Combining the high‐dose/refuge strategy and self‐limiting transgenic insects in resistance management—A test in experimental mesocosms

Abstract The high‐dose/refuge strategy has been the primary approach for resistance management in transgenic crops engineered with Bacillus thuringiensis toxins. However, there are continuing pressures from growers to reduce the size of Bt toxin‐free refugia, which typically suffer higher damage from pests. One complementary approach is to release male transgenic insects with a female‐specific self‐limiting gene. This technology can reduce population sizes and slow the evolution of resistance by introgressing susceptible genes through males. Theory predicts that it could be used to facilitate smaller refugia or reverse the evolution of resistance. In this study, we used experimental evolution with caged insect populations to investigate the compatibility of the self‐limiting system and the high‐dose/refuge strategy in mitigating the evolution of resistance in diamondback moth, Plutella xylostella. The benefits of the self‐limiting system were clearer at smaller refuge size, particularly when refugia were inadequate to prevent the evolution of resistance. We found that transgenic males in caged mesocosms could suppress population size and delay resistance development with 10% refugia and 4%–15% initial resistance allele frequency. Fitness costs in hemizygous transgenic insects are particularly important for introgressing susceptible alleles into target populations. Fitness costs of the self‐limiting gene in this study (P. xylostella OX4139 line L) were incompletely dominant, and reduced fecundity and male mating competitiveness. The experimental evolution approach used here illustrates some of the benefits and pitfalls of combining mass release of self‐limiting insects and the high‐dose/refuge strategy, but does indicate that they can be complementary.


