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Transgene-based genetic sexing methods are being developed for insects of agricultural and public health importance. Male-only rearing has long been sought in sericulture because males show superior economic characteristics, such as better fitness, lower food consumption, and higher silk yield. Here we report the establishment of a transgene-based genetic sexing system for the silkworm, Bombyx mori . We developed a construct in which a positive feedback loop regulated by sex-specific alternative splicing leads to high-level expression of the tetracycline-repressible transactivator in females only. Transgenic animals show female-specific lethality during embryonic and early larval stages, leading to male-only cocoons. This transgene-based female-specific lethal system not only has wide application in sericulture, but also has great potential in lepidopteran pest control.

Background The fall armyworm, Spodoptera frugiperda , is a significant and widespread pest of maize, sorghum, rice, and other economically important crops. Successful management of this caterpillar pest has historically relied upon application of synthetic insecticides and through cultivation of genetically engineered crops expressing insecticidal proteins ( Bt crops). Fall armyworm has, however, developed resistance to both synthetic insecticides and Bt crops, which risks undermining the benefits delivered by these important crop protection tools. Previous modelling and empirical studies have demonstrated that releases of insecticide- or Bt -susceptible insects genetically modified to express conditional female mortality can both dilute insecticide resistance and suppress pest populations. Results Here, we describe the first germline transformation of the fall armyworm and the development of a genetically engineered male-selecting self-limiting strain, OX5382G, which exhibits complete female mortality in the absence of an additive in the larval diet. Laboratory experiments showed that males of this strain are competitive against wild-type males for copulations with wild-type females, and that the OX5382G self-limiting transgene declines rapidly to extinction in closed populations following the cessation of OX5382G male releases. Population models simulating the release of OX5382G males in tandem with Bt crops and non- Bt ‘refuge’ crops show that OX5382G releases can suppress fall armyworm populations and delay the spread of resistance to insecticidal proteins. Conclusions This article describes the development of self-limiting fall armyworm designed to control this pest by suppressing pest populations, and population models that demonstrate its potential as a highly effective method of managing resistance to Bt crops in pest fall armyworm populations. Our results provide early promise for a potentially valuable future addition to integrated pest management strategies for fall armyworm and other pests for which resistance to existing crop protection measures results in damage to crops and impedes sustainable agriculture.

Alternative, biologically-based approaches for pest management are sorely needed and one approach is to use genetically engineered insects. Herein we describe a series of integrated field, laboratory and modeling studies with the diamondback moth, , a serious global pest of crucifers. A \"self-limiting\" strain of (OX4319L), genetically engineered to allow the production of male-only cohorts of moths for field releases, was developed as a novel approach to protect crucifer crops. Wild-type females that mate with these self-limiting males will not produce viable female progeny. Our previous greenhouse studies demonstrated that releases of OX4319L males lead to suppression of the target pest population and dilution of insecticide-resistance genes. We report results of the first open-field release of a non-irradiated, genetically engineered self-limiting strain of an agricultural pest insect. In a series of mark-release-recapture field studies with co-releases of adult OX4319L males and wild-type counterparts, the dispersal, persistence and field survival of each strain were measured in a 2.83 ha cabbage field. In most cases, no differences were detected in these parameters. Overall, 97.8% of the wild-type males and 95.4% of the OX4319L males recaptured dispersed <35 m from the release point. The predicted persistence did not differ between strains regardless of release rate. With 95% confidence, 75% of OX4319L males released at a rate of 1,500 could be expected to live between 3.5 and 5.4 days and 95% of these males could be expected to be detected within 25.8-34.9 m from the release point. Moth strain had no effect on field survival but release rate did. Collectively, these results suggest similar field behavior of OX4319L males compared to its wild-type counterpart. Laboratory studies revealed no differences in mating competitiveness or intrinsic growth rates between the strains and small differences in longevity. Using results from these studies, mathematical models were developed that indicate release of OX4319L males should offer efficacious pest management of . Further field studies are recommended to demonstrate the potential for this self-limiting to provide pest suppression and resistance management benefits, as was previously demonstrated in greenhouse studies.

The Sterile Insect Technique is a species-specific and environmentally friendly method of pest control involving mass release of sterilized insects that reduce the wild population through infertile matings 1 , 2 , 3 , 4 , 5 . Insects carrying a female-specific autocidal genetic system offer an attractive alternative to conventional sterilization methods 6 , 7 while also eliminating females from the release population 7 , 8 , 9 , 10 . We exploited sex-specific alternative splicing in insects to engineer female-specific autocidal genetic systems in the Mediterranean fruit fly, Ceratitis capitata . These rely on the insertion of cassette exons from the C. capitata transformer gene into a heterologous tetracycline-repressible transactivator such that the transactivator transcript is disrupted in male splice variants but not in the female-specific one. As the key components of these systems function across a broad phylogenetic range, this strategy addresses the paucity of sex-specific expression systems (e.g., early-acting, female-specific promoters) in insects other than Drosophila melanogaster . The approach may have wide applicability for regulating gene expression in other organisms, particularly for combinatorial control with appropriate promoters.

