Spatial and temporal variability in host use by Helicoverpa zea as measured by analyses of stable carbon isotope ratios and gossypol residues

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1 Journal of Applied Ecology 2010, 47, doi: /j x Spatial and temporal variability in host use by Helicoverpa zea as measured by analyses of stable carbon isotope ratios and gossypol residues Graham Head 1 *, Ryan E. Jackson 6, John Adamczyk 7, Julius R. Bradley 2, John Van Duyn 2, Jeff Gore 6, Dick D. Hardee 6, B. Rogers Leonard 3, Randall Luttrell 4, John Ruberson 5, J. Walt Mullins 1, Robert G. Orth 1, Sakuntala Sivasupramaniam 1 and Richard Voth 1 1 Monsanto LLC, 800 North Lindbergh Blvd, St. Louis, MO 63167, 2 Entomology Department, North Carolina State University, Box 7630, Raleigh, NC 27695, 3 Louisiana State University AgCenter, 212 A Macon Ridge Rd, Winnsboro, LA 71295, 4 Department of Entomology, University of Arkansas, AGRI 319, Fayetteville, AR 72701, 5 Department of Entomology, University of Georgia, PO Box 748, Tifton, GA 31794, 6 USDA, ARS, SIMRU, PO Box 346, Stoneville, MS 38776; and 7 USDA, ARS, BIRU, 2413 E Highway 83, Weslaco, TX 78596, USA Summary 1. A high dose refuge strategy has been adopted in the USA to manage the risk of Bacillus thuringiensis (Bt) resistance in target pests such as the cotton bollworm (CBW), Helicoverpa zea (Boddie) in transgenic Bt cotton Gossypium hirsutum L. Structured refuges, consisting of non-bt cotton, have been a mandated part of this strategy to produce non-selected insects that are temporally and spatially synchronous with insects from the Bt crop, diluting Bt resistance alleles through mating. However, the bollworm is highly polyphagous and exploits a large number of crop and weedy hosts concurrently with Bt cotton. 2. A study was carried out in five major US cotton-producing states during 2002 and 2003 using the ratios of 13 Cto 12 C in bollworm moths to estimate the proportions of the population originating from C 3 or C 4 plants. A separate study measured gossypol residues in moths from four states in 2005 and 2006, enabling the identification of moths whose natal hosts were cotton rather than other C 3 hosts. 3. C 4 hosts served as the principal source of bollworm moths from mid-to-late June to early September, depending on the state. Beginning in late August early September and lasting 1 4 weeks, the majority of moths exhibited isotopic compositions characteristic of C 3 hosts. During this period, however, the minimum percentage of moths that developed as larvae on C 4 hosts was typically >25%. By mid-september and through October and November, the majority of the bollworm population exhibited C 4 isotopic compositions. 4. Between late June and early August, cotton-derived bollworm moths (moths with gossypol residues) comprised <1% of moths in all states, and remained below this level throughout the season in North Carolina. In other states, cotton-derived moths increased between early August and early September to peak at an average of 19Æ1% of all moths. 5. Synthesis and applications. Data on 13 C 12 C ratios and gossypol residues in CBW moths were used to assess the importance of structured non-bt cotton refuges for the management of Bt resistance risk in H. zea. Weekly estimates of bollworm breeding on cotton, C 3 plants other than cotton and C 4 plants showed that, throughout the season, the majority of bollworm moths caught in pheromone traps adjacent to cotton fields did not develop as larvae on cotton. This result implies that management practices in cotton such as the use of structured cotton refuges will play a relatively minor role particularly compared with maize Zea mays L. in managing potential resistance to Bt cotton in populations of the CBW in the US Cotton Belt. *Correspondence author. graham.p.head@monsanto.com Ó 2010 The Authors. Journal compilation Ó 2010 British Ecological Society

2 584 G. Head et al. Key-words: Bacillus thuringiensis, cotton, cotton bollworm, insect resistance management, refuge Introduction Bollgard Ò (Monsanto Company, St Louis, MO, USA) cotton produces the insecticidal toxin Cry1Ac from Bacillus thuringiensis Berliner (Bt) and was developed to control boll-feeding Lepidoptera such as the cotton bollworm (CBW), Helicoverpa zea (Boddie). As a condition of registration of Bollgard cotton, the US Environmental Protection Agency mandated that structured non-bt cotton refuges be planted in conjunction with Bollgard cotton. These refuges are intended to serve as a source of non-selected individuals of the target pest species whose purpose is to reduce the possibility of two Bt-selected individuals mating, thus slowing the evolution of resistance to thecry1acprotein. However, Bollgard cotton does not produce a high dose of Cry1Ac relative to the tolerance of bollworm (Stone & Sims 1993), meaning that, theoretically, structured cotton refuges would need to be relatively large to delay the evolution of Bt cotton