Field performance of a genetically engineered strain of pink bollworm

Pest insects harm crops, livestock and human health, either directly or by acting as vectors of disease. The Sterile Insect Technique (SIT)--mass-release of sterile insects to mate with, and thereby control, their wild counterparts--has been used successfully for decades to control several pest species, including pink bollworm, a lepidopteran pest of cotton. Although it has been suggested that genetic engineering of pest insects provides potential improvements, there is uncertainty regarding its impact on their field performance. Discrimination between released and wild moths caught in monitoring traps is essential for estimating wild population levels. To address concerns about the reliability of current marking methods, we developed a genetically engineered strain of pink bollworm with a heritable fluorescent marker, to improve discrimination of sterile from wild moths. Here, we report the results of field trials showing that this engineered strain performed well under field conditions. Our data show that attributes critical to SIT in the field--ability to find a mate and to initiate copulation, as well as dispersal and persistence in the release area--were comparable between the genetically engineered strain and a standard strain. To our knowledge, these represent the first open-field experiments with a genetically engineered insect. The results described here provide encouragement for the genetic control of insect pests.     

Towards the genetic control of invasive species

Invasive species remain one of the greatest threats to global biodiversity. Their control would be enhanced through the development of more effective and sustainable pest management strategies. Recently, a novel form of genetic pest management (GPM) has been developed in which the mating behaviour of insect pests is exploited to introduce genetically engineered DNA sequences into wild conspecific populations. These ‘transgenes’ work in one or more ways to reduce the damage caused by a particular pest, for example reducing its density, or its ability to vector disease. Although currently being developed for use against economically important insect pests, these technologies would be highly appropriate for application against invasive species that threaten biodiversity. Importantly, these technologies have begun to advance in scope beyond insects to vertebrates, which include some of the world’s worst invasives. Here we review the current state of this rapidly progressing field and, using an established set of eradication criteria, discuss the characteristics which make GPM technologies suitable for application against invasive pests.     

Site-Specific Cassette Exchange Systems in the Aedes aegypti Mosquito and the Plutella xylostella Moth

Genetically engineered insects are being evaluated as potential tools to decrease the economic and public health burden of mosquitoes and agricultural pest insects. Here we describe a new tool for the reliable and targeted genome manipulation of pest insects for research and field release using recombinase mediated cassette exchange (RMCE) mechanisms. We successfully demonstrated the established ΦC31-RMCE method in the yellow fever mosquito, Aedes aegypti, which is the first report of RMCE in mosquitoes. A new variant of this RMCE system, called iRMCE, combines the ΦC31-att integration system and Cre or FLP-mediated excision to remove extraneous sequences introduced as part of the site-specific integration process. Complete iRMCE was achieved in two important insect pests, Aedes aegypti and the diamondback moth, Plutella xylostella, demonstrating the transferability of the system across a wide phylogenetic range of insect pests.     

害虫遗传防治的研究历史与现状

作为防治或根除重大害虫最为有效的手段之一,害虫遗传防治在世界范围内被广泛采用并取得了巨大成功。本文综述了不育昆虫技术、雌性致死系统和昆虫显性致死技术等经典害虫遗传防治策略的发展历史、技术特点和应用情况。近年来,许多新的分子生物手段被不断提出并整合到害虫遗传防治策略中,包括归巢核酸内切酶基因、锌指核酸酶、转录激活因子样效应因子核酸酶、CRISPR/Cas9系统、Medea元件、Killer-Rescue系统、Wolbachia-细胞质不亲和性系统等。基于这些新的工具手段,许多国家已经在不同程度上启动了下一代害虫遗传防治项目。而我国在该领域的研究相对薄弱,需要在借鉴国外成功经验的同时,进一步加强害虫遗传防治的基础和应用研究,从而实现本地有害生物的可持续治理和外来入侵生物的有效狙击,确保我国未来的粮食和生态安全。     

Pest control by the release of insects carrying a female-killing allele on multiple loci

