Lepidoptera: Noctuidae - BioOne

INSECTICIDE RESISTANCE AND RESISTANCE MANAGEMENT

Why Do F1 Screens Estimate Higher Frequencies of Cry2Ab Resistance in Helicoverpa armigera (Lepidoptera: Noctuidae) Than Do F2 Screens? R. J. MAHON,1,2 S. DOWNES,3 W. JAMES,1

AND

T. PARKER3

J. Econ. Entomol. 103(2): 472Ð481 (2010); DOI: 10.1603/EC09225

ABSTRACT F2 and F1 tests to detect resistance to Cry2Ab in Helicoverpa armigera (Hu¨ bner) (Lepidoptera: Noctuidae) were performed during the 2007Ð2008 summer. F2 tests indicated a resistance frequency of 0.006, which is similar to the published resistance frequencies for this species during the summers spanning 2002Ð2006. In contrast F1 tests indicated a resistance frequency of 0.033. Thus, F1 tests isolated Cry2Ab resistance alleles almost six-fold more frequently than the F2 method. A discrepancy might be expected if the F2 tests detected resistance conferred by more than one locus because F1 tests identify only the form of resistance present in the tester resistant colony. However, if so, F2 tests would detect more, not fewer, cases of resistance. In addition, complementation tests on 10 separate isolates indicate that there is only one common form of resistance. We hypothesized that some “resistance alleles” are homozygous lethal if autozygous (as generated in F2 tests) but not as allozygous homozygotes (as generated in F1 tests). The hypothesis was extended to accommodate the possibility that alleles at linked loci may be homozygous lethal. Neither of two tests of the hypothesis provided evidence that any alleles that confer resistance are associated with severe Þtness costs. Thus we are presently unable to explain the basis of the difference in frequencies between the methods. Because of the simplicity of the F1 tests, it is difÞcult to imagine that it overestimates the frequency of resistance and we therefore accept that this test should provide a more robust method to estimate the frequency of Cry2Ab resistance in H. armigera. KEY WORDS Bt resistance, F2 and F1 screens, Helicoverpa armigera

The F2 screen is an effective way to screen pest populations for rare recessive alleles (Andow and Alstad 1998), and it is a valuable tool for examining wild populations for resistance to insecticidal, genetically modiÞed crops. A major beneÞt of F2 screens is that once a resistant colony is established it becomes possible to employ it to assay the frequency of the resistance using an F1 test. The F1 test involves crossing a Þeld insect (of unknown genotype) to a homozygous resistant insect and screening the F1 offspring for resistance (Gould et al. 1997, Liu et al. 2008, Yue et al. 2008). If the Þeld insect was heterozygous for a resistant allele, then 50% of the F1 should be homozygous for the same allele and thus resistant to a dose of toxin that only homozygous individuals can tolerate. The method is far less labor intensive than F2 tests because it does not require insects to be reared to F2 in the laboratory. In addition, it is common for laboratory reared insects to be more likely to mate as single pairs, thereby increasing the success of crosses to Þeld insects (Blanco et al. 2008). Thus, for the same effort, more alleles can be scored 1 2 3

CSIRO Entomology, GPO Box 1700, Canberra, ACT 2601 Australia. Corresponding author, e-mail: [email protected]. CSIRO Entomology, ACRI Locked Bag 59 Narrabri 2390 Australia.

using the F1 test resulting in more robust estimates of resistance frequency. Premature deployment of F1 tests could provide misleading indications about the incidence of resistance. If, for example, the resistance is recessive, only forms of resistance that are the consequence of mutations at the same locus as that present in the homozygous resistant tester colony will be detected. If alternative forms of resistance resulting from different genes are also present in the Þeld population, they will be detected by F2 tests but remain undetected by F1 tests. However, we expect these two methods to provide the same estimate of frequency for the form of resistance present in the homozygous resistant tester colony. Transgenic cotton containing toxins from Bacillus thuringiensis (Bt) has been grown in Australia since 1996, with the main targets being Helicoverpa armigera (Hu¨ bner) and Helicoverpa punctigera (Wallengren) (Lepidoptera: Noctuidae). The Þrst generation product Ingard (known as Bollgard elsewhere) contained a single toxin produced by the cry1Ac gene, and was grown until 2004. This product provided valuable early season control of larvae but efÞcacy declined as the crop matured (Fitt et al. 1998). From 2004, Ingard was replaced by Bollgard II and its use rapidly increased to

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MAHON ET AL.: F1 AND F2 SCREENS TO DETECT Cry2Ab-RESISTANT Helicoverpa

