Overwintering Cost Associated with Resistance to Transgenic Cotton ...

INSECTICIDE RESISTANCE AND RESISTANCE MANAGEMENT

Overwintering Cost Associated with Resistance to Transgenic Cotton in the Pink Bollworm (Lepidoptera: Gelechiidae) YVES CARRIE`RE, CHRISTA ELLERS-KIRK, AMANDA L. PATIN, MARIA A. SIMS, SUSAN MEYER, YONG-BIAO LIU,1 TIMOTHY J. DENNEHY, AND BRUCE E. TABASHNIK Department of Entomology, the University of Arizona, P.O. Box 210036, Tucson, AZ 85040 Ð2933, USA

J. Econ. Entomol. 94(4): 935Ð941 (2001)

ABSTRACT Fitness costs associated with resistance to transgenic crops producing toxins from Bacillus thuringiensis (Bt) may have important effects on the evolution of resistance. We investigated overwintering costs in pink bollworm, Pectinophora gosypiella (Saunders), strains with different degrees of resistance to Bt cotton. Frequency of resistant individuals in a strain was not associated with induction of diapause or emergence from diapause in early winter. Emergence from diapause in the spring was 71% lower in three highly resistant strains than in two heterogeneous strains from which the resistant strains were derived. This underestimates the overwintering cost because the frequency of the resistance allele was relatively high in the heterogeneous strains. Emergence in the spring in hybrid progeny from crosses between the resistant and heterogeneous strains was greater than in resistant strains but did not differ from susceptible strains, showing that the overwintering cost was recessive to some extent. KEY WORDS Pectinophora gossypiella, Bacillus thuringiensis, diapause, Þtness costs, transgenic cotton

FITNESS COSTS ASSOCIATED with resistance genes expressed in the absence of insecticides affect the evolution of insecticide resistance. The strength and genetic basis of resistance costs determine the equilibrium frequency of resistance alleles before the introduction of an insecticide (Crow 1957, Crow and Kimura 1970). Such initial frequency affects the early rate of resistance evolution (Crow 1957, Tabashnik 1990, Scott et al. 2000) and thus may partly determine the effective life of an insecticide. Resistance costs can also cause a decline of resistance in the absence of exposure to insecticides and contribute to maintaining spatial variation in resistance (Tabashnik and Croft 1982; Daly 1993; Carrie` re et al. 1994, 1995, 1996). Under some circumstances, resistance costs may even contribute to preventing the evolution of resistance (Lenormand and Raymond 1998, Carrie` re and Tabashnik 2001). The frequency of resistance to cotton producing a toxin from Bacillus thuringiensis (Bt) declined in Arizona Þeld populations of the pink bollworm, Pectinophora gosypiella (Saunders), from 1997 to 1999 (Tabashnik et al. 2001). The decline occurred even though use of Bt cotton was high and the proportion of cotton planted to non-Bt cotton refuges remained similar over the 3 yr (Carrie` re et al. 2001). A change in Þtness costs is one of the possible reasons for reversal of resistance (Carrie` re and Tabashnik 2001). Because size of refuges can interact with Þtness costs 1 Current address: US Agricultural Research Station, 1636 Alisal St., Salinas, CA 93905.

to foster declines in resistance (Carrie` re and Tabashnik 2001), knowledge of resistance costs could be useful for designing efÞcient resistance management strategies. This study investigated Þtness costs associated with evolution of resistance to Bt cotton in the pink bollworm. We analyzed the association between estimated frequency of a major allele conferring resistance to Bt cotton and diapause induction and overwintering survival across various pink bollworm strains. We also assessed the extent of the resistance cost by comparing overwintering survival in heterogeneous strains and strains derived from them by selection for resistance to Bt. Finally, we qualitatively assessed dominance of the resistance cost by comparing overwintering survival in a heterogeneous strain, two resistant strains, and in hybrid progeny from the crosses between those strains. Materials and Methods Pink Bollworm Strains and Crosses. Strains were maintained in the laboratory on artiÞcial wheat germ diet at a population size ⬎200 (Patin et al. 1999). Three heterogeneous strains (AZP, MOV97, and SAF97) and three strains resistant to Bt cotton (AZP-RE, AZP-RO, and MOV97-R10) were used in this experiment when in their 15thÐ22nd generation. The AZP strain was started by pooling surviving individuals from 10 Þeld-derived strains that had been exposed to low doses of Cry1Ac (1Ð10 ␮g Cry1Ac per milliliter of diet: Tabashnik et al. 2001). The AZP strain

