Genetics‐based methods for agricultural insect pest management

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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.     

Genetic elimination of field-cage populations of Mediterranean fruit flies

The Mediterranean fruit fly (medfly, Ceratitis capitata Wiedemann) is a pest of over 300 fruits, vegetables and nuts. The sterile insect technique (SIT) is a control measure used to reduce the reproductive potential of populations through the mass release of sterilized male insects that mate with wild females. However, SIT flies can display poor field performance, due to the effects of mass-rearing and of the irradiation process used for sterilization. The development of female-lethal RIDL (release of insects carrying a dominant lethal) strains for medfly can overcome many of the problems of SIT associated with irradiation. Here, we present life-history characterizations for two medfly RIDL strains, OX3864A and OX3647Q. Our results show (i) full functionality of RIDL, (ii) equivalency of RIDL and wild-type strains for life-history characteristics, and (iii) a high level of sexual competitiveness against both wild-type and wild-derived males. We also present the first proof-of-principle experiment on the use of RIDL to eliminate medfly populations. Weekly releases of OX3864A males into stable populations of wild-type medfly caused a successive decline in numbers, leading to eradication. The results show that genetic control can provide an effective alternative to SIT for the control of pest insects.     

害虫遗传不育技术中致死基因的研究与应用

基于遗传修饰手段的昆虫不育技术(SIT)作为一类物种特异、环境友好、科学高效的新兴策略,在害虫防治中具有广阔的应用前景。释放携带显性致死基因昆虫的技术(RIDL)是改进传统SIT的重要手段之一,主要包括四环素调控系统、特异性启动子、性别特异剪接系统和特异性致死基因等重要元件,其中根据不同昆虫的特点选择合适的特异性致死基因对于构建遗传不育品系至关重要。这些致死基因或受到阻遏调控系统的控制、或特异的在雌虫中表达、亦或直接作用于X染色体,导致后代在特定发育阶段或特定性别中条件致死。本文综述了RHG家族(reapr、hid、grim、michelob_x)细胞凋亡基因、转录激活因子t TA及Nipp1Dm、归巢内切酶基因等在害虫遗传不育技术中的研究和应用,讨论了特定致死基因的效应机理和应用特点,并对其可能的发展方向进行了展望。由于不同效应基因的致死作用和调控机理尚未完全明晰,因此深入研究特异致死基因的凋亡机制和在不同物种中的兼容作用,将为害虫遗传防控提供更多的研究思路和手段。     

Genetic control of Plutella xylostella in omics era

Diamondback moth, Plutella xylostella (L.), is a specialist pest on cruciferous crops of economic importance. The large‐scale use of chemical insecticides for the control of this insect pest has caused a number of challenges to agro‐ecosystems. With the advent of the omics era, genetic pest management strategies are becoming increasingly feasible and show a powerful potential for pest control. Here, we review strategies for using transgenic plants and sterile insect techniques for genetic pest management and introduce the major advances in the control of P. xylostella using a female‐specific RIDL (release of insects carrying a dominant lethal gene) strategy. Further, the advantages of gene drive developed in combination with sex determination and CRISPR/Cas9 systems are addressed, and the corresponding prospects and implementation issues are discussed. It is predictable that under the policy and regulation of professional committees, the genetic pest control strategy, especially for gene drive, will open a new avenue to sustainable pest management not only for P. xylostella but also for other insect pests.     

A model framework to estimate impact and cost of genetics-based sterile insect methods for dengue vector control