With recent advances in genetics, many new strategies for pest control have become feasible. This is the second article in which we model new techniques for pest control based on the mass release of genetically modified insects. In this article we model the release of insects carrying a dominant and redundant female killing or sterilizing (FK) allele on multiple genetic loci. If such insects are released into a target population, the FK allele can become widely spread in the population through the males while reducing the population each generation by killing females. We allow the number of loci used to vary from 1 to 20. We also allow the FK allele to carry a fitness cost in males due to the gene insertions. Using a model, we explore the effectiveness and optimal strategies for such releases. In the most ideal circumstances (no density-dependence and released insects equal in fitness to wild ones), FK releases are several orders of magnitude more effective than equal sized sterile male releases. For example, a single release of 19 FK-bearing males for every two wild males, with the released males carrying the FK allele on 10 loci, reduces the target population to 0.002% of no-release size. An equal sized sterile release reduces the target population to 5% of no-release size. We also show how the effectiveness of the technique decreases as the fitness cost of the FK alleles in males increases. For example, the above mentioned release reduces the target population to 0.7% of no-release size if each FK allele carries a fitness cost in males of 5%. Adding a simple model for density-dependence and assuming that each of the released males carries the FK allele on six loci, we show that the release size necessary to reduce the target population to 1/100 of no-release size in 10 generations of releases varies from 0.44:1 to 4:1 (depending on parameter values). We also calculate the optimal number of loci on which to put the FK allele under various circumstances.     

Modelling the effects of inherited sterility for the application of the sterile insect technique

1 The sterile insect technique (SIT) involves the release of large numbers of sterile or partially‐sterile insects into a wild pest population to dilute the number of successful wild matings, with the eventual aim of eradication or area‐wide suppression. General population models, encompassing a wide range of SIT types, were used to derive principles for optimizing the success of SIT, with particular emphasis on the application of partial sterility leading to inherited sterility in the F₁ population. 2 The models show that inherited sterility can only be guaranteed to be more effective than complete sterility if matings between irradiated‐lineage partners are unsuccessful. This is widely assumed but rarely examined experimentally. 3 The models allow the critical overflooding ratio, φ_c, to be calculated for a particular target species, suggesting the release rate required to prevent population increase. Successful eradication using SIT alone should aim for a substantially higher release rate than suggested by φ_c. 4 The models show that pest populations may continue to increase in the first few generations of SIT releases, regardless of release rate, as irradiated‐lineage individuals infiltrate the population. This does not necessarily imply that the SIT programme will be unsuccessful in the longer term. 5For pests with overlapping generations, the models suggest that frequent small releases may be more effective than less frequent large releases, particularly when the average release rate is close to the critical threshold for success.     

Effect of the high dose/refuge strategy on Bt-crop yield dynamics (mathematical simulation)

A mathematical model is presented that describes interactions between Bt-crops and insect pests taking into account the plant growth rate, consumption of plant biomass by pests, suppression of insect proliferation by Bt toxins, emergence of Bt-resistant insects and their mixing with the wild type. It is shown that migration of Bt-susceptible insects from “refuge plots” onto the Bt-crop field invaded by resistance-carrying insects improves the yield dynamics; being high enough, this inflow eventually eliminates the resistant pest population.     

Engineered Repressible Lethality for Controlling the Pink Bollworm, a Lepidopteran Pest of Cotton

The sterile insect technique (SIT) is an environmentally friendly method of pest control in which insects are mass-produced, irradiated and released to mate with wild counterparts. SIT has been used to control major pest insects including the pink bollworm (Pectinophora gossypiella Saunders), a global pest of cotton. Transgenic technology has the potential to overcome disadvantages associated with the SIT, such as the damaging effects of radiation on released insects. A method called RIDL (Release of Insects carrying a Dominant Lethal) is designed to circumvent the need to irradiate insects before release. Premature death of insects’ progeny can be engineered to provide an equivalent to sterilisation. Moreover, this trait can be suppressed by the provision of a dietary antidote. In the pink bollworm, we generated transformed strains using different DNA constructs, which showed moderate-to-100% engineered mortality. In permissive conditions, this effect was largely suppressed. Survival data on cotton in field cages indicated that field conditions increase the lethal effect. One strain, called OX3402C, showed highly penetrant and highly repressible lethality, and was tested on host plants where its larvae caused minimal damage before death. These results highlight a potentially valuable insecticide-free tool against pink bollworm, and indicate its potential for development in other lepidopteran pests.     

Population genetic structure of the biological control agent Macrolophus pygmaeus in Mediterranean agroecosystems