at least 80% of the cotton planted each season. Bollgard II expresses Cry1Ac and Cry2Ab and provides season-long protection against Helicoverpa species. Cry1Ac and Cry2Ab are quite different proteins and do not share a common binding site (Ho¨ fte and Whiteley 1989, English et al. 1994, Liao et al. 2005); thus, Bollgard II should be more impervious to the evolution of resistance by pest species. Using F2 methods, alleles that confer resistance to Cry2Ab have been found in Australian populations of H. armigera and H. punctigera (Mahon et al. 2007a, Downes et al. 2009). The resistance in H. armigera has been characterized: it is recessive, autosomal and due to a single major gene (Mahon et al. 2007b). Published data from F2 screens conducted from 2002Ð2003 to 2005Ð2006 indicate that the frequency of alleles conferring resistance to Cry2Ab in H. armigera is 0.003. Data collected using the same method from 2006 to 2007 (S.D., T.P., and R.J.M., unpublished data) and 2007Ð2008 (reported herein) suggest that the frequency remains at a similar level. Recently, we performed complementation tests between a resistant colony and separate isolates of Cry2Ab resistance in H. armigera. In all of 10 tested isolates obtained by F2 screens, the F1 offspring from crosses to SP15 showed only control levels of mortality against very high concentrations of Cry2Ab in common with the resistant parental strains (Mahon et al. 2008, this study). These results support the notion that the resistance in these isolates is due to alleles at the same locus. We therefore conclude that there is only one common form of Cry2Ab resistance in Australian populations of this species and that the deployment of F1 tests is appropriate for detecting this form of resistance. Since 2004 Ð2005, Monsanto in Australia have performed F1 screens using the SP15 Cry2Ab-resistant strain of H. armigera (Knight 2009). They found that in each season, the frequency of resistance in Þeld populations of H. armigera was 2Ð3 times higher than that found using the F2 tests described by Mahon et al. (2007a). This was of considerable concern as it undermines the assumption that the F2 screen accurately estimates current resistance levels. A simple hypothesis to explain the difference in frequency between F1 and F2 screens is that some alleles that confer resistance carry a severe recessive Þtness cost. Fitness costs are commonly associated with resistance including that relating to Bt toxins in Lepidoptera (e.g., Pectinophora gossypiella (Saunders) [Carrie` re et al. 2001a,b], H. armigera [Bird and Akhurst 2005], Helicoverpa zea (Boddie) [Anilkumar et al. 2008]). However, if this cost was common to all H. armigera “Cry2Ab resistant alleles,” it should be evident in positive F2 tests as a reduction from the expected 6.25% proportion of survivors when one parent was heterozygous for a resistance allele. In contrast, an excess of survivors rather than a deÞciency was often observed in our F2 screens conducted on H. armigera (Mahon et al. 2007a). Our ability to successfully maintain robust colonies of homozygous Cry2Ab resistant insects also does not Þt with this hypothesis.

473

An alternative scenario is that a Þtness cost exists for some alleles (but not all) that is so severe that we would have missed such alleles altogether when using the F2 test, i.e., individuals that are homozygous for alleles of the same origin (autozygous) were not viable. The corollary of this hypothesis is that some alleles, such as that present in SP15 and other autozygous-viable isolates that have successfully produced colonies after isolation in F2 screens, are qualitatively different from many of the alleles circulating in the population that are only viable as homozygotes when the alleles are from different origins (allozygous). A variation of this hypothesis is that the lethal effect is not related to the resistance locus, but rather to loci that ßank the resistance locus. This possibility arises because F2 tests will cause loci linked to the resistance locus and proximal to crossover events in ßanking sections of chromosome to become autozygous. Based on data from Dobzansky and Spasky (1954) who estimated the frequency of lethals and semilethals in whole chromosomes of Drosophila species, Lewontin (1974) calculated that 2.5 lethals occur per genome. If located near the resistance locus, such lethals or semilethals would cause the same effect as a severe Þtness cost, as homozygous resistant individuals would not be represented in the F2. That would not be the case in F1 tests, as loci in the chromosomal region containing the lethal locus would not be made homozygous. Here, we set out to establish if the higher frequencies of SP15-like Cry2Ab resistance alleles in H. armigera detected in F1 tests by Monsanto (Knight 2009) could be replicated in our laboratories. In doing so, we Þrst provide data additional to that reported in Mahon et al. (2008) to further validate the critical assumption that Cry2Ab resistant isolates obtained using F2 screens are allelic. We also tested the hypothesis proposed above to explain the higher frequencies of SP15like Cry2Ab resistance alleles detected using the F1 method, namely, that some alleles which confer resistance are detectable as allozygotes (as per F1 tests) but not as autozygotes (as per F2 tests). Materials and Methods Standardization of Techniques. The work was performed in our Canberra and Narrabri laboratories. The methods and equipment used for setting up and scoring assays, and rearing insects at each laboratory were as uniform as we could achieve. This was ensured by sourcing materials from the same supplier, and having one person train all personnel involved when implementing a standardized set of techniques. Any differences in the techniques used at each site are detailed below, and are unlikely to alter the ability to detect resistance. General Techniques and Laboratory Strains. Rearing. The rearing methods used to maintain H. armigera were the procedures described by Teakle and Jensen (1985) with the following exceptions related to the formulation of the diet. In Canberra, soybean, Glycine max (L.) Merr., ßour was substituted with chickpea ßour and in Canberra and Narrabri, propionic acid

474 Table 1.

JOURNAL OF ECONOMIC ENTOMOLOGY

Vol. 103, no. 2

Summary statistics for comparisons of the responses to Cry2Ab toxin

Group

Cry2Ab resistant genotypes

A B A B A B A B A B

(1) SP116, (2) SP15, (3) SP116 ⫻ SP15 (1) GR, (2) GR ⫻ SP15, (3) GR ⫻ SP116 (1) NA364, (2) NA405, (3) NA364 ⫻ NA405 (1) ANGR, (2) ANGR x NA405, (3) ANGR ⫻ NA364 (1) NA545, (2) NA405, (3) NA545 ⫻ NA405 (1) ANGR, (2) ANGR ⫻ NA405, (3) ANGR ⫻ NA545 (1) NA798, (2) NA405, (3) NA798 ⫻ NA405 (1) ANGR, (2) ANGR ⫻ NA405, (3) ANGR ⫻ NA798 (1) NA286, (2) NA405, (3) NA286 ⫻ NA405 (1) ANGR, (2) ANGR ⫻ NA405, (3) ANGR ⫻ NA286