resistance effectively in bollworm. Supplementary control tactics are frequently required in Bollgard cotton to control bollworms (Mahaffey et al. 1994; Mahaffey, Bradley & Van Duyn 1995; Lambert, Bradley & Van Duyn 1997) and, under conditions of extremely high bollworm populations, structured refuges have been shown to produce inadequate numbers of moths to effectively delay Bt resistance evolution based on bollworm survival estimates in Bollgard (Jackson et al. 2004). Despite these observations, and laboratory data (Ali, Luttrell & Young 2006; Tabashnik et al. 2008a,b) indicating increases in LC 50 values to Cry1Ac protein, there has been no documented change in the efficacy of Bt cotton in the US Cotton Belt (Moar et al. 2008). Therefore, because H. zea is an extremely polyphagous pest with hosts that include numerous crops grown in the cotton belt, especially maize, it was hypothesized that non-transgenic (non-bt) non-cotton crop hosts are contributing significant numbers of Bt-susceptible individuals to the general bollworm population and that this contribution exceeds that of structured cotton refuges (Gould et al. 2002). Gould et al. (2002) showed that wings of bollworm moths that developed as larvae on plants with C 3 physiology had ratios of 13 C: 12 C that differed from ratios of moths that developed as larvae on plants with C 4 physiology. Moths with d 13 C values between )15 and )10 units per mil (&)camefrom C 4 hosts, whereas moths with d 13 C values between )28& and )21& developed on C 3 hosts. Therefore, results from stable carbon isotope analyses can eliminate certain plants as larval food sources but cannot distinguish between plant species that utilize the same photosynthetic pathway. In addition, work by Orth, Head & Mierkowski (2007) showed that it was possible to reliably identify tobacco budworm moths Heliothis virescens (F.) that had fed on cotton as larvae. This technique allows the separation of heliothine moths that fed on cotton as larvae from moths emerging from other C 3 hosts, such as broadleaf weeds, soybeans Glycine max (L.) Merr. or peanuts Arachis hypogea L. In the current study, stable carbon isotope analysis was used to estimate the proportion of the bollworm population that uses C 4 plants as natal hosts in the five US cotton-producing states surveyed by Jackson et al. (2008) in 2002 and This was supplemented by analysis of gossypol residues in bollworm moths in Arkansas (2005 and 2006), North Carolina (2005 only) and Mississippi (2006 only). These two data sources were synthesized to estimate the proportion of the bollworm population that uses cotton, C 3 plants other than cotton, and C 4 plants for larval development over the course of the season. Materials and methods C 3 C 4 HOST STUDY Arkansas, Georgia, Louisiana, Mississippi and North Carolina in the USA were selected as the principal areas for the study of the temporal and spatial production of bollworm from C 3 and C 4 plant hosts in 2002 and Within each of these states, four study locations were established in counties where cotton is an important component of the cropping system. These study locations served as the basis for landscape-level monitoring of bollworm populations, as described by Jackson et al. (2008). The selected states, and counties within these states, were representative of a range of agro-ecosystems associated with cotton production in the mid-south and southeastern USA, with site selection based on annual crop production data from the National Agricultural Statistics Service (see Methods and table 1 of Jackson et al for details). At each study location, a set of crop interfaces was monitored. Each interface was a boundary between a commercial Bollgard cotton field and a commercial field containing either (i) Bollgard cotton, (ii) non-bt cotton or (iii) one of maize, grain sorghum, peanuts or soybeans. These fields were at least 10 ha in size. In total, five or six types of interfaces (pairings of crops) were monitored at each study location, with each interface type replicated four times per location, yielding monitoring sites per state in each of 2002 and Pheromone traps, modified after Hartstack, Witz & Buck (1979), and baited with Zealure (Hercon Ò Luretape Ò Insect Attractant Dispenser; Hercon Environmental Company, Emigsville, PA, USA), were used to monitor numbers of bollworm adults, as reported by Jackson et al. (2008). GOSSYPOL RESIDUE FIELD STUDY This study was conducted on moths collected in Arkansas (2005 and 2006), North Carolina (2005 only) and Mississippi (2006 only). Within each state, pheromone traps were located in regions of high Bollgard and Bollgard II cotton adoption and historically high bollworm population densities, with traps at least several kilometres apart. Pheromone traps, as used for the C 3 C 4 study, were placed adjacent to or near cotton fields and monitored at least weekly from April June through to September or October, depending on state and year. In 2005, 14 and 10 sites were sampled in Arkansas and