Vector-borne diseases impose enormous health and economic burdens and additional methods to control vector populations are clearly needed. The Sterile Insect Technique (SIT) has been successful against agricultural pests, but is not in large-scale use for suppressing or eliminating mosquito populations. Genetic RIDL technology (Release of Insects carrying a Dominant Lethal) is a proposed modification that involves releasing insects that are homozygous for a repressible dominant lethal genetic construct rather than being sterilized by irradiation, and could potentially overcome some technical difficulties with the conventional SIT technology. Using the arboviral disease dengue as an example, we combine vector population dynamics and epidemiological models to explore the effect of a program of RIDL releases on disease transmission. We use these to derive a preliminary estimate of the potential cost-effectiveness of vector control by applying estimates of the costs of SIT. We predict that this genetic control strategy could eliminate dengue rapidly from a human community, and at lower expense (approximately US$ 2~30 per case averted) than the direct and indirect costs of disease (mean US$ 86-190 per case of dengue). The theoretical framework has wider potential use; by appropriately adapting or replacing each component of the framework (entomological, epidemiological, vector control bio-economics and health economics), it could be applied to other vector-borne diseases or vector control strategies and extended to include other health interventions.     

遗传不育技术在蚊媒疾病防控中的应用

疟疾、登革热等重大传染性蚊媒疾病严重危害人类健康,且目前缺乏有效的药物和疫苗,防治埃及伊蚊、冈比亚按蚊等媒介昆虫是控制和消除这些疾病的有效手段。化学杀虫剂的大规模使用在一定程度上控制了疾病的传播,但其抗药性和环境污染等问题也随之而来。分子生物学的飞速发展为昆虫不育技术(SIT)的更新及害虫防治提供了新的策略,由此发展起来的以释放携带显性致死基因昆虫(RIDL)为代表的一系列遗传不育技术为蚊虫种群防控提供了更加有效的选择。本文概述了遗传技术在蚊虫防控中的应用进展,包括蚊虫遗传防治的历史和策略,阐述了RIDL技术体系的原理,同时介绍了相关遗传控制品系和已经开展的田间释放研究,展示了遗传修饰不育技术在蚊媒疾病防治中的巨大潜力。     

昆虫种群的遗传调控

昆虫种群的遗传调控是利用昆虫自身生长发育的关键基因,采用性别控制开关,通过遗传转化使雄虫成为携带导致后代雌虫发育异常或雌性不育的遗传控制复合体（性别开关元件和靶标基因的复合体）．昆虫种群遗传调控是一种基于不育技术的昆虫种群控制系统,具有种类特异、环境友好和便捷高效等特点．目前为止,已经由早期的通过辐射不育方法发展到释放携带显性致死基因昆虫的方法,并在多种昆虫中获得成功．本文综述了昆虫种群遗传调控的发展历程,介绍了昆虫种群遗传调控相关的理论与方法,包括特异的调控元件、致死或缺陷基因和遗传转化体系的应用,并列举了几种昆虫种群遗传调控的实例,最后对于昆虫种群遗传调控系统中存在的问题以及可能的发展方向进行了展望．     

Towards the genetic control of insect vectors: An overview

Insects are responsible for the transmission of major infectious diseases. Recent advances in insect genomics and transformation technology provide new strategies for the control of insect borne pathogen transmission and insect pest management. One such strategy is the genetic modification of insects with genes that block pathogen development. Another is to suppress insect populations by releasing either sterile males or males carrying female‐specific dominant lethal genes into the environment. Although significant progress has been made in the laboratory, further research is needed to extend these approaches to the field. These insect control strategies offer several advantages over conventional insecticide‐based strategies. However, the release of genetically modified insects into the environment should proceed with great caution, after ensuring its safety, and acceptance by the target populations.     

Pest control by the introduction of a conditional lethal trait on multiple loci: potential, limitations, and optimal strategies