% killed at discrimination concn of Cry2Ab

Summary statistics

(1)

(2)

(3)

F/␹2

df

P

2.3 100 1.4 97.2 6.9 68.1 9.6 88.3 11.9 97.0

4.3

0.0 100 4.9 89.4 4.9 63.1 3.4 94.4 7.9 94.8

0.04

2

0.63 2.18 2.19 2.00 2.25 1.19 1.9 1.2

2,6 2,6 2,6 2,6 2,6 2,6 2,6 2,6

0.98a Ñb 0.562c 0.194c 0.193c 0.216c 0.187c 0.367c 0.263c 0.363c

5.6 87.5 9.1 69.4 5.6 98.5 7.1 92.9

(A) Statistics of comparisons of responses to toxin by each resistant strain (SP116, NA364, NA545, NA798, or NA286) versus SP15 or NA405 versus the F1 progeny resulting from crosses of each resistant strain to SP15 or NA405; and (B) by F1 progeny resulting from crosses of each resistant strain to SP15 or NA405 versus the F1 progeny resulting from crosses of each resistant strain to the susceptible strain (GR or ANGR). Group A genotypes were challenged with 2Ð2.5 ␮g of toxin per cm2, and group B genotypes were challenged with 20 Ð25 ␮g of toxin per cm2. a Chi-square test for homogeneity. b Test for homogeneity not appropriate. c ANOVA with genotype as the factor.

(0.08%) was substituted for formalin. Rearing trays were covered and heat-sealed with a perforated lid. Moths were provided with a small pot of 4% honey/ sugar solution that was fed through a cotton wick, and housed in containers that were open at the top and covered with nappy liners or polyester organza cloth secured around their lip. Laboratory Strains. For the F1 screens we used two H. armigera Cry2Ab-resistant colonies, SP15 and NB405, established from positive F2 tests in 2002 and 2005, respectively. Both had been repeatedly outcrossed to a susceptible colony and reselected. The characteristics of SP15 have been described previously (Mahon et al. 2007a,b), and all Cry2Ab resistant isolates seem to exhibit those characteristics. Through repeated selection using Cry2Ab toxin at doses that only permit individuals that are homozygous resistant to thrive, we ensured that all individuals from SP15 and NB405 were of that genotype. Details of the susceptible colonies, GR and ANGR, used in this study are provided in Mahon et al. (2007b). The susceptible strains are largely reared separately from colonies exhibiting resistance to Cry toxins in our laboratories but nevertheless are regularly tested for contamination by Cry2Ab resistant moths. Field Collections. H. armigera were sampled from a range of cultivated and uncultivated hosts in the major cotton regions in eastern Australia. Most samples were collected as one egg per leaf to reduce the possibility of testing more than one individual from the same female parent. Usually, collections were made after we received reports of the presence of eggs in the Þeld, rather than by random sampling. Complementation Tests. Mahon et al. (2008) showed that four Cry2Ab resistant lines isolated using F2 screens between 2003 and 2006 (SP202, SP566, NA405, and NA738) are allelic with SP15. Here, we present data for an additional Þve colonies that were isolated using F2 screens between 2006 and 2008, which proved to be allelic with SP15 or NA405. At the time comple-

mentation tests were conducted, each strain had been outcrossed to a homozygous susceptible lab strain and reselected as follows: SP15 and NA405 was outcrossed two to seven times and selected two to six times; SP116 was outcrossed once and selected twice; NA364, NA545, and NA798 were outcrossed once and selected twice; and NA286 was not outcrossed and was selected twice. When evaluated, each resistant colony was believed to be homozygous for the Cry2Ab resistance allele. The detailed methodology for this work is outlined in Mahon et al. (2008). In brief, the tests involved setting up reciprocal crosses between 1) the Cry2Abresistant colony and the SP15 or NA405 colony, 2) the Cry2Ab-resistant colony and a Cry2Ab-susceptible colony, and, 3) the SP15 or NA405 colony and a Cry2Ab-susceptible colony. To determine whether the characteristics of the captured alleles were similar to those of SP15/NA405, the response to a discriminating concentration of toxin was determined for progeny from the above-mentioned crosses and from the parental strains. Bioassays were conducted in trays which contained rearing diet overlaid with an aqueous solution of toxin. Between 45 and 72 larvae were tested. The discriminating concentration was 2Ð2.5 or 20 Ð25 ␮g of toxin per cm2 depending on the genotype being tested (Table 1). After 7 d, the larvae were scored as being able to undertake coordinated movement (hereafter referred to as “alive”) or dead, moribund, or unable to undertake coordinated movement (hereafter referred to as “killed”), and the growth stage (instar) of all survivors was recorded. Cry2Ab toxin was produced from a clone of the cry2Ab gene of B. thuringiensis variety kurstaki HD-1 in B. thuringiensis. The original clone was provided by L. Masson (National Research Council, Montreal, QC, Canada). The concentration of toxin was estimated by scanning the gel and analyzing the density of the toxin band relative to a bovine serum albumin standard using Scion Image 1.62 software (Scion Corporation, Frederick, MD). The source of Cry2Ab toxin for the