3 Host use by cotton bollworms 585 North Carolina, respectively, while in 2006 the same 14 sites in Arkansas were sampled plus 18 sites in Mississippi. Where possible, sites were the same as those used for the C 3 C 4 study. Moths were stored individually in tubes and kept frozen at )80 C until analysed at Monsanto laboratories at St Louis, Missouri. BOLLWORM REARED ON KNOWN HOSTS In 2002 and 2003, fourth fifth instar bollworm larvae were collected from known crop hosts consisting of maize (n =20), cotton (n =22),peanuts(n = 35), sorghum Sorghum bicolor (L.) Moench (n = 27) and soybeans (n = 8). Larvae were transported to the laboratory and reared to pupation in 470-ml plastic tubs on the respective plant materials obtained from the larval collection site. Pupae were removed from the tubs and placed singly into 30-ml plastic cups. Upon eclosion, moths were placed into 95% ethanol until preparation began for the stable carbon isotope analyses. Ethanol was previously demonstrated to have no effect on carbon isotopic ratios of moth wings (Gould et al. 2002). The d 13 C values from known hosts were analysed with one-way analysis of variance, using single degree of freedom contrasts to evaluate variation between and within the groups of plants with C 3 and C 4 photosynthetic pathways. In 2005 and 2006, similar methods were used to produce moths from known hosts for use in gossypol residue analyses. The only difference in the procedures was that eclosing moths were frozen prior to analysis instead of being stored in alcohol because preliminary studies indicated that storage in alcohol affected subsequent gossypol residue analyses. STABLE CARBON ISOTOPE ANALYSES Subsamples of adult bollworms (up to 100, depending upon the number of moths collected) from pheromone traps at each crop interface within each sampling week during 2002 and 2003 were stored in 95% ethanol. Each left forewing was cut up and placed into a 5 9 mm tin capsule that was tightly folded into a cube. Wing tissue within the tin capsule was converted to CO 2 by micro-dumas combustion using a Carlo Erba NA 1500 CHN Analyser (Carlo Erba Instruments, Milan, Italy) coupled to a Finnigan-MAT Delta C Mass Spectrometer using a Conflo II Interface (Thermo Scientific, Waltham, Massachusetts, USA) at the University of Georgia Stable Isotope Laboratory. The isotope standard reference material was bovine muscle powder (NIST-RM 8414) from the National Institute of Standards and Technology with a precision of approximately ± 0Æ1& for d 13 C. All d 13 C values were recorded relative to V-PDB scale. To determine the percentage of C 3 vs. C 4 -derived moths, calibration curves were generated in both 2002 and 2003 using samples of 100 moths of known composition (0%, 25%, 50%, 75% and 100% C 3 :C 4 moths). A weighted linear regression model was fitted to the data to obtain calibration curves for the percentage of C 3 vs. C 4 -derived moths. Using these calibration curves, the percentage of C 3 vs. C 4 -derived moths was determined for field-collected samples. In total, moths were analysed in 2002 and moths in GOSSYPOL RESIDUE ANALYSES The gossypol-residue analytical method involves the analysis of bound gossypol by creating a Schiff s base using aniline and analysing the resulting dianilino-gossypol complex using high pressure liquid chromatography through an Agrilent Eclipse XDB-C8 column (Agilent Technologies, Santa Clara, California, USA) with a Micromass Quattro Ultima (Waters Corporation, Milford, Massachusetts, USA) triple quadrupole mass spectrometer as the detector (HPLC MS MS) (Orth, Head & Mierkowski 2007). Unlike the stable carbon isotope analyses, gossypol residue analyses were carried out on individual moths. Bollworm moths were freeze-dried for 48 h. The wings were removed and the thorax and abdomen of each moth were weighed. The analyses were carried out as described in Orth et al. (2007). A set of criteria for determining if gossypol was present (whether the test was positive) was determined using a set of cotton reared moths and non-cotton reared moths. The criteria were based on the response ratio and the signal-to-noise ratio for the chromatogram signal, as per Orth et al. (2007), and were derived from the positive and negative controls run for each set. Individual moths collected in the field then were analysed in sets that included method blanks, positive control moths reared on cotton and negative control moths reared on plants other than cotton. In total, 1773 moths were analysed in 2005 and 2749 in STATISTICAL ANALYSIS For the stable carbon isotope analyses, an anova was run on the percentage of moths coming from C 3 vs. C 4 host plants, and pair-wise means comparisons were performed for each state and week within 2002 and The percentage of moths from C 4 hosts from 2002 and 2003 and gossypol data from 2005 and 2006 was analysed with linear mixed model analyses using the REML procedure of