Advances in genetics have made it feasible to genetically engineer insect strains carrying a conditional lethal trait on multiple loci. We model the release into a target pest population of insects carrying a dominant and fully penetrant conditional lethal trait on 1-20 loci. Delaying the lethality for several generations after release allows the trait to become widely spread in the target population before being activated. To determine effectiveness and optimal strategies for such releases, we vary release size, number of generations until the conditional lethality, nonconditional fitness cost resulting from gene insertions, and fitness reduction associated with laboratory rearing. We show that conditional lethal releases are potentially orders of magnitude more effective than sterile male releases of equal size, and that far smaller release sizes may be required for this approach than necessary with sterile males. For example, a release of male insects carrying a conditional lethal allele that is activated in the F4 generation on 10 loci reduces the target populatioin to 10(-4) of no-release size if there are initially two released males for every wild male. We show how the effectiveness of conditional lethal releases decreases as the nonconditional fitness reduction (i.e., fitness reduction before the trait becomes lethal) associated with the conditional lethal genes increases. For example, if there is a 5% nonconditional fitness cost per conditional lethal allele, then a 2:1 (released male:wild male) release with conditional lethal alleles that are activated in the F4 generation reduces the population to 2-5% (depending on the degree of density dependence) of the no-release size. If there is a per-allele reduction in fitness, then as the number of loci is increased there is a trade-off between the fraction of offspring carrying at least one conditional lethal allele and the fitness of the released insects. We calculate the optimal number of loci on which to insert the conditional lethal gene given various conditions. In addition, we show how laboratory-rearing fitness costs, density-dependence, and all-male versus male-female releases affect the efficiency of conditional lethal releases.     

Oral Ingestion of Transgenic RIDL Ae. aegypti Larvae Has No Negative Effect on Two Predator Toxorhynchites Species

Dengue is the most important mosquito-borne viral disease. No specific treatment or vaccine is currently available; traditional vector control methods can rarely achieve adequate control. Recently, the RIDL (Release of Insect carrying Dominant Lethality) approach has been developed, based on the sterile insect technique, in which genetically engineered ‘sterile’ homozygous RIDL male insects are released to mate wild females; the offspring inherit a copy of the RIDL construct and die. A RIDL strain of the dengue mosquito, Aedes aegypti, OX513A, expresses a fluorescent marker gene for identification (DsRed2) and a protein (tTAV) that causes the offspring to die. We examined whether these proteins could adversely affect predators that may feed on the insect. Aedes aegypti is a peri-domestic mosquito that typically breeds in small, rain-water-filled containers and has no specific predators. Toxorhynchites larvae feed on small aquatic organisms and are easily reared in the laboratory where they can be fed exclusively on mosquito larvae. To evaluate the effect of a predator feeding on a diet of RIDL insects, OX513A Ae. aegypti larvae were fed to two different species of Toxorhynchites (Tx. splendens and Tx. amboinensis) and effects on life table parameters of all life stages were compared to being fed on wild type larvae. No significant negative effect was observed on any life table parameter studied; this outcome and the benign nature of the expressed proteins (tTAV and DsRed2) indicate that Ae. aegypti OX513A RIDL strain is unlikely to have any adverse effects on predators in the environment.     

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.     

Genetic sexing through the use of Y-linked transgenes

Sterile insect technique (SIT)-based pest control programs rely on the mass release of sterile insects to reduce the wild target population. In many cases, it is desirable to release only males. Sterile females may cause damage, e.g., disease transmission by mosquitoes or crop damage via oviposition by the Mediterranean fruit fly (Medfly). Also, sterile females may decrease the effectiveness of released males by distracting them from seeking out wild females. To eliminate females from the release population, a suitable sexual dimorphism is required. For several pest species, genetic sexing strains have been constructed in which such a dimorphism has been induced by genetics. Classical strains were based on the translocation to the Y chromosome of a selectable marker, which is therefore expressed only in males. Recently, several prototype strains have been constructed using sex-specific expression of markers or conditional lethal genes from autosomal insertions of transgenes. Here, we describe a novel genetic sexing strategy based on the use of Y-linked transgenes expressing fluorescent proteins. We demonstrate the feasibility of this strategy in a major pest species, Ceratitis capitata (Wiedemann), and discuss the advantages and disadvantages relative to other genetic sexing methods and potential applicability to other species.     

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.     