April 2010


F1 and F2 tests was dried and ground corn leaf material. This material was provided by Monsanto Company (St. Louis, MO) as a lyophilized maize, Zea mays L., leaf powder containing the transgenically expressed B. thuringiensis crystal protein Cry2Ab2, at a concentration of 6 mg/g powder. Frequencies of Cry2Ab Alleles Obtained Using the F1 Versus F2 Method. Here, we used the same methods reported in Mahon et al. (2007a) to estimate frequencies of alleles conferring resistance to Cry2Ab in H. armigera in 2007Ð2008. This work was conducted in our Narrabri laboratories as part of the Bt resistance monitoring program supported by the Australian Cotton Industry (Downes et al. 2007). We also used F1 tests on the same Þeld populations to estimate frequencies of alleles conferring resistance to Cry2Ab in H. armigera in 2007Ð2008. This work was conducted in our Canberra and Narrabri laboratories. For both screens, assays to identify resistant insects were conducted using the protocols published in Mahon et al. (2007a). A detailed justiÞcation of the development of these methods, including the source of Cry2Ab toxin and concentration used, is reported therein. In brief, the assays were conducted in 45-well (2.7-cm2) trays that contained rearing diet that was overlaid with an aqueous suspension of toxin at a selected concentration. After the addition of one neonate larvae per well, trays were heat sealed and maintained at 25 ⫾ 2⬚C and 45Ð55% RH. We aimed to expose 90 neonate larvae (two 45-well trays) to Cry2Ab toxin for each line. After 7 d, the larvae were scored as being alive or killed, and the growth stage (instar) of all survivors was recorded. F2 Method. Eggs collected from Þeld hosts of H. armigera were reared to pupae in the laboratory. Pupae were sexed, and on emergence, single male and female moths were placed in an individual 850-ml plastic container with a dilute honey solution. Eggs laid on gauze placed across the opening of the container were collected every 1Ð2 d. If eggs proved to be fertile, ⬇135 hatching larvae were reared to establish isofemale lines. On pupation, individuals were sexed and equivalent numbers of males and females were placed in a 5-liter white round plastic container and allowed to mate with their siblings in bulk. F2 offspring generated from these parents were either challenged with a discriminating dose of toxin applied as a surface treatment or reared on uncontaminated diet as a control to assess Þtness of the cohort of larvae. If either Þeld-collected insect carried a “resistant allele,” we would expect 1/16th, or 6.25%, of the toxin-exposed larvae to be homozygous for that allele and thus survive and grow to at least third instar by day 7 (Andow and Alstad 1998). If resistance exhibited dominance, we would expect a greater proportion than 6.25% to survive. However, we have found that all 10 isolates of Cry2Ab resistance examined to date (Mahon et al. 2008, this study) are due to a common gene and resistance is recessive (Mahon et al. 2007a,b). F1 Method. This technique makes use of colonies of resistant insects in a similar fashion to that used by

475

Gould et al. (1997) to determine the frequency of resistance in Heliothis virescens (F.). Field-collected eggs were reared to pupae and male and female pupae were placed in groups in separate cages. As moths emerged, individual males were placed into 850-ml plastic containers with two virgin females from a resistant colony (Canberra: SP15, Narrabri: NA405). Similarly, individual females were placed in an 850-ml plastic container with two males from a resistant colony. If fertile eggs were obtained from such crosses, F1 offspring were exposed to toxin at a dose that would permit only homozygous resistant insects to grow to at least third instar by 7 d. If the Þeld-derived individual tested in this process was heterozygous for resistance, we would expect that ⬇50% of the larvae to be homozygous for resistance and therefore to thrive. In the unlikely event that we collected and tested homozygotes from the Þeld, the frequency of survivors would be close to 100%. Direct Test for Severe Fitness Costs. For a subset of the F1 tests involving Þeld collected males mated to SP15 females in Canberra, we used the Þeld males in a further test; one that simulated a F2 test. This approach is similar to that of Blanco et al. (2008) who evaluated the efÞciency of the F2 screen to detect Cry1Ac resistance in H. virescens. For this subset, once it became clear that a male crossed to a SP15 female produced fertile eggs, the male was recovered and placed in a new cage with two virgin GR females. Eggs from the Þeld male crossed to the SP15 female were hatched and the F1 test proceeded as described above. When the result of the F1 screen was known, if it revealed that the male carried a gene for resistance, we retained the eggs from the same Þeld male crossed to the GR females. Eggs from the GR females were allowed to hatch and resultant larvae were reared to adults and mated among themselves as per the F2 method. Up to 90 of these F2 offspring were exposed to the discriminating dose as neonates and up to 45 were reared on uncontaminated diet as a control to assess Þtness of the cohort of larvae. This process mimicked the standard F2 procedure except that instead of two Þeld insects initiating the test, we used a male known to carry a resistant gene and the female was known to be homozygous susceptible. Indirect Test for Severe Fitness Costs. The direct test detailed above was only attempted for a portion of the F1 tests involving Þeld-derived male insects and was not practical for any tests involving females. Therefore, a mating scheme was developed for the group of isolates not involved with direct tests to further explore the hypothesis that some Þeld-derived alleles that conferred resistance could not be detected as homozygotes. For this set of isolates, survivors from positive F1 tests were retained for further analysis. These survivors must have been allozygous with one resistance allele derived from the resistant colony (here designated Rc) and one derived from the Þeld insect (Rf), i.e., individuals were RcRf. The survivors from individual isolates of resistance were mass mated to the GR