0022-0493/01/0935Ð0941$02.00/0 䉷 2001 Entomological Society of America

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was reared without exposure to Bt after that initial bout of selection. The SAF97 and MOV97 strains originated from Safford and Mohave valleys, respectively, in southeastern and northwestern Arizona. They were also reared without exposure to Bt. The AZP-R strain was produced by selecting the F5 progeny of AZP with a concentration of 10 ␮g Cry1Ac per milliliter of diet (Tabashnik et al. 2001). Part of the F6 and F7 progeny of AZP-R was selected with a concentration of 100 ␮g Cry1Ac per milliliter of diet, which respectively yielded the AZP-RE and AZP-RO strains. The AZP-RE and AZP-RO strains were thereafter selected every other generation with a concentration of 100 ␮g Cry1Ac per milliliter of diet. MOV97R10 was initiated by selecting the F10 progeny from MOV97 and subsequent progeny in every other generation with a concentration of 10 ␮g Cry1Ac per milliliter of diet. When used in the present experiments, the AZP-RO and AZP-RE strains had been selected once at a low Cry1Ac concentration (between one and 10 ␮g Cry1Ac per milliliter of diet), once at 10 ␮g Cry1Ac per milliliter of diet, and 5Ð 6 times at a concentration of 100 ␮g Cry1Ac per milliliter of diet. The MOV97-R10 strain had been selected Þve times at a concentration of 10 ␮g Cry1Ac per milliliter of diet. The AZP-RO, AZP-RE, and MOV97R10 strains survive on Bt cotton, although resistance is incomplete (Liu et al. 1999, Tabashnik et al. 2001; Y.C., unpublished data). Frequency of the Resistance Allele. The frequency of the resistance allele was estimated in all strains before induction of diapause (see below), except for the cross between AZP-RE and AZP for which frequency was approximated by averaging estimates obtained for AZP-RE and AZP. Newly hatched larvae from each strain were placed individually in cups containing a wheat germ diet with either 10 ␮g Cry1Ac per ml diet or 0 ␮g Cry1Ac per ml diet. For each strain, 40 larvae (four replicates of 10) were fed on Bt diet and non-Bt diet. Mortality was assessed by counting fourth instar larvae and pupae surviving after 21 d of feeding in darkness at 29 ⫾ 2⬚C (Patin et al. 1999). Only individuals homozygous for a major resistance allele survive a concentration of 10 ␮g Cry1Ac per milliliter of diet (Tabashnik et al. 2001). Frequency of the resistance allele in a strain was thus estimated by taking the square root of adjusted survival at 10 ␮g Cry1Ac per milliliter of diet (survival at 10 ␮g/ml/ survival at 0 ␮g/ml). Induction of Diapause. For each cross (AZP-RE ⫻ AZP or AZP-RO ⫻ AZP), eggs were Þrst obtained separately for the two possible mating types (resistant males ⫻ susceptible females; susceptible males ⫻ resistant females). The number of males and females used in the four mating types varied between 40 and 70. Eggs obtained from the two mating types for a cross were pooled in equal proportion to provide the progeny used in subsequent experiments. At least 100 moths supplied the eggs for the AZP, AZP-RE, AZPRO, MOV97, MOV97-R10, and SAF97 strains. Eggs were laid by females on cotton pads (⬇1.5 by 1.5 cm). These pads were placed on wheat germ diet