Biological control of agricultural pests relies on knowledge of agroecosystem functionality, particularly when affected by the use of mass‐produced biological agents. Incorporating pre‐ and/or post‐release information such as genetic diversity and structure on these agents using molecular‐based approaches could advance our knowledge of how they perform in agroecosystems. We evaluated the population genetics of Macrolophus pygmaeus, the most widely used predatory mirid against many arthropod pests of greenhouse crops in the Mediterranean region, using the mitochondrial Cytb sequence and microsatellite data, and population genetics and phylogeny approaches. We investigated commercially mass‐produced insects (i.e., commercial insects either mass‐reared in the laboratory for many generations, or purchased by farmers and released in the greenhouses) and “wild” insects (i.e., that occur naturally outside or are collected in nature for release in the greenhouses). The mirids were mainly collected in agroecosystems in which solanaceous plants are grown in northern Spain, southern France and Greece. Both molecular markers and approaches distinguished 2 genetically differentiated populations. The less genetically diverse population, hereafter named the “commercial” strain included all individuals from laboratory mass‐rearings and most releases of commercially bred individuals. The most genetically diverse population mainly comprised individuals originating from noncultivated environments, or from releases of “wild” individuals. Rare examples of hybridization between M. pygmaeus from the 2 populations were observed and asymmetric gene flow was revealed. These findings provide new insights into what happens to M. pygmaeus released in the agroecosystems we studied, and show that it is possible to monitor some commercial strains.     

Landscape refuges delay resistance of the European corn borer to Bt-maize: a demo-genetic dynamic model

We constructed a reaction-diffusion model of the development of resistance to transgenic insecticidal Bt crops in pest populations. Kostitzin’s demo-genetic model describes local interactions between three competing pest genotypes with alleles conferring resistance or susceptibility to transgenic plants, the spatial spread of insects being modelled by diffusion. This new approach makes it possible to combine a spatial demographic model of population dynamics with classical genetic theory. We used this model to examine the effects of pest dispersal and of the size and shape of the refuge on the efficiency of the “high-dose/refuge” strategy, which was designed to prevent the development of resistance in populations of insect pests, such as the European corn borer, Ostrinia nubilalis Hübner (Lepidoptera, Crambidae). We found that, with realistic combinations of refuge size and pest dispersal, the development of resistance could be considerably delayed. With a small to medium-sized farming area, contiguous refuge plots are more efficient than a larger number of smaller refuge patches. We also show that the formal coupling of classical Fisher–Haldane–Wright population genetics equations with diffusion terms inaccurately describes the development of resistance in a spatially heterogeneous pest population, notably overestimating the speed with which Bt resistance is selected in populations of pests targeted by Bt crops.     

Dissimilarity of contemporary and historical gene flow in a wild carrot (Daucus carota) metapopulation under contrasting levels of human disturbance: implications for risk assessment and management of transgene introgression

Background and Aims

Transgene introgression from crops into wild relatives may increase the resistance of wild plants to herbicides, insects, etc. The chance of transgene introgression depends not only on the rate of hybridization and the establishment of hybrids in local wild populations, but also on the metapopulation dynamics of the wild relative. The aim of the study was to estimate gene flow in a metapopulation for assessing and managing the risks of transgene introgression.

Methods

Wild carrots (Daucus carota) were sampled from 12 patches in a metapopulation. Eleven microsatellites were used to genotype wild carrots. Genetic structure was estimated based on the F_ST statistic. Contemporary (over the last several generations) and historical (over many generations) gene flow was estimated with assignment and coalescent methods, respectively.

Key Results

The genetic structure in the wild carrot metapopulation was moderate (F_ST = 0·082) and most of the genetic variation resided within patches. A pattern of isolation by distance was detected, suggesting that most of the gene flow occurred between neighbouring patches (≤1 km). The mean contemporary gene flow was 5 times higher than the historical estimate, and the correlation between them was very low. Moreover, the contemporary gene flow in roadsides was twice that in a nature reserve, and the correlation between contemporary and historical estimates was much higher in the nature reserve. Mowing of roadsides may contribute to the increase in contemporary gene flow. Simulations demonstrated that the higher contemporary gene flow could accelerate the process of transgene introgression in the metapopulation.

Conclusions

Human disturbance such as mowing may alter gene flow patterns in wild populations, affecting the metapopulation dynamics of wild plants and the processes of transgene introgression in the metapopulation. The risk assessment and management of transgene introgression and the control of weeds need to take metapopulation dynamics into consideration.     