476


colony (homozygous susceptible, SS) in reciprocal crosses (male survivors were crossed to female GR and visa versa) and their offspring would be of two genotypes, RcS and Rf S in a ratio of 1:1. Pupae of the same sex from reciprocal crosses were pooled and the sexes allowed to emerge in separate cages to ensure the virginity of females. As adults emerged, they were set up in cages as single pairs, one male and one female per cage. Up to 25 such cages were prepared for each isolate. Three types of pairings were possible: RcS ⫻ RcS, RcS ⫻ Rf S, and Rf S ⫻ Rf S and were expected to occur in a ratio of 1:2:1, respectively. The latter pairing, Rf S ⫻ Rf S was the only type able to generate the targeted Rf Rf genotype in the next generation and if that particular Rf proved to be homozygous lethal, should not produce resistant insects. Other pairings would be able to produce RcRf (known to be resistant and viable as it was detected as survivors in the F1 test) or RcRc.(the genotype of the resistant colony). Therefore, these crosses would produce survivors when their neonate offspring were exposed to a discriminating dose of Cry2Ab. Because only one quarter of the mating types (single pairs) were informative, up to 20 single pairs were scored to maximize the probability of assaying offspring from at least one informative pair. Informative single pairs would generate three genotypes, RfRf, RfS, and SS, in the ratio of 1:2:1. For this and the other mating types, one quarter of the offspring should be homozygous resistant. By exposing up to 90 neonate offspring from each pair to a discriminating concentration of Cry2Ab we ensured that there was a high probability that all genotypes would be represented in the sample. If all single pairs resulting from a male that scored positive in the F1 test produced resistant individuals, we could infer the existence of viable Rf Rf genotypes for the allele isolated from that positive F1 test. Statistical Analyses. Unless otherwise stated, analyses were conducted on raw data that were not corrected for control mortality. We used Bayesian inference (Brunk 1975) to estimate the expected allelic frequency and the 95% credibility intervals for F1 and F2 screens. The expected Bt resistance allele frequency in the population was estimated for F2 screens using equation 1 from Andow and Alstad (1998), and for F1 screens using equation 4 from Yue et al. (2008). Bayesian methods determine the probability that the present experimental results give an estimated p within some credibility of the parameter estimate. The 95% credibility intervals for our estimated frequencies were determined for F2 screens using equation 2, and for F1 screens using equation 15, from Andow and Alstad (1999). We calculated the probability that Bayesian estimates from the F1 screen and F2 screen are the same using the methods developed in Wenes et al. (2006). It is possible to assume an isofemale line carries only susceptible alleles when it actually had resistance alleles. It can occur, for example, when a line starts with a resistance allele, but the allele is lost or reduced to

Vol. 103, no. 2

a low frequency in subsequent generations and becomes difÞcult to detect. We followed the methods developed by Andow and Alstad (1998), Stodola and Andow (2004), and Yue et al. (2008) for calculating the probability of a false negative (PNo) for a line in the F1 and F2 screens. For F2 screens PNo depends on the number of F1 males and F1 females that contribute to the F2 generation, the number of F2 offspring screened per F1 female, and the nonscreen mortality of F2 larvae. Our counts of F1 parents only include those that were screened. For each isofemale line, the actual numbers of offspring tested were included in our calculation of the number of F2 offspring assayed per F1 female. For F1 screens PNo depends on the number of F1 offspring screened, the nonscreen mortality of F1 larvae, and the actual (and unknown) number of Bt-RR F1 larvae. For each isofemale line the actual numbers of offspring tested were included in our calculation of the number of F2 offspring assayed per F1 female. For both screens, nonscreen mortality of larvae was measured for each isofemale line. We reared a random selection of 20 Ð 45 F1 or F2 neonates (for F1 or F2 screens, respectively) on artiÞcial diet in the same room as their siblings which were undergoing the screens. After 7 d, we counted the killed individuals plus those that did not reach at least third instar and divided this value by the total number of individuals reared. Results Complementation Tests. Mortality among F1 progeny exposed to toxin from crosses between the resistant parental strains, SP116, NA364, NA545, NA798, and NA286, and SP15 or NA405, and the parental strains themselves, was low and there was no signiÞcant difference among them (Table 1). In contrast, the mortality responses of F1 progeny from crosses between the resistant parental strains, SP116, NA364, NA545, NA798, NA286, NA405, and SP15, compared with mortality responses of F1 progeny from crosses between the resistant parental strains and a susceptible strain, were highly signiÞcant for each strain (in all cases, F ⬎ 9.14 and P ⬍ 0.039). In all cases, the mortality of F1 progeny from crosses between the resistant colonies, including SP15, and the Bt-susceptible laboratory strain were not signiÞcantly different from those for the susceptible strain itself (Table 1). The resistance exhibited by F1 larvae of crosses between the resistant colonies and SP15/NA405, together with the lack of dominance exhibited by F1 progeny of the crosses to the susceptible strain, indicate that mutations present in a single gene are responsible for the Cry2Ab resistance present in SP15, NA405, SP116, NA364, NA545, NA798, and NA286. Frequencies From the F2 Screens Versus the F1 Screens. The F2 tests performed in 2007/08 estimated Cry2Ab resistance alleles in H. armigera to be four of 772 tested. One of these isolated cases (NA286) was included in the complementation tests reported above. We presume that the other three cases are also SP15-like alleles. Thus, in 2007/08 the F2 screens

April 2010


q2

477

Table 2. Results from the direct test of severe fitness costs for five males that proved to be heterozygous for resistance to Cry2Ab in F1 tests

0.04 Isolate

q1 = q2

116 596 597 953 101

0.02

␹2 Þt to Corrected % third % third No. F2 expected 1/16 % third instars on instars on tested proportion instars on Cry2Ab control (probability) Cry2Ab 180 89 83 167 179

2.2 20.22 8.43 4.79 5.59

14.68 100 92.86 86.37 69.05

15.13 20.22 9.08 5.57 8.09

1.53 (0.232) 41.22 (⬍0.001) 3.11 (0.07) 2.42 (0.12) 0.91 (0.33)

If the resistant allele was Þt, the expected proportion of F2Õs to survive and reach at least third instar was 0.0625.