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in 500-ml cups, and maintained at a photoperiod of 12:12 (L:D) h and 29⬚C/15⬚C (⫾2⬚C) light/temperature cycle, which favors diapause induction in pink bollworm (Adkisson et al. 1963). Diet cups with egg pads were placed in two identical growth chambers. One growth chamber received eggs from AZP, AZPRE, AZP-RO, and the two crosses; the other, eggs from AZP, AZP-RE, MOV97, MOV97-R10, and SAF97. Two weeks after hatching, larvae (most in third instar) were transferred to new 500-ml diet cups at a density of 100 larvae per cup. Four weeks after hatching, the growing conditions were changed to a photoperiod of 11:13 (L:D) h and 24⬚C/15⬚C (⫾ 2⬚C) light/temperature cycle to further stimulate diapause induction. From that time, the diet cups were checked once a week and the pupae (nondiapausing individuals) were counted and removed. Pupation rate strongly diminished 10 wk after hatching. The remaining fourth-instar larvae (presumably diapausing) were transferred to plastic boxes (39 by 24 by 16 cm deep) containing ⬇10 cm of autoclaved soil from an organic cotton Þeld. A thin layer of autoclaved cotton debris obtained from a cotton gin was scattered on the soil surface to provide potential overwintering sites (Rice and Reynolds 1971). The top and bottom of the boxes were Þtted with vinyl-screened openings (top openings: 29 by 17 cm; bottom opening: 20 by 13 cm) to allow rain to percolate through the boxes. Diapausing larvae from each strain were arbitrarily distributed in equal number between at least two, and among a maximum of six, diapausing boxes. With the exception of one strain (AZP-RO had only 28 diapausing larvae), between 66 and 295 larvae were transferred to boxes for each strain. The boxes were maintained for 5 d after larval transfer at 24⬚C and a photoperiod of 12:12 (L:D) h to allow settlement of larvae in overwintering sites. The boxes were then placed arbitrarily on the soft bare soil of a screened insectary in Tucson on 18 October 1999. October is a period of high diapause induction in pink bollworm larvae (Bull and Adkisson 1960, Bariola and Henneberry 1980, Walhood et al. 1981). Each box was monitored for moth emergence once per week (between 18 October and three December; 14 February and 27 June) or twice per week (between 3 December and 14 February). Each box was watered with 1.75 liter of water to stimulate emergence from diapause (Watson et al. 1973) on 28 May and 15 June before ending the experiment on 27 June 2000. Statistical Analyses. Because emergence from diapause was bimodal (see Results), two different response variables were analyzed: proportion of the transferred larvae that emerged from diapause in the winter (NovemberÐJanuary), and proportion emergence in the spring (FebruaryÐMay). Proportion emergence from diapause was arcsine-square root transformed before analyses to improve normality and homogeneity of variance (Sokal and Rohlf 1981). Temperature diverged for a period of 2 d between the two growth chambers during the period of diapause induction. Thus, an analysis of variance

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CARRIE` RE ET AL.: COST OF RESISTANCE TO BT COTTON IN PINK BOLLWORM

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Fig. 1. Distribution of emergence from diapause in pink bollworm in 1999Ð2000.

(ANOVA) with Strain nested within Growth chamber was used to assess whether variation between growth chambers affected emergence from diapause in the winter or spring. Growth chamber and Strain were considered as a Þxed and random effect, respectively, which implies that synthetic error terms were used to test the growth chamber effect (JMP 1998). Regression analyses were used to assess the association between frequency of the resistance allele in a strain and proportion emergence in the winter or spring. Proportion emergence in boxes originating from larvae raised in the same growth chamber was averaged for each strain and used as observation units in these regression analyses (thus two estimates for winter or spring emergence were available for AZP and AZP-RE and one estimate for the other strains). The association between frequency of the resistance allele and proportion emergence from diapause was nonlinear for the spring cohort. Therefore, linear and polynomial regression (JMP 1998) were used to evaluate the association between frequency of the resistance allele and winter or spring emergence from diapause, respectively. To qualitatively assess dominance of the overwintering cost, we used planned contrasts between means to compare proportion emergence in the spring among the three types of strains derived from AZP (heterogeneous [AZP], crosses [AZP-RE ⫻ AZP and AZP-RO ⫻ AZP], and resistant [AZP-RE and AZPRO]). Average proportion emergence across all boxes for a combination of strain and growth chamber was the observation unit, and results for the AZP and AZP-RE strain raised in the two growth chambers were included in this analysis. Regression analyses were used to evaluate the association between frequency of the resistance allele in a strain and proportion induction of diapause. As before, incidence of diapause was estimated for each combination of strain and growth chamber, by averaging the proportion of fourth-instar larvae produced

in all the 500-ml diet cups containing individuals from a strain in a given chamber. Thus, two estimates of diapause induction in AZP and AZP-RE were used in the regression analysis, whereas one estimate was available for the other strains.