| INTRODUCTION
The damage caused by invertebrate pests accounts for 10%-15% of agricultural production, costing approximately US$8 billion in the United States (Metcalf, 1996), US$17.7 billion in Brazil (Oliveira, Auad, Mendes, & Frizzas, 2014), and US$359.8 million in Australia (Murray, Clarke, & Ronning, 2013). One approach to control pests and maintain sustainable agricultural yields is through the use of biopesticides such as Bacillus thuringiensis (Bt). Bt is extremely valuable in modern agriculture. This utility results from the insecticidal crystal (Cry) proteins that have high specificity to particular insect groups and hence low toxicity to nontarget organisms (Schnepf et al., 1998). The application of these insecticidal proteins through conventional spray formulations and in transgenic crops can provide effective pest management while maintaining agro-ecosystem biodiversity (Bravo, Likitvivatanavong, Gill, & Soberon, 2011). Nineteen crops and over 60 million hectares of land have been cultivated with biotech crops expressing Bt toxins (James, 2014). However, despite the success of genetically modified (GM) crops, a range of pest species have developed increased levels of resistance to Bt biopesticides and to the Cry toxins expressed in GM crops (Gassmann, Petzold-Maxwell, Keweshan, & Dunbar, 2011;Kruger, Van Rensburg, & Van den Berg, 2011;Storer, Kubiszak, Ed King, Thompson, & Santos, 2012;Tabashnik, Gassmann, Crowder, & Carrière, 2008;Tabashnik, Van Rensburg, & Carrière, 2009;Zhang et al., 2011Zhang et al., , 2012. While current resistance management strategies have been effective in a range of species (Carrière, Crowder, & Tabashnik, 2010), there is still scope for improvement and development.
The cornerstone of resistance management for GM crops is the high-dose/refuge strategy, an approach mandated in several countries. In the high-dose/refuge strategy, one part of target pest population is exposed to high concentrations (high doses) of toxins produced by Bt crops, rendering resistance functionally recessive. When the inheritance of resistance is recessive, only homozygous-resistant individuals (RR genotype) survive on Bt crops. Another proportion of the pest population is maintained in nearby refuges of non-Bt host plants, providing a reservoir of susceptible alleles (from RS and SS genotypes).
If the resistance allele frequency is low, homozygous-resistant pests surviving on Bt crops will be relatively rare, while susceptible pests will be abundant and readily available to mate with resistant individuals. Progeny from such matings will be heterozygous for resistance alleles and phenotypically susceptible to high-dose Bt crops, thereby hindering the evolution of resistance. Theoretical models and empirical observations have shown that the high-dose/refuge strategy is an effective approach to delay or prevent the development of resistance when the above conditions are met (Alphey, Coleman, Bonsall, & Alphey, 2008;Alstad & Andow, 1995;Caprio, Faver, & Hankins, 2004;Gould, 1998;Gryspeirt & Gregoire, 2012;Huang, Andow, & Buschman, 2011;Hutchison et al., 2010;Tyutyunov, Zhadanovskaya, Bourguet, & Arditi, 2008).
The high-dose/refuge strategy cannot be applied without regard to its basic assumptions. Certain genetic and ecological conditions need to hold true before it can be used to delay the evolution of resistance. These include the following: low initial resistance allele frequency; effectively recessive resistance; and efficient dispersal to refugia. The latter condition includes both random mating between the resistant and susceptible genotypes as well as random oviposition on Bt crop and in refugia (Burd, Gould, Bradley, Van Duyn, & Moar, 2003;Frutos, Rang, & Royer, 2008;Liu et al., 2001;Tellez-Rodriguez et al., 2014). Theoretical models and practical experience have shown that violation of these assumptions of the high-dose/ refuge strategy can lead to rapid evolution of resistance (Alstad & Andow, 1995;Campagne et al., 2016;Caprio et al., 2004;Georghiou & Taylor, 1977;Gould, 1998;Gryspeirt & Gregoire, 2012;Hutchison et al., 2010;Tyutyunov et al., 2008). In addition, if growers fail to plant refugia, then evolution of resistance to GM crops can also be rapid (Farias et al., 2014;Kruger et al., 2011;Monnerat et al., 2015;Storer et al., 2010). Thus, recent incidences of the evolution of resistance to Bt toxins in GM crops can largely be traced to failure of the basic assumptions, that is low doses or nonrecessive resistance (Gassmann et al., 2011;Storer et al., 2012) or to the fact that farmers are not adhering to the mandatory refuge planting requirements (Tabashnik, Brevault, & Carrière, 2013).
Alternative approaches may include the use of transgenic insects to mitigate resistance and to reduce pest population size directly.
Here, we will address experimentally whether the release of transgenic insects to suppress insect population size is compatible with the high-dose/refuge strategy and can improve its resilience. Recent advances in genetic engineering have enabled the development of transgenic insects carrying a repressible female-specific lethal gene (Thomas, Donnelly, Wood, & Alphey, 2000). In a strategy mimicking sterile insect technique programmes, the release of large numbers of transgenic males can reduce target populations, as there will be no viable offspring arising from mating of wild females and transgenic males (Alphey, Bonsall, & Alphey, 2009;Alphey, Coleman, Donnelly, & Alphey, 2007;Gentile, Rund, & Madey, 2015;Thomas et al., 2000). As these transgenes are designed to reduce insect fitness and will decline in frequency postrelease, this transgenic approach has been termed "self-limiting" (Gould, Huang, Legros, & Lloyd, 2008). In addition to suppressing pest population sizes, the mass release of self-limiting transgenic males can affect the genetic make-up of pest populations if lethality is targeted only at females, that is female-specific self-limiting transgenes. For example, alleles conferring susceptibility to insecticides carried by the transgenic population can be introgressed into the target population through the male line. Deterministic models of the mass release of self-limiting males show that this technology can be a valuable tool in slowing the evolution of resistance (Alphey et al., 2007(Alphey et al., , 2009. Given the importance of the high-dose/refuge strategy for managing the evolution of resistance in modern agriculture, a significant advance would be to understand how best to combine refugia with the use of transgenic insects bearing female-specific self-limiting genes. Theoretically, the mass release of the self-limiting males could facilitate the planting of smaller refugia while still preventing the evolution of resistance (Alphey et al., 2007(Alphey et al., , 2009. With increasing release ratios of the self-limiting insects, the mass release of the genetically engineered males could even reverse resistance development (Alphey et al., 2007(Alphey et al., , 2009. Smaller refuge sizes may be particularly attractive to farmers who are reluctant to tolerate large refugia or where it is difficult to enforce compliance. The mass release of the self-limiting males could also potentially help tackle issues like nonrandom mating between resistant and susceptible individuals as a result of different development times and population structure (Cerda & Wright, 2004;Liu, Tabashnik, Dennehy, Patin, & Bartlett, 1999). Local mass release of the self-limiting insects might also, for example, eradicate resistant populations before they become widespread.
Building on previous work on the high-dose/refuge strategy and the self-limiting insects, we will investigate the interaction between the release of self-limiting transgenic insects and the high-dose/refuge strategy in mitigating the evolution of resistance in model experimental system using the diamondback moth (DBM), Plutella xylostella.
DBM is a well-known and widespread pest of cruciferous crops.
Globally, it imposes management costs of US$1.3 billion-US2.3 billion and causes yield losses estimated at US$2.7 billion per annum worldwide (Furlong, Wright, & Dosdall, 2013;Zalucki et al., 2012). Control failure of DBM is a major concern in agriculture, as this species has developed resistance to almost every insecticide applied in the field as well as resistance to microbial Bt sprays (Sarfraz & Keddie, 2005;Tabashnik, 1994). Diamondback moth is also a well-established model for evaluating novel resistance management strategies (Raymond et al., 2007;Zhao et al., 2005). Genetic markers for resistance to the Bt toxin Cry1Ac in our resistant line have been well established (Baxter et al., 2011) and this protein can be incorporated into artificial diet at doses that render resistance functionally recessive. Transgenic strains of DBM with female-specific self-limiting constructs have been developed (Jin et al., 2013). Evidence of population suppression by the DBM self-limiting system has been observed in caged continuous generation studies and low numbers of released self-limiting males have been shown to slow the evolution of resistance to Bt in transgenic crucifers (Harvey-Samuel et al., 2015).
Using DBM populations with known frequencies of Cry1Acresistance alleles, we tested the compatibility of self-limiting DBM releases with the high-dose/refuge strategy in single-generation and multi-generation experiments. We investigated whether the release of Cry-susceptible self-limiting insects could slow or reverse the evolution of resistance at a range of refuge sizes, release ratios, and initial frequencies of resistance. To compare experimental results to previous theoretical and experimental work, we also characterized the fitness costs associated with transgenic constructs and resistance alleles in our experimental set-up.