0.0

0.0

0.02

0.04

q1

Fig. 1. Joint 95% credibility region for resistance allele frequency estimated by the F2 screen (q1) and the F1 screen (q2). The diagonal line is the hypothesis q1 ⫽ q2. The line does not intersect the 95% credibility region. The point is the joint maximum likelihood estimate, and the lines passing through the point are the 95% credibility intervals for each independent estimate of the allele frequency.

yielded a frequency of SP15-like Cry2Ab resistance alleles in H. armigera of 0.006 with 95% credibility intervals of 0.002 and 0.013. The proportions of alive insects in the F2 screen ranged from 4.4 to 10.1% and were not signiÞcantly different from the 6.25% expected under the scenario of recessive resistance (in all cases, ␹2 ⬍ 2.1, df 1, P ⬎ 0.15). The probability that at least one of the lines was erroneously classiÞed as susceptible was 0.011. Thus, the probability that all possible resistance alleles in the 193 parental lines were detected was 0.989. The F1 tests performed in 2007/08 estimated SP15like Cry2Ab resistance alleles in H. armigera to be 13 of 432 tested in Canberra and 9 of 284 tested in Narrabri. The frequency of detection of positive alleles do not differ signiÞcantly between the laboratories (␹2 ⫽ 0.01, df ⫽ 1, P ⫽ 0.92). Pooling the data from the two laboratories yields a frequency of SP15-like Cry2Ab resistance alleles in H. armigera of 0.033 with 95% credibility intervals of 0.021 and 0.047. The probability that at least one of the lines was erroneously classiÞed as susceptible was 0.003. Thus, the probability that all possible resistance alleles in the 356 parental lines were detected was 0.997. The 5.5-fold difference between the F1 and F2 tests is highly signiÞcant (␹2 test for homogeneity ⫽ 13.62; P ⫽ 0.0002). The joint 95% credible region for the two estimated allele frequencies shows that the F1 estimate is statistically different from the F2 estimate (Fig. 1). The probability that the two estimates are from the same population is 0.0001. Direct Test for Severe Fitness Costs. The nature of the F1 test ensures that if the Þeld insect used in the single pair mating is heterozygous for resistance, 50% of the F1 offspring tested will be homozygous resistant; that proportion would approach 100% if the Þeld in-

sect was homozygous resistant. Our direct tests for severe Þtness costs assumed that the former was the case. In those tests that scored positive for resistance in Canberra, 57.8% of the tested control (not exposed to toxin) larvae reached third or fourth instar by day 7, whereas in those exposed to toxin 27% reached that stage. For tests conducted in Narrabri a greater frequency (95.4%) of control larvae tested reached third or fourth instar by day 7, whereas 51.0% of those exposed to toxin reached that stage. We assume that the difference in maturity rates of the controls from the two laboratories was due to variation in the temperature at which larvae were reared; however, relative to the controls at each location, approximately half of the selected proportion reached third or fourth instar at scoring. The proportion of larvae that reached third and fourth instar never approached levels which indicated the tested insect was homozygous for resistance. Fourteen males that were shown to be heterozygous for resistance in F1 tests were recovered and mated to GR females to simulate a F2 test. Eight of these males either died before their second mating or failed to fertilize a GR female. The remaining six males were successfully tested and Þve produced larvae in the F2 generation that survived the discriminating concentration of Cry2Ab (Table 2). For one of the simulated F2 tests (isolate 186) is not represented in Table 2 because the F2 test on neonates failed. As an alternative, F2 larvae were reared to adults that were then subjected to progeny tests by mating individual moths (either male or female) to one or two virgin SP15 of the other sex. Seventeen such tests on isolate 186 yielded fertile offspring, and based on survival rate and the proportion of third plus fourth instar larvae, it was possible to assign a genotype to each F2 moth. Twelve proved to be homozygous susceptible, four were determined to be heterozygotes and one homozygous resistant. In the one assigned a genotype of homozygous resistant, of the 43 neonates tested, two died and the remainder reached either third or fourth instar. Thus, like the Þve other isolates tested in the simulated F2 tests (Table 1), the progeny tests demonstrated that isolate 816 was also able to produce autozygous homozygotes.

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Table 3.

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Results from the indirect test of severe fitness costs using survivors of F1 tests

Isolate

No. families tested

Probability of missing a negative

No. negative families

No. positive families

% third instars on Cry2Ab

% third instars on control

Corrected % third instars on Cry2Ab

180 327 378 637 750 1339 1684 1685

9 14 9 13 20 12 17 16

0.083 0.031 0.083 0.037 0.001 0.046 0.017 0.021

0 0 0 0 0 0 0 1

9 14 9 13 20 12 17 15

20.15 23.98 18.13 13.75 21.94 17.04 22.42 17.75

77.8 80.08 62.39 61.26 90.33 67.49 81.97 70.42

25.89 29.95 29.06 22.45 24.29 25.25 27.35 25.21

We present the probability of not testing a cross between one quarter of the mating combinations (i.e., missing a negative) which depends on the number of single pairs successful screened for each isolate. The survival of larvae tested on Cry2Ab toxin and the control (no toxin) was calculated by dividing the number of larvae reaching at least third instar by the total larvae tested for all assays on that particular isolate. The “corrected” percentage of third instars on Cry2Ab was calculated as the proportion of larvae ⱖ third instar on toxin/proportion on uncontaminated diet.