genstat (V11.1) (VSN International, Hemel Hempstead, UK). Data were excluded from the C 4 moth analyses at the beginning and end of each season if fewer than three states were represented. In 2002, data were analysed for the period from 1 July to 9 September, while in 2003, the period extended from 2 June to 22 September. Because data from 2 years were included in the gossypol analysis, time was expressed as Julian week rather than calendar date. For the gossypol analyses, data were excluded prior to Julian week 27 (late June) and after week 36 (early September) because (a) gaps existed for some state years outside of this period, and (b) for some weeks fewer than 35 moths were analysed per state year. Truncating the data in this way excluded data points that were likely to be imprecise estimates of gossypol-residue incidence, especially given the low overall percentage of moths positive for gossypol (see Results). To understand the patterns in the data, a limited number of specific unplanned comparisons were made using the Fisher s protected LSD test. As this test is considered overly generous in declaring differences as significant when used with unplanned comparisons (Quinn & Keough 2002), then logically if the difference between the largest and smallest of a set of means is not significantly different, there is confidence that there are no real differences in the data. SYNTHESIS OF STABLE CARBON ISOTOPE RATIO AND GOSSYPOL RESIDUE ANALYSES The weekly average C 3 C 4 data were integrated with the gossypol residue data to provide estimates by Julian weeks of the percentage of moths whose larval hosts were cotton, C 3 crops other than cotton, or C 4 crops. The C 3 crops other than cotton parameter was calculated as the difference between % of moths from C 3 crops and % with gossypol residues. Because the seasonal pattern of both C 3 C 4 host use and gossypol residues for North Carolina was different from the other states for which there was data (see Results), these calculations were made separately for North Carolina from the rest of the mid-south and southeastern states.

4 586 G. Head et al. For North Carolina, both types of data were available from Julian week 28 (early July) through to week 38 (mid-september), while for the other states, the data set started at week 22 (late May) and ended at week 40 (early October). Results ISOTOPIC SEGREGATION OF BOLLWORM ADULTS FROM C 3 AND C 4 HOSTS d 13 C values of wings from moths reared on maize, cotton, peanuts, sorghum and soybeans ranged from 12Æ31& to 10Æ96&, 31Æ51& to 23Æ20&, 28Æ45& to 25Æ35&, )18Æ63& to )10Æ66& and 27Æ64& to 26Æ71& respectively. A clear distinction was present between moths that developed as larvae on hosts with C 3 isotopic compositions (cotton, peanuts and soybeans) and those from hosts with C 4 isotopic compositions (maize, sorghum) (F 1,107 = 3773, P <0Æ0001). While there were some significant differences within both of the C 3 and C 4 crop groups, these were very small compared to the difference between the C 3 and C 4 groups. Specifically, maize and sorghum differed within the C 4 group (F 1,107 =6Æ35, P =0Æ013) and, within the C 3 group, cotton differed from both peanuts (F 1,107 =11Æ35, P =0Æ001) and soybeans (F 1,107 =11Æ43, P = 0Æ001). The results from these analyses confirmed those of Gould et al. (2002), demonstrating that a moth with a d 13 C value of )20& or less fed on a plant utilizing C 3 photosynthesis, and a moth with a d 13 C value of )15& or above fed on a plant utilizing the C 4 photosynthetic pathway. DISCRIMINATION OF MOTHS FROM COTTON VS. OTHER C 3 HOSTS Using moths from known hosts and the discrimination criteria of Orth et al. (2007), all positive controls (i.e. cotton-reared moths) were classified correctly as positive for gossypol, indicating that the method has a low rate of false negatives. However, a small percentage of the negative control moths reared on non-cotton hosts were incorrectly judged as positive for gossypol using these criteria, indicating that the method yields a low rate of false positive results but this was consistently <5%. Therefore, estimates of the percentage of cottonderived moths in bollworm populations based on these analyses may slightly overestimate true values. REGIONAL HOST UTILIZATION BY BOLLWORM ThepercentageofmothsfromC 3 vs. C 4 hosts was determined by comparing the results of stable carbon isotope analyses of composite samples (from each trap on each date) with calibration curves generated from moths of known composition. The calibration curves were similar for the 2 years; the respective regression equations for 2002 and 2003 were: % C 4 = 180Æ65 7Æ076* d 13 C, and % C 4 = 179Æ25 6Æ842* d 13 C. In both years, the regression model fitted the data very well with R 2 values of 98Æ6% and 97Æ1% respectively (2002, n =10, P <0Æ001; 2003, n = 10, P <0Æ001). The percentage of bollworm adults that developed as larvae on C 3 and C 4 hosts was not affected by the crop interface where