Results Insects surviving on diet in the laboratory were fourth-instar larvae, pupae, or moths. Averaged across strains, 9.1 ⫾ 1.4% (⫾SE, n ⫽ 92) of the surviving insects were diapausing fourth instars, showing that the experimental conditions were not optimal for diapause induction. Moth emergence in the Þeld was bimodal (Fig. 1). Many moths emerged in November, few in December, and none in January. Moth emergence increased from February to April and tapered off in May. The high moth emergence in November indicates that many of the larvae transferred to the diapausing boxes were not in a profound state of diapause. Nevertheless, percentage induction of diapause in cohorts of pink bollworm larvae increases progressively from late August to mid-October (Bull and Adkisson 1960, Bariola and Henneberry 1980, Beasley 1997). Moreover, the present pattern of emergence from diapause (Fig. 1) is qualitatively similar to what was observed in cohorts of pink bollworm larvae collected early in the Þeld (Þgure 10B in Henneberry and Jech 1999). Thus, the current results are most relevant to describe overwintering survival in larval cohorts that enter diapause early in the fall. Emergence from Diapause in the Winter. Variation between growth chambers did not affect proportion emergence from diapause in the winter (F ⫽ 1.22; df ⫽ 1, 8.28; P ⫽ 0.30). The proportion of winter emergence differed among strains within the growth chambers (F ⫽ 5.15; df ⫽ 8, 16; P ⫽ 0.0026). There was no consistent association, however, between frequency of the resistance allele and emergence from diapause

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Table 1. Mean proportion of larvae emerging from diapause (arcsine-square root transformed) in the winter (November–January) and spring (February–May) as a function of estimated frequency of a major resistance allele in pink bollworm strains reared in two growth chambers Straina

Growth chamber

Allele frequency

Nb

AZP AZP-RE AZP-RO F1-E F1-O AZP AZP-RE MOV97 MOV97-R10 SAF97

1 1 1 1 1 2 2 2 2 2

0.42 0.99 1 0.67 0.71 0.42 0.99 0.43 0.96 0.46

2 2 2 2 2 2 6 2 3 3

Proportion Proportion winter spring emergence emergence 0.49 0.37 0.27 0.35 0.50 0.34 0.47 0.31 0.24 0.23

0.24 0.072 0 0.21 0.38 0.10 0.11 0.20 0.046 0.21

a F1-E and F1-O respectively designate the cross between AZP and AZP-RE and between AZP and AZP-RO. b Number of diapausing boxes used to estimate mean proportion of emergence from diapause.

in winter (Table 1: slope ⫽ ⫺0.012; t ⫽ ⫺0.09; df ⫽ 1, 9; P ⫽ 0.93). Emergence from Diapause in the Spring. Proportion emergence in the spring was not affected by variation between growth chambers (F ⫽ 0.51; df ⫽ 1, 8.43; P ⫽ 0.50), although strains within chambers differed in spring emergence (F ⫽ 3.36; df ⫽ 8, 16; P ⫽ 0.019). Spring emergence was relatively high in the hybrid progeny (Fig. 2), which resulted in a nonlinear association between allele frequency and proportion emergence from diapause in the spring. The coefÞcient for the quadratic term in the multiple regression between allele frequency and proportion spring emergence was negative and signiÞcantly different from zero (slope ⫽ ⫺2.18; t ⫽ ⫺3.14; df ⫽ 1, 7; P ⫽ 0.016). Thus, the general relationship between resistance allele frequency and overwintering survival was concave down (Fig. 2), suggesting the presence of an