Improving the biological control of leafminers (Diptera: Agromyzidae) using the sterile insect technique

The leafminer Liriomyza trifolii (Burgess) (Diptera: Agromyzidae) is a worldwide pest of ornamental and vegetable crops. The most promising nonchemical approach for controlling Liriomyza leafminers in greenhouses is regular releases of the parasitoid Diglyphus isaea (Walker) (Hymenoptera: Eulophidae). In the current study, we examine the hypothesis that the use of D. isaea for biological control of leafminers in greenhouse crops may be more practical and efficient when supplemented with additional control strategies, such as the sterile insect technique (SIT). In small cages, our SIT experiments suggest that release of sterile L. trifolii males in three sterile-to-fertile male ratios (3:1, 5:1, and 10:1) can significantly reduce the numbers of the pest offspring. In large cage experiments, when both parasitoids and sterile males were released weekly, the combined methods significantly reduced mine production and the adult leafminer population size. Moreover, a synergistic interaction effect between these two methods was found, and a model based on our observed data predicts that because of this effect, only the use of both methods can eradicate the pest population. Our study indicates that an integrated pest management approach that combines the augmentative release of the parasitoid D. isaea together with sterile leafminer males is more efficient than the use of either method alone. In addition, our results validate previous theoretical models and demonstrate synergistic control with releases of parasitoids and sterile insects.     

Mating rates between sterile and wild codling moths (Cydia pomonella) in springtime: a simulation study

The sterile insect technique (SIT) can be a powerful method for pest control without the negative environmental effects of conventional pesticides. The goal is to induce pest population collapse by arranging conditions where wild females mate only with sterile males and thus do not produce offspring. In applying the SIT, it can be important to understand both how subtle alterations of sterile and wild insect behaviour alter the effectiveness of the SIT in different applications, and how this is reflected in the data gathered through associated monitoring devices, often pheromone traps.Our work in this paper is motivated by the use of SIT against orchard pests, particularly the codling moth (Cydia pomonella). We investigate how individual behaviours affect the mating rate between wild females and sterile males, and the corresponding sterile to wild trap catch ratio, through a preliminary individual-based model. Our analysis suggests that the sterile males may not be effective at interfering with mating between wild moths during springtime releases, while at the same time monitoring information gathered from trap catches may give no indication of reduced effectiveness of the SIT.     

Conceptual framework and rationale

The sterile insect technique (SIT) has been shown to be an effective and sustainable genetic approach to control populations of selected major pest insects, when part of area-wide integrated pest management (AW-IPM) programmes. The technique introduces genetic sterility in females of the target population in the field following their mating with released sterile males. This process results in population reduction or elimination via embryo lethality caused by dominant lethal mutations induced in sperm of the released males. In the past, several field trials have been carried out for mosquitoes with varying degrees of success. New technology and experience gained with other species of insect pests has encouraged a reassessment of the use of the sterility principle as part of integrated control of malaria vectors. Significant technical and logistic hurdles will need to be overcome to develop the technology and make it effective to suppress selected vector populations, and its application will probably be limited to specific ecological situations. Using sterile males to control mosquito vector populations can only be effective as part of an AW-IPM programme. The area-wide concept entails the targeting of the total mosquito population within a defined area. It requires, therefore, a thorough understanding of the target pest population biology especially as regards mating behaviour, population dynamics, dispersal and level of reproductive isolation. The key challenges for success are: 1) devising methods to monitor vector populations and measuring competitiveness of sterile males in the field, 2) designing mass rearing, sterilization and release strategies that maintain competitiveness of the sterile male mosquitoes, 3) developing methods to separate sexes in order to release only male mosquitoes and 4) adapting suppression measures and release rates to take into account the high reproductive rate of mosquitoes. Finally, success in area-wide implementation in the field can only be achieved if close attention is paid to political, socio-economic and environmental sensitivities and an efficient management organization is established taking into account the interests of all potential stakeholders of an AW-IPM programme.     