the moths were collected. Based on an anova for each location and date, <1% of all comparisons were statistically significant, indicating that the composition of bollworm populations is not determined by the pattern of local hosts. Therefore, the crop-interface factor was included aspartoftherandom model in the mixed model analyses of stable carbon isotope data. The results of stable carbon isotope analyses for each state in 2002 and 2003 are presented in Figs 1 and 2 respectively. These data parallel the CBW adult trap catches in Figs 3 and 4ofJacksonet al. (2008) for the same set of study sites. In both years, the residuals for the C 4 data were symmetrical but not normally distributed and this non-normality was not corrected by any of the standard data transformations. Consequently, the C 4 data were analysed on the original scale but care was taken to avoid over-interpretation of the data to minimize the risk of drawing spurious conclusions (Zar 1984, pp ). In both years, the linear mixed model analysis indicated the presence of a state by week interaction (2002 F 34,753 =19Æ26, P <0Æ001; 2003 F 58,1237 =23Æ13, P <0Æ001), i.e. the pattern of seasonal changes in stable carbon isotope ratio differed among states. The major difference among states in 2002 was that the decline from high (>80%) C 4 moth percentage occurredseveralweekslaterinnorthcarolinathaninallof the other states (Fig. 1). There was also some evidence of a rebound in % C 4 moths late in the season in some states (Fig. 1). The 2003 data covered a longer period of time than the 2002 data and provided more information on the seasonal patterns of C 4 host use. As in 2002, the pattern of seasonal change in host use during 2003 occurred several weeks later in North Carolina than in all other states, with the period of high C 4 host use being from mid-july to early September (Fig. 2). In contrast, the other states exhibited this period of high C 4 host use from mid-to-late June to mid-august. During May and early June, bollworms used both C 3 and C 4 plants as larval hosts. The proportions of bollworms developing on C 4 hosts during this period ranged from 0% in Georgiain2003to83Æ3% in Louisiana in 2002, and averaged 35Æ8% across states and years (Figs 1 and 2). During this period, moth catches were low (Jackson et al. 2008), indicating that at a regional level, bollworm populations were low and were coming from a mixture of C 3 and C 4 plants. The percentage of bollworm adults with C 4 isotopic ratios began to increase rapidly by mid-june to early July, nearing 100% during this period in all states and years (Figs 1 and 2). At the same time, bollworm trap catches increased by up to an order of magnitude (Jackson et al. 2008), with both events coinciding with emergence of moths from maize and sorghum. During this period, moth populations were high and almost exclusively derived from C 4 hosts. These observations are consistent with recorded larval populations on these hosts (Jackson et al. 2008). Subsequently, the proportion of moths from C 4 hosts fell, commonly being in the vicinity of 50% of the collected

5 Host use by cotton bollworms 587 (a) (d) (b) (e) (c) Fig. 1. Percentage of Helicoverpa zea adults that developed on C 3 and C 4 natal hosts in five US states in 2002 with the highlighted period in which adults would be expected to emerge from Bollgard cotton. bollworm adults from mid-to-late August. Trap catches also fellduringthesameperiodtolevelsapproximatelythesameas prior to the emergence of adults from maize and sorghum. The majority of bollworm adults collected in late August and early September across the study area developed as larvae on C 3 hosts, while from early September to early November there was another marked increase in the percentage of moths with C 4 isotopic compositions. Percentages of bollworm adults from C 4 hosts ranged from 25% to 90% during this end-ofseason period, with 24 of 31 weeks collections yielding >50% of moths with C 4 isotopic compositions. From late August onwards, bollworm trap catches were low and generally declining (Jackson et al. 2008). At a regional level, these data indicate that bollworm populations were low from late August onwards and derived from a mixture of C 3 and C 4 plants, with more C 3 plants being used during late August and early September than later in the season. REGIONAL USE OF COTTON AS A HOST CROP The linear mixed model analysis of the gossypol residue analyses from 2005 and 2006 (Fig. 3) showed that there were significant differences in the seasonal pattern of cotton contribution to bollworm populations among states, i.e. there was a significant interaction (F 24,301 =4Æ27, P <0Æ001). The major difference was that the percentage of cotton-derived moths from North Carolina did not vary over the course of the season (P >0Æ05), averaging 0Æ96% of moths, while the cotton contribution to bollworm collections increased as the season progressed for the other three data sets.