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.

The Sterile Insect Technique (SIT) used to control insect pests relies on the release of large numbers of radiation-sterilized insects. Irradiation can have a negative impact on the subsequent performance of the released insects 1 , 2 , 3 , 4 and therefore on the cost and effectiveness of a control program 5 . This and other problems associated with current SIT programs could be overcome by the use of recombinant DNA methods and molecular genetics 6 , 7 , 8 , 9 , 10 , 11 , 12 . Here we describe the construction of strains of the Mediterranean fruit fly (medfly) harboring a tetracycline-repressible transactivator (tTA) that causes lethality in early developmental stages of the heterozygous progeny but has little effect on the survival of the parental transgenic tTA insects. We show that these properties should prove advantageous for the implementation of insect pest control programs.

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.

The cotton pest, pink bollworm (Pectinophora gossypiella (Saunders)), is a significant pest in most cotton-growing areas around the world. In southwestern USA and northern Mexico, pink bollworm is the target of the sterile insect technique (SIT), which relies on the mass-release of sterile pink bollworm adults to over-flood the wild population and thereby reduce it over time. Sterile moths reared for release are currently marked with a dye provided in their larval diet. There are concerns, however, that this marker fails from time to time, leading to sterile moths being misidentified in monitoring traps as wild moths. This can lead to expensive reactionary releases of sterile moths. We have developed a genetically marked strain that is engineered to express a fluorescent protein, DsRed2, which is easily screened under a specialised microscope. In order to test this marker under field conditions, we placed wild-type and genetically marked moths on traps and placed them in field cages. The moths were then screened, in a double-blind fashion, for DsRed2 fluorescence at regular intervals to determine marker reliability over time. The marker was shown to be robust in very high temperatures and generally proved reliable for a week or longer. More importantly, genotyping of moths on traps by PCR screening of the moths was 100% correct. Our findings indicate that this strain - and fluorescent protein markers in general - could make a valuable contribution to SIT.

The mass release of transgenic insects carrying female lethal self‐limiting genes can reduce pest insect populations. Substantial releases are also a novel resistance management tool, since wild type alleles conferring susceptibility to pesticides can dilute resistance alleles in target populations. However, a potential barrier is the need for large‐scale area‐wide releases. Here, we address whether localized releases of transgenic insects could provide an alternative means of population suppression and resistance management, without serious loss of efficacy. We used experimental mesocosms constituting insect metapopulations to explore the evolution of resistance to the Bacillus thuringiensis toxin Cry1Ac in a high‐dose/refugia landscape in the insect Plutella xylostella. We ran two selection experiments, the first compared the efficacy of “everywhere” releases and negative controls to a spatially density‐dependent or “whack‐a‐mole” strategy that concentrated release of transgenic insects in subpopulations with elevated resistance. The second experiment tested the relative efficacy of whack‐a‐mole and everywhere releases under spatially homogenous and heterogeneous selection pressure. The whack‐a‐mole releases were less effective than everywhere releases in terms of slowing the evolution of resistance, which, in the first experiment, largely prevented the evolution of resistance. In contrast to predictions, heterogeneous whack‐a‐mole releases were no more effective under heterogeneous selection pressure. Heterogeneous selection pressure did, however, reduce total insect population sizes. Whack‐a‐mole releases provided early population suppression, indistinguishable from homogeneous everywhere releases. However, insect population densities tracked the evolution of resistance in this system, as phenotypic resistance provides access to additional diet containing the toxin Cry1Ac. Thus, as resistance levels diverged between treatments, carrying capacities and population sizes increased under the whack‐a‐mole approach. Synthesis and applications. Spatially density‐dependent releases of transgenic insects, particularly those targeting source populations at a landscape level, could suppress pest populations in the absence of blanket area‐wide releases. The benefits of self‐limiting transgenic insects were reduced in spatially localized releases, suggesting that they are not ideal for “spot” treatment of resistance problems. Nevertheless, spatially homogeneous or heterogeneous releases could be used to support other resistance management interventions. Spatially density‐dependent releases of transgenic insects, particularly those targeting source populations at a landscape level, could suppress pest populations in the absence of blanket area‐wide releases. The benefits of self‐limiting transgenic insects were reduced in spatially localized releases, suggesting that they are not ideal for “spot” treatment of resistance problems. Nevertheless, spatially homogeneous or heterogeneous releases could be used to support other resistance management interventions.