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overwintering cost mainly affecting the most resistant strains (see below). There was no difference in spring emergence between the AZP strain and the hybrid progeny from the crosses (t ⫽ 1.43; df ⫽ 1, 4; P ⫽ 0.22), but the resistant strains (AZP-RE and AZP-RO) had signiÞcantly lower proportion of spring emergence than the hybrid progeny (t ⫽ ⫺2.97; df ⫽ 1, 4; P ⫽ 0.041). This suggests that the overwintering cost was somewhat recessive (see Discussion). The mean transformed proportion emergence from diapause in the spring was 0.17 for the AZP strain, whereas it was 0.061 for the resistant strains derived from AZP by selection for resistance to Bt (AZP-RE and AZP-RO: see Table 1). The ratio of survival between those related resistant and susceptible strains was therefore 0.36. The ratio of overwintering survival for the MOV97-R10 and MOV97 strain was 0.23 (Table 1). The average of these two ratios is 0.29 ⫾ 0.065 (SE), which indicates an overwintering cost ⬎71% (see Discussion) associated with resistance to Bt under the conditions of this experiment. Induction of Diapause. The cost of resistance could result from a lower ability of resistant than susceptible larvae to sustain a prolonged overwintering period. Alternatively, pleiotropic effects of the resistance allele could have disrupted diapause induction, such that resistant larvae would have entered a less profound diapause state than susceptible larvae (see for example Carrie` re and Roff 1995, Carrie` re et al. 1995). According to the second hypothesis, the resistant larvae would show the highest emergence in the winter (a trend not found) and the lowest in the spring (as found above). A linear regression between allele frequency and proportion diapause within a strain was used to assess the second hypothesis, assuming that proportion of diapause within a strain is an index of diapause intensity. No association was found between frequency of the resistance allele and incidence of diapause across the strains (slope ⫽ 2.61; t ⫽ 0.42; df ⫽

Fig. 2. Proportion spring emergence (arcsine-square root transformed) in pink bollworm strains as a function of estimated frequency of a major allele conferring resistance to Bt cotton (data in Table 1).

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1, 9; P ⫽ 0.68), indicating that the overwintering cost of resistance was not due to interference of the resistance mechanism with diapause induction. Discussion In the southwestern United States, some pink bollworm moths ßy late in the fall or in early winter when cotton is no longer available (Bariola 1978, Beasley and Adams 1995). Pink bollworm moths can survive up to 2Ð3 mo in the laboratory under cool conditions (Butler and Foster 1979), although it seems unlikely that moths emerging in early winter would live long enough to colonize cotton later in the spring. Thus, because pink bollworm is essentially monophagous on cotton in Arizona, only the moths emerging in the spring in the current study would have reproduced successfully. Proportion emergence in the spring was substantially lower in resistant than heterogeneous strains. Although the resistant strains were almost Þxed for the resistance allele, frequency of the allele was 0.42Ð 0.43 in the heterogeneous strains used to estimate magnitude of the resistance cost. Thus, because the heterogeneous strain comprised some resistant larvae (see below), the estimate of 71% for the overwintering cost represents an underestimate. There was no association between frequency of resistance to Bt and proportion diapause induction or early winter emergence. This indicates that the mechanisms that confer resistance to Bt cotton in pink bollworm have no pleiotropic effects on those traits. Unfortunately, the high frequency of the resistance allele in the heterogeneous strains prevents us from precisely estimating the dominance and magnitude of the overwintering cost. The similarity between spring emergence in the hybrid and heterogeneous strains and the difference between the resistant and heterogeneous strains indicate that the cost was recessive to some extent. We can obtain a range for the size of the cost as follows: Assume that the frequency of the resistance allele (R) in the resistant and heterogeneous strains was 1.0 and 0.425, respectively (see Table 1). With Hardy-Weinberg equilibrium, the heterogeneous strains would have comprised 0.181, 0.488, and 0.331 of RR, RS, and SS larvae, respectively (S being the wild type allele). Because there were many RS individuals in the heterogeneous strains, the denominator of the ratio between spring emergence in the resistant and heterogeneous strains will take the largest value for a totally dominant cost and the smallest for a recessive cost. Respectively deÞning the expected overwintering survival of the resistant, heterozygous, and susceptible genotypes as E (RR), E (RS) and E (SS), we can solve the equation 0.29 ⫽ E (RR)/[E (RR) 0.181 ⫹ E (RS) 0.448 ⫹ E (SS) 0.331] to obtain a range for the magnitude of the overwintering cost. For the dominant (E [RR] ⫽ E [RS]) and recessive (E [RS] ⫽ E [SS]) cases, the estimated value of the ratio E (RR)/E (SS) is 0.12 and 0.25, respectively. These ratios represent the maximum and minimum cost (i.e., 88% and 75%) expected under the