| Experimental conditions and insect populations
All insect populations were reared at 25°C (±1°C) and 45% (±5%) relative humidity, with a 12:12 light/dark cycle. The rearing procedure of DBM followed published protocols (Martins et al., 2012). The construction of the self-limiting DBM (OX4319L, Oxitec Ltd) has also been described (Jin et al., 2013). In brief, the self-limiting system has also been implemented in our Bt-susceptible line using sequences from the self-limiting gene derived from the doublesex (dsx) gene of pink bollworm (Jin et al., 2013). Sex-alternate splicing of this dsx sequence allows the development of a female-specific lethal genetic system that is repressible by provision of tetracycline, or suitable analogues, in the larval feed (Jin et al., 2013). The OX4319L moths are denoted as genotype LL, where "L" represents the OX4319L construct insertion (Jin et al., 2013), and are all homozygous-susceptible to Cry1Ac toxin (genotype SS).
Exogenous B. thuringiensis Cry1Ac was purified from Escherichia coli JM109 cells carrying the plasmid pGem1Ac, a gift of Dr Neil Crickmore (University of Sussex), following published protocols (Cornforth, Matthews, Brown, & Raymond, 2015). The purified Cry1Ac toxin was incorporated into artificial diet (F9221B, Frontier Agricultural Sciences) to make toxin diet, at doses (0.5 μg/ml) sufficient to cause near-recessive resistance (Supporting Information: toxin bioassays). Our resistant population, designated VB-R, was constructed from a Cry1Ac-resistant population NO-QAGE (Baxter et al., 2005;Heckel, Gahan, Liu, & Tabashnik, 1999) and a susceptible population Vero Beach, which is the genetic background of the self-limiting population (VB, Oxitec Ltd). The VB-R population was constructed by backcrossing a hybrid population of VB and NO-QAGE into VB, and selecting for resistance to Cry1Ac for three generations. To create a Cry1Ac-susceptible population with a similar genetic background, we reared VB-R without toxin selection for five generations (before resistance became fixed); thereafter, we genotyped mated pairs of males and females using the length polymorphism marker for Cry1Ac resistance (Baxter et al., 2011). Our susceptible population VB-S was then established using 20 pairs of homozygous-susceptible individuals. PCR conditions for genotyping homozygous-susceptible alleles were 5 min at 95°C, 30 × (30 s at 94°C, 30 s at 63°C, 1 min at 72°C), 10 min at 72°C, using primers abcc2F (5′-GGACGTGATCCCGGTGGGCAGCG-3′) and abcc2R (5′-CGTGCGGCAGCTTAGTGTAC-3′). Both the VB-R and VB-S populations were nontransgenic (ww genotype, where "w" represents wild type or absence of the "L" construct).
Single and multiple generations, with the same basic design, investigated the impact of transgenic male release on the evolution of resistance to Bt toxins (Table 1, details below). Homozygous-susceptible LL male pupae were introduced into resistant populations with confirmed resistance allele frequencies. Following LL male releases, resistant populations were exposed to toxin selection and refuge treatment.
Population size (number of pupae) and resistant frequencies were monitored throughout the experiments.

