- Email: [email protected]
The effects of diet on the detection of resistance to Cry1Ac toxin in Australian Helicoverpa armigera Hübner (Lepidoptera: Noctuidae)
Pesticide Biochemistry and Physiology 97 (2010) 55–59
Contents lists available at ScienceDirect
Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
The effects of diet on the detection of resistance to Cry1Ac toxin in Australian Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) Robin V. Gunning a,*, Graham D. Moores b a b
Tamworth Agricultural Institute, New South Wales Department of Industry and Investment, 4 Marsden Park Road, Calala, NSW 2340, Australia Rothamsted Research, Harpenden, Herts AL5 2JQ, UK
a r t i c l e
i n f o
Article history: Received 29 December 2008 Accepted 17 December 2009 Available online 24 December 2009 Keywords: Helicoverpa armigera Cry1Ac Resistance Bollgard II cotton
a b s t r a c t The effects of raw or heat-denatured soybean flour in an artificial diet on the detection of Cry1Ac resistance in Helicoverpa armigera were examined. Resistant neonate larvae reared on denatured soybean flour diet showed resistance factors of 7980 and 16,901 at the LC50 and LC99.9 levels, respectively. By comparison, resistance could not be detected in neonate larvae reared on raw flour diet. Third instar larvae reared on denatured flour diet showed resistance factors of 322 and 21,190 at the LC50 and LC99.9 levels. Resistance was not detected in third instar larvae reared on raw flour diet. There was 68% survival of resistant neonate larvae on Bollgard II cotton leaf feeding assays, compared to 100% mortality in a susceptible strain. We conclude that detection of CRY1Ac resistance in H. armigera from Australia can be masked, if an artificial diet gives chronic exposure to potent, protease inhibitors present in raw soy flour. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction During the last 50 years, world-wide use of synthetic insecticides to control insect pests has led to both insecticide resistance and environmental problems [1]. The introduction of genetically engineered crops incorporating the use of insecticidal proteins of Bacillus thuringiensis (Bt), has provided growers with the opportunity to significantly reduce the use of synthetic insecticides whilst managing key Lepidopteran pests. These transgenic crops, such as cotton, are now grown in many parts of the world. In Australia, China, the Indian sub-continent and Africa, transgenic cottons are largely targeted against the cotton bollworm, Helicoverpa armigera (Hübner). Within Australia, transgenic cotton varieties encoding for the expression of the single Bt delta-endotoxin Cry1Ac (INGARDÒ) for control of Helicoverpa spp. were introduced in 1996. In recognising the potential threat of resistance developing, through the continued exposure of a single endotoxin, INGARD was replaced with a two-gene variety (Bollgard IIÒ), encoded for expression of both Cry1Ac and Cry2Ab toxins. In Australia, Bollgard II was commercially released in 2003. Even with the introduction of two-gene technology the continuous expression of Bt toxins in transgenic cotton presents an enduring threat of resistance selection, a risk increased by variable Cry1Ac expression in Australia [2]. The key to managing any resistance threat is the capacity to detect any change in resistance frequency. Since the introduction of Bt cotton in Australia, there have been various Bt resistance * Corresponding author. Fax: +61 2 6763 1222. E-mail address: [email protected] (R.V. Gunning). 0048-3575/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2009.12.004
monitoring programmes for Helicoverpa spp. on Bt cotton [3–5] and H.T. Dang (unpublished). Laboratory selection has demonstrated the capacity of H. armigera in Australia to develop resistance against Cry1Ac [6] and Cry1Ac resistance has also been detected in a field derived strain [7,8]. Significant H. armigera field resistance to Cry1Ac in transgenic cotton was recently reported from China [9] and field resistance to Cry1Ac in some USA cotton populations of Helicoverpa zea has also been reported [10]. Throughout its history, Australian H. armigera has been shown to evolve a range of mechanisms to confer resistance to insecticides [11–13]. Therefore it is vital that any resistance monitoring programme developed for transgenic crops must not only be able to detect resistance at low frequencies but also be able to detect resistance resulting from any number of potential mechanisms: target-site resistance, through changes in the receptor, a resistance mechanism against toxins based on a systemic immune-induction and metabolic resistance (through increased titres of non-specific esterase in the mid-gut) have been reported for Cry1Ac resistance in Australian H. armigera [6–8,14]. As Bt toxins are ingested by Helicoverpa spp. larvae, feeding bioassays are necessary. In the Australian Bt resistance monitoring programmes for cotton populations of H. armigera, Cry1Ac toxin is administered to larvae in an artificial diet based on either soybean or chickpea flour. However, there is a dichotomy of procedures at this point; some laboratories heat-treat legume components of diets, [7,8] whilst other laboratories involved in Bt resistance monitoring work use or have used, raw soybean or raw chickpea flour in Helicoverpa spp. diets. [3–6,15,16].