6 588 G. Head et al. (a) (d) (b) (e) (c) Fig. 2. Percentage of Helicoverpa zea adults that developed on C 3 and C 4 natal hosts in five US states in 2003 with the highlighted period in which adults would be expected to emerge from Bollgard cotton. Between weeks 27 and 31 (late June to early August), the percentage of cotton-derived moths was low in all four data sets and did not vary significantly (P >0Æ05), with an average of 0Æ72% over this period. Between early August to early September (Julian weeks 32 36), Arkansas 2005 and 2006 and Mississippi in 2006 all showed significant increases in the cotton contribution (P < 0Æ05), peaking at between 10Æ2% and 30Æ2% of moths. Specific comparisons were made between the 2 years of data from Arkansas and between the two states in 2006 to clarify these seasonal patterns. This showed that while there were some differences between the data sets, this was primarilyrelatedtowhethertheseasonalpeakoccurredin1week or the next, with the same overall seasonal pattern. In summary, the seasonal pattern in the percentage of cotton-derived moths was for the cotton contribution to be low from late June to early August for Arkansas 2005 and 2006 and Mississippi in 2006, followed by an increase to a peak in mid-to-late August. Over the three data sets, peak incidence of cotton contribution averaged 19Æ1%. However, over the course of the season, an average of only 3Æ1% of all moths were classified as cottonderived, indicating that at a regional level cotton contributes only a very small proportion of the overall adult bollworm population. SYNTHESIS OF STABLE CARBON ISOTOPE AND GOSSYPOL RESIDUE ANALYSES As described above, the results of the stable carbon isotope analyses and gossypol residue analyses were both consistent across years and relatively large regions. Therefore, these two

7 Host use by cotton bollworms 589 (a) (c) (b) (d) Fig. 3. Percentage of Helicoverpa zea adults that developed on cotton in (a) Arkansas 2005, (b) North Carolina 2005, (c) Arkansas 2006 and (d) Mississippi types of data can be integrated into an overall summary of bollworm host use as shown in Fig. 4. From this, it is clear that cotton contributes relatively few moths to regional populations across the cotton-growing areas of the mid-south and southeastern USA. Throughout the season, C 4 plants always host a larger proportion of bollworm larvae than does cotton. In addition, both early in the season and after approximately mid-august, C 3 plants other than cotton also host as many if not more bollworm larvae than does cotton. Discussion REGIONAL HOST UTILIZATION BY BOLLWORM The results reported in Figs 1 and 2 support those of Gould et al. (2002) with regard to mixed use of C 3 and C 4 host plants by bollworm larvae early in the growing season. In addition, bollworm larvae have been documented early in the season feeding primarily on weed hosts, and also on whorl-stage maize and sorghum (Neunzig 1969; Stadelbacher et al. 1986; Kogan et al. 1989). The majority of reported weed hosts utilize the C 3 photosynthetic pathway (Neunzig 1969; Kogan et al. 1989). Thus, because C 3 crop hosts (cotton, peanuts and soybeans) are not attractive to bollworm during this period, most of the early season bollworm population must have developed as larvae on C 3 weed hosts, as confirmed here (Figs 1, 2 and 4). The predominance of moths emerging from C 4 hosts from mid-june to mid-july is consistent with results from regional larval surveys in the same seasons (Jackson et al. 2008), which showed that maize and sorghum were the principal crop hosts until early July in the mid-south and lower southeast and late July in North Carolina (Jackson et al. 2008). These results also support those of Gould et al. (2002), who demonstrated that most bollworm moths collected during July and early August in Louisiana and Texas from 1997 to 1999 originated from C 4 hosts. There is also agreement between the carbon isotope analyses for the late August to early September period and the larval surveys from mid-july to mid-august through early September (Jackson et al. 2008); both sources of data show that C 3 hosts (cotton, peanuts and soybeans) supported higher numbers of bollworm from mid-july to mid-august. During this period, however, the percentage of bollworm moths with C 4 isotopic compositions ranged from 22% to 48%, with the exception of 2002 collections in Arkansas where 10 26% of moths developed as larvae on C 4 hosts. These results confirm those of Jackson, Bradley & Van Duyn (2003) who demonstrated bollworm larval production from commercial maize as late as 21-August. The increase in the proportion of C 4 moths after early September did not follow the conventional wisdom that moths collected during this period originate primarily from cotton and soybeans (Brazzel et al. 1953). Gould et al. (2002) suggested that these moths with d 13 C compositions characteristic of a C 4 host were likely to be long-distance migrants that originated from maize in the upper Midwest but no evidence to support this hypothesis has been published since its original suggestion. However, weather patterns could allow such migration to occur. This process would carry Bt resistance genes from the maize belt to southern bollworm populations but the effects would likely be offset by the diversity of non-bt hosts in the