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present experimental conditions if fully resistant and susceptible strains had been used. Therefore, the 71% cost that was estimated here is an underestimate and the value of this cost would have been increasingly underestimated with a greater level of dominance of the cost. We detected overwintering costs associated with resistance to Bt cotton. In addition, the resistant strains have higher mortality on non-Bt cotton than susceptible strains (unpublished data). These resistance costs are in contrast to what occurs in the laboratory. After 17Ð22 generations in the laboratory, frequency of the resistance allele was 0.46 in the SAF97 strain, 0.42 in AZP, and 0.43 in MOV97 (Table 1). However, allele frequency estimated earlier in the same strains (after 7Ð12 generations in the laboratory) was 0.16, 0.16, and 0.47, respectively (Tabashnik et al. 2001; T.J.D., unpublished data). Thus, frequency of the resistance allele apparently increased in two strains and declined in another over ⬇10 generations. This indicates that the resistance allele is neutral in the laboratory and subjected to random drift. The possible difference in occurrence of costs between the laboratory and the more natural conditions used in the aforementioned experiments provides evidence that expression of resistance costs in pink bollworm depends on environmental conditions. Fluctuation in costs of resistance due to environmental variation is one of the factors that could explain the decline in frequency of the resistance allele that occurred in the Þeld in the last three years (Tabashnik et al. 2001, Carrie` re and Tabashnik 2001). Fitness costs associated with resistance genes in the absence of insecticides are common. They can affect a large number of characters, including life-history traits (Carrie` re et al. 1994, Hollingsworth et al. 1997, Alyokhin and Ferro 1999), diapause induction (Carrie` re et al. 1995), mating ability (Campanhola et al. 1991, Groeters et al. 1993, Alyokhin and Ferro 1999), and predator avoidance (Rowland 1991 a, 1991b). Interestingly, costs affecting overwintering survival are often very high (but see Daly and Fitt 1990): 70% in Culex pipiens (Chevillon et al. 1997), 95% in Lucilia cuprina (McKenzie 1990), 50% in Leptinotarsa decemlineata (Alyokhin and Ferro 1999), and at least 71% in pink bollworm (this study). A caveat in this experiment is that a low number of larvae entered diapause in the AZP-RO strain (n ⫽ 28). Such a low number could have resulted in slight overestimation of the overwintering cost in the AZPderived strains, because any overwintering proportion less than ⬇3% would not have been detected with such a low number of larvae (3% ⫻ 28 ⫽ 0.84). Nevertheless, the cost estimated for the Mohave-derived strains was similar to the one obtained for the AZPderived strains, suggesting that low sample size in AZP-RO was not an important problem. Induction of diapause in this experiment was clearly lower than expected under the photoperiod and temperature used (Adkisson et al. 1963). Such low diapause induction may partly result from the high quality of the artiÞcial diet that was used in our study (see Adkisson

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et al. 1963), or from genetic differences between pink bollworm populations used in the two studies. Controlling relative humidity to low levels, however, substantially increases incidence of diapause in pink bollworm (Y.C., unpublished data). This indicates that moisture is a vital factor in diapause induction in pink bollworm (Fife 1949, Tauber et al. 1998). The early winter emergence in pink bollworm in Arizona (Bariola 1978, Henneberry and Jech 1999) likely represents maladaptive timing of diapause. The pink bollworm was introduced to Arizona less than a century ago (Noble 1969), in a desert environment that may be quite different from its ancestral habitat. Whether ecological and evolutionary constraints prevent pink bollworm populations from evolving better timing of diapause in Arizona, or whether the actual maladaptive diapausing behavior is transient, deserves further investigation. Moreover, a study of the effect of climatic and cultural variation on expression of overwintering and other resistance costs in pink bollworm could improve our ability to predict the dynamics of evolution of resistance (Carrie` re and Tabashnik 2001). Ecological, physiological and population genetics studies are under way to answer those questions.

Acknowledgments This research was supported by Grant 99Ð35302Ð8300 and 01Ð35302Ð09976 from the USDA NRI program.

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