| Single-generation experiment
These experiments assessed the effect of the susceptible self-limiting DBM in resistance management at a range of refuge sizes. We hypothesized that the use of susceptible self-limiting DBM will have a greater effect on slowing the evolution of resistance at smaller refuge sizes. The single-generation experiments were timed so that wildtype adults and transgenic males would emerge from their pupae over the same period (24-48 hr) and compete for mates in experimental cages. The eggs produced within each replicate cage were allocated to Cry1Ac toxin diet or toxin refugia where larvae experienced selection for resistance. These experiments sought to control for any differences in development time between wild-type and transgenic insects (and between Cry1Ac-resistant and Cry1Ac-susceptible insects) but otherwise allowed genetic background to affect mating behaviour.
Experiments were set up with 200 individuals of the wild-type population with a 15% resistance allele frequency (R). The population was reared for at least two generations prior to selection starting and frequencies were confirmed with PCR, using methods described above. In the transgenic LL male release treatment, 200 LL male pupae were added to each replicate, so that the release ratio was 2:1 OX4319L males to wild-type nontransgenic males. Here, we crossed a refuge size treatment (10% and 20% Cry1Ac toxin-free refugia) with a transgenic treatment (with and without LL male release), each replicated three times (Table 1). Refugia were based on the percentage of egg population: refugia eggs were reared separately on toxin-free diet, while remaining eggs were reared on toxin diet (0.5 μg/ml) to pupation. For every replicate, pupae survivors from both the selection diet and refuge diet were collected and pooled for bioassays in the following generation (N = 90 larvae and three Cry1Ac doses including 0.131, 0.262 and 0.524 μg/ml) to assess for differences in resistance to Cry1Ac.

| Three-generation experiments
To investigate the value of the self-limiting DBM in resistance management over multiple generations, we designed two multi-generation selection experiments with weekly releases of LL males (Table 1).
Populations with 4% and 15% resistance allele initial frequencies were generated as above. After confirming the resistant frequency with PCR, we started the first experiment (15% resistance allele frequency) with two treatments (with and without LL male release) and four replicates (400 pupae) in each treatment. In the release treatment, male pupae were introduced into the experimental populations twice a week at approximately a 6:1 ratio (LL male to pupal survivors from each cage, assuming 1:1 sex ratio in cage survivors) for 12 weeks.
Eggs were collected every two days with 10% of the eggs placed onto toxin-free refuge diet. The diet infestation was staggered every two days to build gradually a continuous population with overlapping generations. Thus, genotype differences in development time or mating success are allowed to influence results, adding more realism than in single-generation experiments.
The experimental populations were bio-assayed every generation to measure the proportion of homozygous-resistant (RR) individuals in the population. Survival data-the numbers of pupae surviving the selection diet and refuge diet-were collected weekly. To test whether the release of transgenic insects was capable of reversion, that is, decreasing the resistance allele frequency in the face of selection, the experiment was repeated with another population with initial resistance allele frequency at 4%.

| Life history and fitness cost experiments
To evaluate the fitness costs of the self-limiting gene and the resistance allele, we measured life history traits and mating competitiveness of the aforementioned P. xylostella populations. All males denoted as LL and Lw were homozygous-susceptible at the resistance locus (SS), and all VB-S and VB-R individuals were nontransgenic (ww). We confirmed that the VB-R population used in this experiment was fixed for resistance by PCR screening of 96 individuals. Single-pair mating of LL male × SS female, VB-S individuals (SS), VB-R individuals (RR) and SS × RR genotype was set up to measure fecundity, egg hatch rate and larval survival until pupation. Single pairs were mated in 106 pots. The number of eggs laid on cabbage juice-infused green cloths (3 cm × 3 cm) from the single pairs was counted manually for all pots, and eggs were allowed to hatch in situ (Raymond et al., 2007). Twenty freshly emerged neonates from each mating pot were randomly selected to grow on artificial diet until pupation. After scoring survival, pupae developed from single-pair pots were used in mate competition experiments. In these experiments 10 nontransgenic SS males competed with the same number of LL males, RR males, or hemizygoussusceptible OX4319L males (LwSS) for mating with 10 SS females.
LL males were also competed with hemizygous Lw males for mating with SS females. As the self-limiting gene contains a dominant heritable, fluorescent DsRed2 protein marker (Jin et al., 2013), pupae can be sorted using a binocular microscope with Nightsea™ light source (excitation 510-540 nm) and 600-nm filter. Mating success of either LL or Lw males in competition with SS males was scored based on the