973项目“重大农业害虫猖獗危害的机制及可持续控制的基础研究”简介

2006年9月,国家重点基础研究发展计划(973)农业领域2006年度项目“重大农业害虫猖獗危害的机制及可持续控制的基础研究”经科技部批准正式立项启动。该项目以全系统管理思想为指导,在基因、个体、种群、生态系统等不同层次,阐明我国重大农业害虫种群分化与暴发的分子基础,解析害虫与寄主作物及天敌间的相互作用机制,建立害虫监测与预警系统,提出重大农业害虫可持续控制的新途径和新方法,为我国农业减灾、经济的可持续发展奠定科学基础。项目的主要研究内容包括:害虫生长发育与生殖调控的分子机制;害虫对环境胁迫的适应机制;杀虫药剂诱导害虫再猖獗的机制;害虫与寄主植物的协同进化;天敌与害虫的互作及控害机制;作物-害虫-天敌食物网关系及其调控机理;重大害虫区域性暴发监测与预警。项目的总体目标为:阐明害虫生长发育、种群分化的分子基础,揭示害虫种群调节的内在机制;解析作物、害虫及天敌间的互作机制,丰富和发展植物-害虫-天敌协同进化理论;阐明主要害虫区域性灾变机理,发展害虫预警新技术;发展与环境相容的、增强自然控害功能的新技术,提出重大农业害虫可持续控制的新途径和新方法;凝炼一支害虫控制基础研究的创新团队,丰富和发展我国害虫管理的科学理论与实践,提升我国有害生物防控的原始创新和集成创新能力,扩大国际影响。     

High nucleotide diversity and limited linkage disequilibrium in Helicoverpa armigera facilitates the detection of a selective sweep

Insecticides impose extreme selective pressures on populations of target pests and so insecticide resistance loci of these species may provide the footprints of ‘selective sweeps''. To lay the foundation for future genome-wide scans for selective sweeps and inform genome-wide association study designs, we set out to characterize some of the baseline population genomic parameters of one of the most damaging insect pests in agriculture worldwide, Helicoverpa armigera. To this end, we surveyed nine Z-linked loci in three Australian H. armigera populations. We find that estimates of π are in the higher range among other insects and linkage disequilibrium decays over short distances. One of the surveyed loci, a cytochrome P450, shows an unusual haplotype configuration with a divergent allele at high frequency that led us to investigate the possibility of an adaptive introgression around this locus.     

Beyond Bt resistance of pests in the context of population dynamical complexity

Complexity in ecological systems often prevents long-term predictions about changes in population size and properties of the population dynamics. Mathematical modeling of such complex system behaviors can provide a rough idea of scenarios of the population dynamics. We use the reaction–diffusion model [Medvinsky, A.B., Morozov, A.Y., Velkov, V.V., Li, B.-L., Sokolov, M.S., Malchow, H., 2004. Modeling the invasion of recessive Bt-resistant insects: an impact on transgenic plants. J. Theor. Biol. 231, 121–127] to study the impact of pests resistant to toxins produced by genetically modified plants on the dynamics of the plant–insect system. Using genetically modified crops is an effective pest management tool for world-wide growers. However, there is a concern that pests may develop resistance to Bt toxins, which are a product of Bacillus thuringiensis genes introduced into genetically modified Bt plants. We show by computer simulations that the Bt plant–Bt-resistant insect dynamics resulting from the invasion of the Bt-resistant pests leads to variety of complex changes in the plant–insect biomass, which underlie the dependence of the Bt plant biomass on the duration of the insect reproduction period. We demonstrate that the plant and insect biomass can undergo both regular and irregular oscillations. We show that the character of such oscillations essentially depends on local insect fluxes resulting from inhomogeneous spatial distributions of the insects. In order to characterize the insect diffusion fluxes we introduce a new parameter, the diffusion number Dn. We show that the dependence between a value of Dn and regularity/irregularity of the plant–insect biomass oscillations is governed by a region in the model parameter space. In one of the regions the growth of the value of the diffusion number correlates with the transformation of regular oscillations into irregular ones, while in the neighboring region of the model parameter space the dependence between the character of the plant–insect oscillations and the value of the diffusion number Dn is more complex. The oscillations are irregular if the values of Dn are between 0.05 and 0.25. On either side of this interval the plant–insect oscillations are regular. The complex character of the response of the Bt crop–pest system to the invasion of Bt-resistant insects can lead to significant complications in attempts to regulate the system dynamics.     

Managing insecticide resistance by mass release of engineered insects

Transgenic crops producing insecticidal toxins are now widely used to control insect pests. The benefits of this method would be lost if resistance to the toxins spread to a significant proportion of the pest population. The primary resistance management method, mandatory in the United States, is the high-dose/ refuge strategy, requiring toxin-free crops as refuges near the insecticidal crops, and the use of toxin doses sufficiently high to kill insects heterozygous for a resistance allele, thereby rendering resistance functionally recessive. We propose that mass-release of harmless susceptible (toxin-sensitive) insects could substantially delay or even reverse the spread of resistance. Mass-release of such insects is an integral part of release of insects carrying a dominant lethal (RIDL), a method of pest control related to the sterile insect technique. We show by mathematical modeling that specific RIDL strategies could form an effective component of a resistance management strategy for plant-incorporated protectants and other toxins.     

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.     