8 590 G. Head et al. (a) that non-cotton crop hosts of bollworm contribute significantly to populations throughout the season in the mid-south and southeast (Jackson et al. 2008). Based on bollworm larval productivity estimates for individual states, a minimum of 10 41% of collected moths developed as larvae on C 4 hosts when moth production would be expected from cotton.in addition, the gossypol analyses make clear that a much higher percentage of collected moths originated from non-cotton C 3 hosts because of moth production levels from soybeans, peanuts and other C 3 hosts. Thus, the data presented here and by Jackson et al. (2008) indicate that, at both broad and fine scales, C 4 hosts are driving bollworm population dynamics rather than cotton. Consequently, bollworm populations will be more strongly influenced by management practices in C 4 cropsratherthanincotton. (b) Fig. 4. Synthesis of the stable carbon isotope and gossypol residue data for (a) mid-south and south-eastern states, and (b) North Carolina only, showing seasonal trends in % moths whose natal hosts were cotton, a C 3 crop other than cotton, or a C 4 crop. south and the introduction of two-gene Bt maize products (see below). Another hypothesis considered by Gould et al. (2002) was the existence of one or more unreported native C 4 hosts of bollworm, but this scenario is unlikely as the numbers of moths collected during this period were moderate to high (Figs 3 and 4, Jackson et al. 2008). Another possible explanation is that many of the late-season moths with C 4 isotopic compositions developed as larvae on post-harvest maize and sorghum volunteers (B. R. Leonard, pers. comm.). Overall, the greatest contribution to bollworm populations in all regions and seasons was from the C 4 crops maize and sorghum. This is consistent with observations of related species such as H. armigera in China (Wu, Guo & Gao 2002) where maize and other C 4 crops predominate as hosts compared to cotton. The seasonal homogeneity in C 4 host use by bollworms across the south and south-eastern states of the USA (Figs 1 and 2) indicates also that moth movement is substantial, bringing resistance management benefits through the mating of Bt-selected individuals from cotton with unselected individuals from C 4 crops. These results strongly support those from regional field assessments of bollworm larval productivity from cotton and non-cotton crop hosts, which demonstrated IMPLICATIONS OF ALTERNATIVE HOST USE BY BOLLWORM FOR IRM Bacillus thuringiensis cotton and Bt maize crops have been grown in the USA for 14 years. The initial generation of these insect-protected products expressed a single Bt gene. The first Bt cotton product was Bollgard Ò cotton containing the Cry1Ac protein, giving the crop protection against lepidopteran cotton pests such as H. zea, H. virescens and the pink bollworm Pectinophora gossypiella (Lepidoptera: Gelechiidae). ThefirstBtmaizeproductscontainedtheCry1Abproteinand were targeted against lepidopteran maize pests such as the European corn borer Ostrinia nubilalis (Lepidoptera: Crambidae). A subsequent generation of Bt crops has since been released in the USA that express two or more Bt genes each targeted at these same lepidopteran pest complexes in cotton and maize (so-called pyramids). In 2003, Bollgard II Ò cotton was introduced which contains the Cry1Ac protein present in Bollgard along with an additional Bt protein Cry2Ab2. Similarly, in 2005 Widestrike Ò cotton, containing the Cry1Ac and Cry1F proteins, was released. In 2008, YieldGard VT Pro maize, which contains the Cry1A.105 and Cry2Ab2 proteins, was approved for commercial use (also known as event MON 89034; EPA Registration Number , 10 June 2008, Currently, in the US, almost all Bt cotton expresses two cry genes, being either Bollgard II or Widestrike, and the original single gene Bollgard cotton will be completely phased out by the end of A comparable process is underway in maize; the majority of US maize currently contains at least the cry1ab gene but pyramided products are expected to replace single gene products within 3 5 years. Studies of cross-resistance between the various Cry proteins in the CBW (Anilkumar et al. 2008) indicate that cross-resistance could be expected between Cry1Ab and Cry1Ac but not to the Cry2A toxins because of differences in amino acid sequences and modes of action. This lack of cross-resistance between Cry1Ac and Cry2 proteins has also been found in H. virescens, H. armigera and P. gossypiella, leading Anilkumar et al. (2008) to conclude that the two Cry genes pyramided