One of the isolates (596) yielded a high (20%) proportion of F2 larvae that survived and reached at least third instar and that proportion was signiÞcantly greater than the expected 6.25% (Table 2). In the remainder of tests the proportion did not signiÞcantly depart from the expected value. Thus individuals that were homozygous for all six Cry2Ab resistance alleles detected in the F1 screen were also detected as allozygotes and we conclude that none incurred a severe cost to Þtness. Indirect Test: Analysis of Survivors of the F1 Test. Eight isolates were subjected to the indirect test and the results are presented in Table 3. Because we examined from 9 to 20 single pairs, the simple probability of missing one of the RfS ⫻ Rf S matings by chance alone varied with the number tested and this value is included in the table. Seven of the eight isolates only produced “positive families” (i.e., contained survivors that reached third instar by day 7). However, one of the 16 families tested from isolate 1685 was exceptional as it failed to produce a survivor, which may indicate that the allele from this isolate could not be made homozygous. An unexpected level of mortality was observed (32%) in the control for this assay where neonates from the same cohort were reared in the absence of toxin, was of concern. However, this level of mortality was not inconsistent with the hypothesis of a severe Þtness cost as 25% mortality would be expected if the hypothesized lethal gene expressed itself posthatching. This family was further analyzed via progeny testing control male survivors by mating them to SP15. If the original mating type was indeed Rf S ⫻ Rf S, the possible genotypes of the survivors were Rf R f, R f S, or SS in the ratio of 1:2:1, respectively. Six control males successfully fertilized SP15 females and their neonates were tested. RfRf males would be expected to produce only RcRf offspring and thus all would be resistant, Rf S males would be expected to produce 50% resistant offspring, whereas SS females would produce no resistant offspring. Two out of the six males tested proved to be homozygous resistant. For the Þrst, 43 neonates were tested on toxin-contaminated diet, one died but the remainder reached third instar or greater after 7 d. For the second male, 44 neonates were tested, two died but the remainder

reached third instar. Thus the apparent exceptional family also contained homozygous resistant individuals, but by chance they were not detected among the tested sample. Such a chance event was made more likely by the unusual level of mortality in that family. In addition, in performing so many assays (110), the chance of one or more exceptions became more probable. As we would expect one quarter of all neonates screened in the indirect test to be homozygous for resistance, if Þtness costs were associated with either Rf or Rc alleles, we would expect a reduction in the proportion achieving at least third instar. Table 3 lists the proportion reaching third instar, and they are indeed all ⬍25%. However, if that proportion is adjusted by correcting for the proportion of control insects that were not exposed to toxin but were assayed in parallel, and which reached third instar, then there is a good Þt to the expected 1:4 (Table 3). The power to detect a recessive Þtness cost in Rf alone may not be high, as only one fourth of the crosses would be informative, but as only two of the nine isolates tested exhibited an adjusted proportion of ⬍25%, any Þtness costs would necessarily be small. Discussion In theory, F1 screens and F2 screens should yield similar frequencies of resistance genes. An exception might occur if frequencies detected using F2 screens reßected mutations present in more than one gene but in this situation we would expect F1 tests to yield lower not higher resistance frequencies. The complementation tests reported herein support our previously published notion that the SP15-like Cry2Ab resistance detected in F2 isolates of H. armigera is due to alleles at the same locus. However, our results from screens conducted in 2007Ð2008 conÞrm the reports by Knight (2009) from 2004 to 2007 that frequencies of SP15-like Cry2Ab resistance genes in H. armigera detected by F1 tests are signiÞcantly greater than those detected on the sample populations using F2 tests. The most striking result was the extent of the difference between the frequencies. The observed 5.5-fold difference is highly signiÞcant and invites several questions.

April 2010


Why Do the F1 Versus F2 Tests Provide Different Frequencies? We tested the hypothesis that the difference in frequencies among these closely related tests was due to the presence of recessive lethal alleles at the locus conferring resistance or at other loci that are linked to the Cry2Ab resistant gene. Two tests, one designated “direct” and one “indirect” were conducted to reveal lethal effects. For the direct test, if the hypothesis was correct, the difference in frequencies revealed by the F1 and F2 0.033 ⫺ 0.006 tests (Table 2) imply that 82% ⫻ 100 0.033 of resistance alleles are autozygous lethal. Therefore we would expect only 18% of the F2 tests involving males that were known to carry a resistance gene to yield resistant insects. We observed that all six of the males tested were able to produce viable homozygous resistant larvae. Under the hypothesis, the chance that all six were positive is improbable (P ⫽ (0.18)6 ⫽ ⬍0.001). This result suggests that the hypothesis should be rejected. This conclusion is reinforced by the results of the indirect tests whereby we infer that all of eight unique isolations of “resistant alleles” can exist in a homozygous form without an individual incurring a signiÞcant Þtness cost. It is possible that some alleles (if not all) suffer from recessive Þtness costs that limit the generation of homozygous resistant insects in F2 tests. However, there are a number of sources of information that indicate that this hypothesis is unlikely. First, the indirect tests reported herein indicate that for all isolates, when exposed to toxin, the frequency of homozygous resistant individuals was near to the expected frequency (25%). That 25% of individuals would consist of a mix of allozygous (Rf Rc) and autozygous (RfRf ;RcRc) individuals so there is no evidence that any genotype suffered recessive Þtness costs. Second, none of the Cry2Ab isolates detected in the F2 screens generated signiÞcantly fewer than the expected 6.25% of larvae that reach at least third instar, and in many of the isolates a greater proportion than the expected proportion reached this stage (Mahon et al. 2007a, this study). This result is not expected under the hypothesis of a recessive Þtness costs. Further, we have considerable experience with Cry2Ab resistant colonies of H. armigera (e.g., SP15 and, NB405) including measures of the relative responses of different genotypes as part of complementation tests (Mahon et al. 2008, this study). These experiments were not designed to detect Þtness costs but would have shown any marked lack of Þtness under laboratory conditions if they were present. Additionally, the survival and growth rate of RR, RS, and SS genotypes are comparable when reared on whole laboratory-grown or portions of Þeld-grown conventional or Bollgard II cotton and thus did not indicate the presence of Þtness costs (Mahon and Olsen 2009). In addition, although there was variability among the genotypes in their pupae weights, time to emergence, longevity, percentage fertile and number of eggs per female, there was no evidence that resistant genotypes