Population differentiation and intraspecific genetic admixture in two Eucryptorrhynchus weevils (Coleoptera: Curculionidae) across northern China

Increasing damage of pests in agriculture and forestry can arise both as a consequence of changes in local species and through the introduction of alien species. In this study, we used population genetics approaches to examine population processes of two pests of the tree‐of‐heaven trunk weevil (TTW), Eucryptorrhynchus brandti (Harold) and the tree‐of‐heaven root weevil (TRW), E. scrobiculatus (Motschulsky) on the tree‐of‐heaven across their native range of China. We analyzed the population genetics of the two weevils based on ten highly polymorphic microsatellite markers. Population genetic diversity analysis showed strong population differentiation among populations of each species, with F _ST ranges from 0.0197 to 0.6650 and from −0.0724 to 0.6845, respectively. Populations from the same geographic areas can be divided into different genetic clusters, and the same genetic cluster contained populations from different geographic populations, pointing to dispersal of the weevils possibly being human‐mediated. Redundancy analysis showed that the independent effects of environment and geography could account for 93.94% and 29.70% of the explained genetic variance in TTW, and 41.90% and 55.73% of the explained genetic variance in TRW, respectively, indicating possible impacts of local climates on population genetic differentiation. Our study helps to uncover population genetic processes of these local pest species with relevance to control methods.     

Admixture analysis of stocked brown trout populations using mapped microsatellite DNA markers: indigenous trout persist in introgressed populations

Admixture between wild and captive populations is an increasing concern in conservation biology. Understanding the extent of admixture and the processes involved requires identification of admixed and non-admixed individuals. This can be achieved by statistical methods employing Bayesian clustering, but resolution is low if genetic differentiation is weak. Here, we analyse stocked brown trout populations represented by historical (1943–1956) and contemporary (2000s) samples, where genetic differentiation between wild populations and stocked trout is weak (pairwise F_ST of 0.047 and 0.053). By analysing a high number of microsatellite DNA markers (50) and making use of linkage map information, we achieve clear identification of admixed and non-admixed trout. Moreover, despite strong population-level admixture by hatchery strain trout in one of the populations (70.8%), non-admixed individuals nevertheless persist (7 out of 53 individuals). These remnants of the indigenous population are characterized by later spawning time than the majority of the admixed individuals. We hypothesize that isolation by time mediated by spawning time differences between wild and hatchery strain trout is a major factor rescuing a part of the indigenous population from introgression.     

Survey of Heritable Endosymbionts in Southern Mexico Populations of the Fruit Fly Species Anastrepha striata and A. ludens

Heritable endosymbiotic bacteria associated with insects are ubiquitous and taxonomically diverse. Many of these endosymbionts influence the fitness of their hosts and/or manipulate their host reproduction. Exploiting the effects of endosymbionts on hosts for pest control is a growing research area, but requires knowledge of endosymbionts associated with the target pest population. In this study, we used molecular methods to screen southern Mexico populations of two species of tephritid fruit fly pests, Anastrepha ludens and A. striata, for heritable bacteria. The only heritable endosymbiont found was Wolbachia in A. striata. Based on multilocus sequence typing and phylogenetic analyses, this Wolbachia strain is new and belongs to the Wolbachia supergroup B. Wolbachia strains previously reported in members of the genus Anastrepha in South America belong to supergroup A. We discuss the potential implications for pest control of the presence of a different Wolbachia strain in southern Mexico.     

Modeling resistance to genetic control of insects

The sterile insect technique is an area-wide pest control method that reduces pest populations by releasing mass-reared sterile insects which compete for mates with wild insects. Modern molecular tools have created possibilities for improving and extending the sterile insect technique. As with any new insect control method, questions arise about potential resistance. Genetic RIDL^®1 (Release of Insects carrying a Dominant Lethal) technology is a proposed modification of the technique, releasing insects that are homozygous for a repressible dominant lethal genetic construct rather than being sterilized by irradiation. Hypothetical resistance to the lethal mechanism is a potential threat to RIDL strategies' effectiveness. Using population genetic and population dynamic models, we assess the circumstances under which monogenic biochemically based resistance could have a significant impact on the effectiveness of releases for population control. We assume that released insects would be homozygous susceptible to the lethal genetic construct and therefore releases would have a built-in element of resistance dilution. We find that this effect could prevent or limit the spread of resistance to RIDL constructs; the outcomes are subject to competing selective forces deriving from the fitness properties of resistance and the release ratio. Resistance that is spreading and capable of having a significant detrimental impact on population reduction is identifiable, signaling in advance a need for mitigating action.