetic gene drive constructs could, in principle, provide the basis for highly efficient interventions to control disease vectors and other pest species. This efficiency derives in part from leveraging natural processes of dispersal and gene flow to spread the construct and its impacts from one population to another. However, sometimes (for example, with invasive species) only specific populations are in need of control, and impacts on non-target populations would be undesirable. Many gene drive designs use nucleases that recognise and cleave specific genomic sequences, and one way to restrict their spread would be to exploit sequence differences between target and non-target populations. In this paper we propose and model a series of low threshold double drive designs for population suppression, each consisting of two constructs, one imposing a reproductive load on the population and the other inserted into a differentiated locus and controlling the drive of the first. Simple deterministic, discrete-generation computer simulations are used to assess the alternative designs. We find that the simplest double drive designs are significantly more robust to pre-existing cleavage resistance at the differentiated locus than single drive designs, and that more complex designs incorporating sex ratio distortion can be more efficient still, even allowing for successful control when the differentiated locus is neutral and there is up to 50% pre-existing resistance in the target population. Similar designs can also be used for population replacement, with similar benefits. A population genomic analysis of CRISPR PAM sites in island and mainland populations of the malaria mosquito Anopheles gambiae indicates that the differentiation needed for our methods to work can exist in nature. Double drives should be considered when efficient but localised population genetic control is needed and there is some genetic differentiation between target and non-target populations.     

Wolbachia and cytoplasmic incompatibility in mosquitoes

Wolbachia are maternally inherited bacteria that induce cytoplasmic incompatibility in mosquitoes, and are able to use these patterns of sterility to spread themselves through populations. For this reason they have been proposed as a gene drive system for mosquito genetic replacement, as well as for the reduction of population size or for modulating population age structure in order to reduce disease transmission. Here, recent progress in the study of mosquito Wolbachia is reviewed. We now have much more comprehensive estimates of the parameters that can affect the spread of Wolbachia through natural populations from low starting frequencies, and for waves of spread to be maintained in the face of partial barriers to gene flow. In Aedes albopictus these dynamics are extremely favourable, with very high maternal transmission fidelity and levels of incompatibility recorded. Correspondence between measurements taken in the lab and field is much better than in the Drosophila simulans model system. Important research goals are also discussed, including Wolbachia transformation, interspecific transfer and the elucidation of the mechanisms of incompatibility and rescue; all will be aided by a wealth of new Wolbachia genome information.     

A branching process for the early spread of a transposable element in a diploid population

Transposable elements (TEs) face significant challenges upon transfer into a new host population, invariably beginning their invasion with only a single element. The fate of this element is a product of its internal properties, the population dynamics of the host species, and genetic drift. We present a continuous-time multi-type branching process to model the early stages of TE spread. The model incorporates seasonal population size changes and is applicable to diploid hosts for prevalences up to 10%. We reproduce standard results of TE population dynamics and show that population growth may have a greater influence on reducing TE loss probability than a transpositional burst. These results are applied to the planned use of a TE to drive an antimalarial gene into an Anopheles gambiae population. The model favors a transgenic release immediately following the dry season when the An. gambiae population begins to grow. Increasing the number of transgenic hosts released has the greatest influence on reducing the probability of TE loss. Following release, the rate at which the TE increases its proportion in the population is most sensitive to its replicative transposition rate. The model recommends a replicative transposition rate greater than 0.1 per TE per generation to satisfy public health goals.     

Cytoplasmic feminizing elements in a two-population model: infection dynamics, gene flow modification, and the spread of autosomal suppressors

We investigate the dynamics of a cytoplasmic parasitic element with feminizing effect in a two-population model. We assume that the host species has a ZZ/ZW sex determination system. Our analysis reveals that the feminizer and the W chromosome can stably coexist by dominating different populations if the transmission rate differs significantly between the populations and migration is sufficiently weak. In the equilibrium of coexistence, genetic influx at any host autosomal locus is strongly enhanced in the population where infection is prevalent but not modified in the other population. We further explore conditions for the spread of autosomal suppressor genes that reduce transmission of feminizing elements to the cost of host viability, and compute their equilibrium frequencies. Our results confirm the hypothesis that selfish genetic elements convert infected host populations into genetic sinks, thereby restricting the spread of transmission suppressors.     

Feasible Introgression of an Anti-pathogen Transgene into an Urban Mosquito Population without Using Gene-Drive

Background

Introgressing anti-pathogen constructs into wild vector populations could reduce disease transmission. It is generally assumed that such introgression would require linking an anti-pathogen gene with a selfish genetic element or similar technologies. Yet none of the proposed transgenic anti-pathogen gene-drive mechanisms are likely to be implemented as public health measures in the near future. Thus, much attention now focuses instead on transgenic strategies aimed at mosquito population suppression, an approach generally perceived to be practical. By contrast, aiming to replace vector competent mosquito populations with vector incompetent populations by releasing mosquitoes carrying a single anti-pathogen gene without a gene-drive mechanism is widely considered impractical.