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were disadvantaged relative to the susceptible genotype. Thus, under laboratory conditions when feeding on conventional cotton there is no evidence that the resistance is associated with recessive or dominant Þtness costs. If (so far undetected) dominant Þtness costs are responsible for failure to detect homozygous resistant genotypes in F2 tests, such alleles must beneÞt from positive selection to persist in the population at a frequency of 0.033 which presumably is far greater than the mutation rate. Some selection for Cry2Ab resistance may occur late in the season in Bollgard II Þelds when the titer of Cry1Ac declines below toxic levels but these opportunities are limited (Mahon and Olsen 2009). Which Test, F1 or F2, Provides the More Accurate Estimate of Resistance? We contend that the F1 test provides the most appropriate estimate of the frequency of SP15-like Cry2Ab resistance genes in H. armigera because it involves one less mating event and generation than the F2 screen. Although we failed to determine the mechanism that causes the difference in test results, it is conceivable that homozygotes can be lost at some stage in the F2 test, particularly if Þtness costs are allele-speciÞc, but it is difÞcult to conceive how the F1 tests could exaggerate the frequency. Thus, through previously accepting the F2 results we significantly underestimated the frequency of Cry2Ab resistance in H. armigera. What Are the Consequences of the Higher Cry2ab Frequencies for the Evolution of Resistance? The most obvious consequence of accepting the F1 results, and therefore that the frequency of Cry2Ab genes is 0.033 rather than 0.006, is that Þeld-scale resistance is likely to evolve in a shorter time than hitherto expected. Helicoverpa species are innately tolerant of Cry toxins (Liao et al. 2002); thus, the titer of Cry toxins within Bt cotton do not present a “high” dose to susceptible H. armigera and H. punctigera. Furthermore, Bollgard II is essentially the same product as Ingard (known as Bollgard beyond Australia) with the addition of the cry2Ab gene and its controlling sequences (APVMA 2003). Thus, the well documented decline in titer of Cry1Ac transgenic varieties of cotton with time (Greenplate 1999, Adamczyk et al. 2001, Rochester 2006, Dong, and Li 2007, Olsen et al. 2005) is likely to also occur within Bollgard II. On average over a season, the abundance of H. armigera pupae underneath Ingard crops was ⬇60% of that found below conventional cotton (Baker et al. 2008). Thus, although SP15-like isolates are fully susceptible to Cry1Ac, there are clearly opportunities for such insects to survive in Bollgard II crops. If Cry2Ab resistance genes occur at a rate of 0.033 in Þeld populations, homozygous resistant individuals will be at a frequency of (0.033)2 ⫽ 0.001, or one in 1,000 individuals. Although this genotype remains relatively rare, moderate to heavy egg lays can result in ⬎50 H. armigera eggs per meter row of cotton; thus, a 0.5- by 0.5-km Þeld of Bollgard II may contain ⬎12,500 homozygous resistant eggs.

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Are Other Resistance Monitoring Programs That USe F2’s Similarly Underestimating Gene Frequencies? Many estimates of resistance frequencies have been made using the standard F2 technique described in Andow and Alstad (1998), including Cry1Ab resistance in the sugarcane borer, Diatraea saccharalis (F.) (Huang et al. 2007) and Cry3Aa resistance in the beetle Chrysomela tremulae F. (Ge´ nissel et al. 2003). Following the work of Ge´ nissel and colleagues, Wenes et al. (2006) used F1 screens to detect Cry3Aa resistance in the poplar beetle and found similar resistance frequencies to those detected using the F2 results. Similarly, an F1 screen for Cry1b resistance in D. saccharalis (Yue et al. 2008) revealed a comparable estimate to that found by the F2 test. Thus, perhaps the difference in F2 and F1 frequencies we observed is an isolated phenomenon restricted to Cry2Ab resistance in Helicoverpa armigera. Nevertheless, it behoves others charged with resistance management to employ F1 tests once they have isolated viable resistant colonies and satisfy themselves that that colony represents the only common form of resistance in the populations of interest. Acknowledgments Judy Nobilo, Karen Stanford, Norm Winters, Joel Armstrong, and Karen Olsen provided expert technical assistance. D. Andow generously provided the programs that he developed to calculate resistance frequencies, credibility intervals, PNo, and the differences between F1 and F2 frequencies and also provided advice on F1 and F2 screen statistics. Monsanto Australia provided the Cry2Ab corn powder used for some of the reported work. The Australian Cotton Research and Development Corporation and Commonwealth ScientiÞc and Industrial Research Organization Þnancially supported the research.

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