| Statistical analyses and experimental design
To assess the potential discriminatory power of the experiments, we simulated discrete generations of DBM classified by sex and genotype (at L/w and S/R loci), assuming a constant proportion of released LLSS males to emerging males (initial males or, after the first generation, emerging males of any genotype) and random mating. Where known, parameter values were set to match experimental protocols.
These simulations were adapted from a previously published discretegeneration deterministic model of this genetic system in a generic pest insect (Alphey et al., 2007(Alphey et al., , 2009 Mating success was analysed with a chi-squared goodness-of-fit tests, which compared the expected frequency of L and R alleles under random mating with observed frequencies. All model assumptions were checked with graphical analysis of error distribution assumptions.

| Single-generation experiment
We predicted that larger refuge sizes and the addition of transgenic males would slow the evolution of resistance. However, given the increased population size associated with larger refugia, we anticipated that the release of transgenic insects would have more impact at smaller refuge sizes. After one discrete generation, at 10% refuge size, one replicate in the release treatment had only five pupal survivors. The replicate went extinct in the following generation and was excluded from bioassays, but was included in the population size analysis. As predicted, the larger refuge size (20%) led to a lower frequency of phenotypic resistance, that is, frequency of RR genotype inferred from bioassay results, compared to replicates with 10% refuge size (Figure 1a, likelihood ratio test = 10.04, p = .0015). At 10% refuge size, the addition of transgenic males also lowered the proportion of phenotypic resistance compared to replicates without LL male release treatment (Figure 1a, likelihood ratio test = 8.10, p = .0044).
However, at 20% refuge size, there was no significant difference between the release and nonrelease treatments (Figure 1a, likelihood ratio test = 0.34, p = .56).
The release of transgenic males was also expected to suppress population size by killing female progeny (Alphey et al., 2007(Alphey et al., , 2009. We define total survivors as the number of surviving pupae pooled from Cry1Ac-containing diet and refuge diet across replicates. Given an initial R allele frequency of 15%, after one discrete generation, neither refuge size (F 1,10 = 0.025, p = .88) nor the release of transgenic males (F 1,9 = 0.0008, p = .98) had an impact on the total survivors ( Figure 1b).
The single-generation design is less realistic and has less power than the multiple generation experiment below. In addition to

| Three-generation experiments
While the single-generation experiment showed an effect on resistance frequency at lower refuge size, it was weaker than that predicted by theory. Here, we hypothesized that a higher release ratio of transgenic males in a continuous generation experiment should produce a more robust impact on both population size and resistance frequency as transgene frequencies are expected to increase over time in target populations under continuous release. In the first multi-generation experiment, initial conditions were the following: initial resistance allele frequency of 15%, and a 10% refuge size, and a release ratio of 6:1 transgenic: wild-type males. Under these conditions, the release of transgenic males significantly reduced phenotypic resistance com-

| Life history and fitness cost experiments
We assessed the fitness cost of the self-limiting gene and the resistance allele in single-pair crosses and mate competition experiments.
In the single-pair mating experiment, successful mating was defined as mating that resulted in more than 10 eggs. Only eggs from successful matings were counted and used to estimate fecundity and hatch rate as mating efficiency was assessed in competition experiments.
The genotype of mating partners had a strong impact on fecundity