Methodology/Principal Findings

Here we use Skeeter Buster, a previously published stochastic, spatially explicit model of Aedes aegypti to investigate whether a number of approaches for releasing mosquitoes with only an anti-pathogen construct would be efficient and effective in the tropical city of Iquitos, Peru. To assess the performance of such releases using realistic release numbers, we compare the transient and long-term effects of this strategy with two other genetic control strategies that have been developed in Ae. aegypti: release of a strain with female-specific lethality, and a strain with both female-specific lethality and an anti-pathogen gene. We find that releasing mosquitoes carrying only an anti-pathogen construct can substantially decrease vector competence of a natural population, even at release ratios well below that required for the two currently feasible alternatives that rely on population reduction. Finally, although current genetic control strategies based on population reduction are compromised by immigration of wild-type mosquitoes, releasing mosquitoes carrying only an anti-pathogen gene is considerably more robust to such immigration.

Conclusions/Significance

Contrary to the widely held view that transgenic control programs aimed at population replacement require linking an anti-pathogen gene to selfish genetic elements, we find releasing mosquitoes in numbers much smaller than those considered necessary for transgenic population reduction can result in comparatively rapid and robust population replacement. In light of this non-intuitive result, directing efforts to improve rearing capacity and logistical support for implementing releases, and reducing the fitness costs of existing recombinant technologies, may provide a viable, alternative route to introgressing anti-pathogen transgenes under field conditions.     

Evolutionary dynamics of a selfishly spreading gene that stimulates sexual reproduction in a partially sexual population

The ability of selfishly spreading DNA sequences to invade host populations is intimately bound up with sex. In the absence of sexual reproduction, an element that lowers the fitness of its host and which is initially found in only some of the population will inevitably be lost by natural selection. This will occur even if the element can spread selfishly in the genomes of those individuals which initially possessed it. Here, we create a model in which such a gene is introduced into a population in which individuals sometimes reproduce sexually and sometimes asexually. The element can raise the level of sexuality in its bearers. There is selection against those individuals with the gene (i.e. it is selfish), and a further selective cost to sexual reproduction. The dynamics of the model that arises from these simple assumptions are remarkably complex, with fixation or loss of the selfish gene, unstable and stable equilibria, and effective neutrality all being possible dependent on the parameter values. A selfish gene that increases the level of sexuality of its bearers will tend to have a higher likelihood of invading a host population, and faster spread, but a lower likelihood of spreading to fixation, than an equivalent gene with no effect on sex.     

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.     

The Population Dynamics of Maternal-Effect Selfish Genes

We use population genetic methods to describe the expected population dynamics of the selfish-gene chromosomal factor, Medea (maternal-effect dominant embryonic arrest), recently discovered in flour beetles, genus Tribolium. In the absence of deleterious effects on gross fecundity, Medea factors spread to fixation for all degrees of maternal-effect lethality greater than zero and the rate of spread is proportional to the strength of the maternal-effect. The rate of spread when rare is very slow, on the order of the frequency squared p(2), but this can be accelerated to order p when there is density regulation at the level of families as is known to occur for some genetic strains of flour beetles. When there are general deleterious effects of Medea on fecundity, affecting all offspring genotypes in addition to the genotype-specific maternal effect, then a stable interior polymorphism is possible. The location of the interior equilibrium and the probability of loss or fixation are sensitive to the degree of dominance of these fecundity effects.     

The distribution and spread of naturally occurring Medea selfish genetic elements in the United States

Selfish genetic elements (SGEs) are DNA sequences that are transmitted to viable offspring in greater than Mendelian frequencies. Medea SGEs occur naturally in some populations of red flour beetle (Tribolium castaneum) and are expected to increase in frequency within populations and spread among populations. The large‐scale U.S. distributions of Medea‐4 (M⁴) had been mapped based on samples from 1993 to 1995. We sampled beetles in 2011–2014 and show that the distribution of M⁴ in the United States is dynamic and has shifted southward. By using a genetic marker of Medea‐1 (M¹), we found five unique geographic clusters with high and low M¹ frequencies in a pattern not predicted by microsatellite‐based analysis of population structure. Our results indicate the absence of rigid barriers to Medea spread in the United States, so assessment of what factors have limited its current distribution requires further investigation. There is great interest in using synthetic SGEs, including synthetic Medea, to alter or suppress pest populations, but there is concern about unpredicted spread of these SGEs and potential for populations to become resistant to them. The finding of patchy distributions of Medea elements suggests that released synthetic SGEs cannot always be expected to spread uniformly, especially in target species with limited dispersal.     

Testing transposable elements as genetic drive mechanisms using Drosophila P element constructs as a model system

The use of transposable elements (TEs) as genetic drive mechanisms was explored using Drosophila melanogaster as a model system. Alternative strategies, employing autonomous and nonautonomous P element constructs were compared for their efficiency in driving the ry⁺ allele into populations homozygous for a ry^- allele at the genomic rosy locus. Transformed flies were introduced at 1%, 5%, and 10% starting frequencies to establish a series of populations that were monitored over the course of 40 generations, using both phenotypic and molecular assays. The transposon-borne ry⁺ marker allele spread rapidly in almost all populations when introduced at 5% and 10% seed frequencies, but 1% introductions frequently failed to become established. A similar initial rapid increase in frequency of the ry⁺ transposon occurred in several control populations lacking a source of transposase. Constructs carrying ry⁺ markers also increased to moderate frequencies in the absence of selection on the marker. The results of Southern and in situ hybridization studies indicated a strong inverse relationship between the degree of conservation of construct integrity and transposition frequency. These finding have relevance to possible future applications of transposons as genetic drive mechanisms. This revised version was published online in July 2006 with corrections to the Cover Date.     

Regulating the expression of gene drives is key to increasing their invasive potential and the mitigation of resistance

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.