9 Host use by cotton bollworms 591 in Bollgard II cotton should be a viable approach to managing potential resistance to Cry1Ac. However, given the cross-resistance linkage for Cry1Ab and Cry1Ac in H. zea, selection for resistance to Cry1Ac in the US Cotton Belt could come from either direct selection in cotton or from indirect selection in maize from the use of products containing Cry1Ab. For a pest such as the CBW, which utilizes both hosts but prefers maize and for much of the season breeds much more extensively on maize then cotton (Jackson et al. 2008) the Cry1 selection pressure should be greater from maize than cotton, other things being equal. Collectively, the greater area of maize than cotton and the greater bollworm productivity on maize than cotton are the likely causes of the patterns reported here. Larval production estimates (Jackson et al. 2008) and the data presented here demonstrate that non-cotton crop hosts produce a much greater proportion of the Bt-susceptible moths than do structured cotton refuges. Likewise, any bollworm management in cotton will have a minor impact on Bt resistance and population dynamics of bollworms compared to management in maize and sorghum. Based in part on these data, the US EPA determined that structured non-bt cotton refuges would not be required for pyramided cotton products including Bollgard II and WideStrike in regions of the USA where heliothines are the primary target pest because of the contribution of non-cotton host plants as sources of refuge. Similar findings were encountered in China where Helicoverpa armigera production from non-cotton hosts such as maize, peanuts, wheat and soybeans was shown to provide sufficient numbers of Bt-susceptible individuals to effectively delay resistance to Bt cotton, despite near 100% adoption of Bt cottonwiththecry1ac gene and no use of non-bt cotton refuges (Wu et al. 2002; Wu, Guo & Head 2006). Likewise, Ravi et al. (2005) suggested that structured refuges for Bt cotton may not be necessary in India because of the overwhelming contributions of H. armigera from non-cotton crop hosts including the pulses pigeon pea Cajanus cajan and chickpea Cicer arietinum, and grains such as maize and sorghum. Thus, it appears that the population dynamics and Bt-resistance status of Helicoverpa species in these cases is driven primarily by crop hosts other than cotton, and that non-bt cotton refuges will be relatively unimportant for Bt resistance management. These data point to the importance of rigorous studies of target pest host use and population ecology to underpin decisions on implementation strategies for insecticide-resistance management. ThebroadhostusebybollwormsintheUSA,andtherapid introduction of pyramided Bt cotton products, makes it unsurprising that no change in the efficacy of Bt cotton has been detected in the USA over the past 14 years (Moar et al. 2008). However, based on laboratory assays conducted before and after the introduction of Bt cotton in the US, Tabashnik et al. (2008a,b) have claimed that Cry1Ac resistance is present in bollworm populations from the cotton belt. The data presented here on alternative host use, and the relatively low use of Bt maize in the cotton belt, make the claims of Tabashnik et al. (2008a,b) unlikely. Furthermore, these claims have been disputed by Moar et al. (2008) on methodological grounds and because the variability in response interpreted by Tabashnik et al. (2008a,b) as resistance has not been observed to lead to any change in the efficacy of Bt cotton. On the other hand, where Bt cultivars are released in maize and other major crop hosts of Helicoverpa species, in-crop resistance management will be an important issue. For example, high levels of adoption of Bt maize in the US, particularly in southern regions where bollworms can over-winter, could result in strong selection pressure for resistance to Cry1A proteins in the CBW. Therefore, resistance management programmes in maize systems including the use of structured non-bt maize refuges will be important for the management of Bt resistance in the bollworm. In recognition of the resistance risk created by the joint selection from Bt maize and Bt cotton, the required refuge for the first-generation single-gene maize products in the USA was 50% in cotton areas compared to 20% in non-cotton areas (EPA 2001). However, as described above, the current trend in the US is for first-generation, single-gene Bt maize products to be replaced by pyramided Bt genes with different modes of action. These pyramided products are recognized as having reduced resistance risk compared with single gene products, particularly where the Bt proteins have a low probability of cross-resistance (Roush 1998; McKenzie 1996; Zhao et al. 2003). Because of this reduced resistance risk, the United States Environmental Protection Agency determined that the appropriate refuge size for MON was 20% in cotton-producing areas, and not 50% as previously set for the single-gene Bt maize products ( reviews/006514/ htm). Acknowledgements The authors would like to thank USDA-ARS and Monsanto Company for project funding. John Rogers, Research Connections and Consulting, Brisbane, Australia, assisted with the preparation of this manuscript. References Ali, M.I., Luttrell, R.G. & Young, S.Y. 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