| DISCUSSION
Here, we have investigated the role of transgenic insect releases in mitigating levels of resistance and suppressing population growth in DBM. We found good support for population suppression and resistance reduction with the combined use of the high-dose/refuge strategy and self-limiting transgenic DBM (Alphey et al., 2007(Alphey et al., , 2009). The most straightforward evidence was that the transgenic DBM males were able to suppress both population size and resistance development ( Figure 2).
Here, we found effects on the evolution of resistance in this even though refuge size (10%) and the initial resistance allele frequency (15%) in this work were substantially smaller and higher, respectively, than are typical in the field . Moreover, in comparison with conventional sterile insect technique programmes, which could release typically 10, or even up to 50 sterile males to one wildtype male (Dyck, Hendrichs, & Robinson, 2005;Lees, Gilles, Hendrichs, Vreysen, & Bourtzis, 2015), the release ratio of 6:1 of the self-limiting DBM is relatively modest.
Given the success of the self-limiting DBM, several factors could potentially limit the effect of the transgenic males. Our data demonstrated that the release of the self-limiting males had a greater impact on resistance frequency at smaller refuge size (Figure 1a). Overall, transgenic males release could slow the evolution of resistance in repeated experiments, albeit at a reduced rate than that predicted by theory (Alphey et al., 2007(Alphey et al., , 2009). According to the published models, a lower release ratio was associated with effective resistance management consequences when refugia are larger and R allele frequencies lower than in our experiments (Alphey et al., 2007(Alphey et al., , 2009. At 10% refuge size and 10% initial R allele frequency, the release ratio of 1:1 was capable of slowing resistance development (Alphey et al., 2007(Alphey et al., , 2009), but in our experiment we released five or six transgenic males to every wild-type male to achieve similar effects.
As a consequence, we examined whether unforeseen impacts of transgenes and resistance alleles on life history traits (not reflected in the models) might explain this discrepancy.
Homozygous-resistant individuals had reduced fitness as a result of lower fecundity and fertility (Figure 4a,b). Homozygous-resistant individual males also had reduced mating success with susceptible females ( Figure 4d). In the mating competition experiment, the males and females were introduced into the mating cages as emerged adults; it is unlikely that the mating success of tested males was correlated with different development times and population structure (Liu et al., 2001). Male mating success in the studied system may be associated with reduced number of matings, as seen in previous experiments with the NOQA, the DBM line that provided the resistance alleles for our population (Groeters, Tabashnik, Finson, & Johnson, 1993). Reduced fitness for RR individuals improves resistance management generally , but nonrandom mating could obstruct the effectiveness of the high-dose/refuge strategy (Gould, 1998;Tabashnik et al., 2009).
Our results showed that the self-limiting males were less competitive than wild-type males in terms of accessing wild-type females ( Figure 4d) and that these matings resulted in fewer hatched eggs relative to wild-type counterparts (Figure 4b). The fitness of the transgenic males was greatly reduced, as very few progeny were produced and survived from the mating. In addition, heterozygous transgenic males, which are responsible for introgressing susceptible alleles into the population at large, showed incompletely dominant fitness costs associated with transgenes ( Figure 4d). If transgenes reduce the fitness of heterozygous males, then the potential for introgression of pesticide susceptibility alleles will be limited and the genetic consequences of release will approximate that of "bisex-lethal" strains rather than female-specific lethal. The process of building up the L allele frequency through releases over multiple generations, and its consequences for population suppression, will be attenuated by high dominant fitness costs. Critically, the efficacy of self-limiting transgenic insects as tools in resistance management (above and beyond their use in population suppression) will be partly dependent on the dominance and degree of fitness costs associated with transgenes.
These fitness costs are higher than previously described for this  et al., 2015). In contrast to that study, we introduced toxin-free refugia, which can substantially increase the reproductive potential of a population when resistance frequencies are low. In addition, in this study experiments used artificial diet, which imposes minimal mortality on early instars, whereas B. oleracea can cause substantial mortality on neonates, rising to 70% for genotypes resistant to Cry toxins (Raymond et al., 2011). Both these factors would facilitate population suppression on broccoli plants.
It is difficult to assess how relevant experiments conducted in caged insect population are for real-world resistance dynamics. We hope that mate competition experiments in the laboratory capture sufficient naturalistic behaviour to be able to reflect what might happen in the field. The effects of relatively small population sizes can clearly impose some limitations and create additional variability when gene frequencies are low. Nevertheless, we have constructed experimental conditions that pose a very challenging scenario for resistance management. Frequencies of resistance alleles were high, refugia sizes were small and the release ratios low (Dyck et al., 2005). For diamondback moth on artificial diet, the fitness costs of resistance were relatively modest and resistant insects had survival rates of up to 100% on diet containing very high levels of Cry toxins, a situation that does not occur in the field, even in insect species prone to evolve resistance to Bt toxins readily (Tellez-Rodriguez et al., 2014). In addition, while fitness costs of transgenes resulted in reduced mate competitiveness, in comparison to previous experiments with P. xylostella (index of competitive ability estimated at 0.09, where equal fitness =1), (estimated at 0.09 in this study, where equal fitness with wild type = 1), competitive ability was higher than that observed for Aedes aegypti (0.008-0.31; Carvalho et al., 2015), suggesting that our experiments are not unrealistic in this regard. Thus, even under relatively stringent experimental conditions, our results suggest that the self-limiting DBM is a promising strategy, compatible with the high-dose/refuge strategy.