Pest control by genetic manipulation of sex ratio

We model the release of insects carrying an allele at multiple loci that shifts sex ratios in favor of males. We model two approaches to sex ratio alteration. In the first (denoted SD), meiotic segregation (or sperm fertility) is distorted in favor of gametes carrying the male-determining genetic element (e.g., Y-chromosome). It is assumed that any male carrying at least one copy of the SD allele produces only genotypically male offspring. In the second approach (denoted PM), the inserted allele alters sex ratio by causing genetically female individuals to become phenotypically male. It is assumed that any insect carrying at least one copy of the PM allele is phenotypically male. Both approaches reduce future population growth by reducing the number of phenotypic females. The models allow variation in the number of loci used in the release, the size of the release, and the negative fitness effect caused by insertion of each sex ratio altering allele. We show that such releases may be at least 2 orders of magnitude more effective than sterile male releases (SIT) in terms of numbers of surviving insects. For example, a single SD release with two released insects for every wild insect and a 5% fitness cost per inserted allele could reduce the target population to 1/1000th of the no-release population size, whereas a similar-sized SIT release would only reduce the population to one-fifth of its original size. We also compare these two sex ratio alteration approaches to a female-killing (FK) system and the sterile male technique when there are repeated releases over a number of generations. In these comparisons, the SD approach is the most efficient with equivalent pest suppression achieved by release of approximately 1 SD, 1.5-20 PM, 2-70 FK, and 16-3,000 SIT insects, depending on conditions. We also calculate the optimal number of SD and PM allele insertions to be used under various conditions, assuming that there is an additional genetic load incurred for each allelic insertion.     

Mutations and their use in insect control

Traditional chemically based methods for insect control have been shown to have serious limitations, and many alternative approaches have been developed and evaluated, including those based on the use of different types of mutation. The mutagenic action of ionizing radiation was well known in the field of genetics long before it was realized by entomologists that it might be used to induce dominant lethal mutations in insects, which, when released, could sterilize wild female insects. The use of radiation to induce dominant lethal mutations in the sterile insect technique (SIT) is now a major component of many large and successful programs for pest suppression and eradication. Adult insects, and their different developmental stages, differ in their sensitivity to the induction of dominant lethal mutations, and care has to be taken to identify the appropriate dose of radiation that produces the required level of sterility without impairing the overall fitness of the released insect. Sterility can also be introduced into populations through genetic mechanisms, including translocations, hybrid incompatibility, and inherited sterility in Lepidoptera. The latter phenomenon is due to the fact that this group of insects has holokinetic chromosomes. Specific types of mutations can also be used to make improvements to the SIT, especially for the development of strains for the production of only male insects for sterilization and release. These strains utilize male translocations and a variety of selectable mutations, either conditional or visible, so that at some stage of development, the males can be separated from the females. In one major insect pest, Ceratitis capitata, these strains are used routinely in large operational programs. This review summarizes these developments, including the possible future use of transgenic technology in pest control.     

Insect quality control: synchronized sex,mating system,and biological rhythm

The sterile insect technique (SIT) is a method of eradicating insects by releasing mass-reared sterilized males into fields to reduce the hatchability of eggs laid by wild females that have mated with the sterile males. SIT requires mass-production of the target insect, and maintenance of the quality of the mass-reared insects. The most important factor is successful mating between wild females and sterile males because SIT depends on their synchronized copulation. Therefore, understanding the mating systems and fertilization processes of target insects is prerequisite. Insect behavior often has circadian rhythms that are controlled by a biological clock. However, very few studies of relationships between sterile insect quality and circadian rhythm have been performed compared with the amount of research on the mating ability of target insects. The timing of male copulation attempts with receptivity of females is key to successful mating between released males and wild females. Therefore, we should focus on the mechanisms controlling the timing of mating in target insects. On the other hand, in biological control projects, precise timing of the release of natural enemies to attack pest species is required because behavior of pests and control agents are affected by their circadian rhythms. Involving both chronobiologists and applied entomologists might produce novel ideas for sterile insect quality control by synchronized sex between mass-reared and wild flies, and for biological control agent quality by matching timing in activity between predator activity and prey behavior. Control of the biological clocks in sterile insects or biological control agents is required for advanced quality control of rearing insects.