56
R.V. Gunning, G.D. Moores / Pesticide Biochemistry and Physiology 97 (2010) 55–59
This work investigates whether denaturing soybean flour or using a raw soybean flour in an artificial diet results in changes to the observed mortality in a CRY1Ac feeding bioassay.
tion). MVP contains a modified Cry1Ac protein produced by a recombinant strain of the bacterium Psuedomonas fluorescens. MVP contains only Cry1Ac toxin in an inert formulation [18,19].
2. Experimental methods
2.4. Diet bioassays
2.1. Insects
For Cry1Ac bioassays, two forms of the artificial diet were prepared. The standard diet (described above) incorporating heatdenatured soybean flour and a diet using raw soybean flour. Feeding bioassays with Cry1Ac, using neonate and third instar larvae were performed using both diets. Cry1Ac was overlayed onto the diet by surface treatment. Neonate and third instar (30–40 mg) larval bioassays were performed in 128-well trays (CD International, USA). Blocks of diet (0.5 g) (made from denatured or non-denatured soybean proteins), were placed into each well and compressed to fit. The resulting surface was approximately 1 cm2. Six concentrations of toxin were used with dilutions made in Triton-X detergent (Roche) (0.1%), to obtain uniform spreading onto the diet [20]. Each well was treated with solution (30 ll). Diet in control wells was treated with Triton-X (0.1%) only. Neonate or third instar larvae were transferred singly into wells, which were then covered with vented adhesive lids (CD International, USA). Thirty larvae were used for each replicate at each dose. The trays were held at 25 °C at 70% RH in natural light. For neonates, mortality was recorded at 7 days. In accordance with previous MVP bioassay methods on Helicoverpa spp. third instar larvae were given fresh, untreated food after 4 days (N.W. Forrester, L.J. Bird, unpublished) and transferred to 32-well trays to allow sufficient space for the larva to grow. Mortality was recorded at 10 days. Larvae were considered to be alive if growth had occurred and they were actively feeding. Data were analysed by probit analysis [21]. In order to relate artificial diet bioassay data to survival on Bt Cry proteins expressed by transgenic cotton, feeding bioassays were carried out on Bollgard II cotton. Neonate resistant and susceptible larvae (not exposed to artificial diet) were placed on 5 cm diameter cotton leaf discs cut from leaves of irrigated, field grown non-Bt cotton (Delta18RF) and irrigated, field grown Bollgard II (Delta 12BRF) expressing both Cry1Ac and Cry2Ab. (Bollgard II was expressing Cry1Ac toxin at a rate of 8–12 ppm and Cry2Ab 55–88 ppm, respectively (EnvirologiX QuantiPlate™ Kits). Thirty-two neonate, first instar larvae were sealed into vented, air tight polystyrene 5 cm diameter petri dishes (Falcon) (4 larvae per petri dish), and allowed to feed on the cotton leaf discs (5 cm diameter) for 4 days (25 °C). Each experiment was replicated 21 times. Initial mortality was assessed at this time. Larvae were considered alive, if actively feeding and growing. Surviving larvae were retained on the standard artificial diet (heat-denatured soybean flour), and further mortality (if any) was assessed 10 days after first exposure to the cotton leaf material. Data were analysed by Prism 5 Software (GraphPad, USA), means and 95% confidence limits were calculated.
A Bt susceptible strain of H. armigera (GR), originally obtained from CSIRO Entomology was used as the susceptible strain for these studies. This strain has been in culture in CSIRO’s Canberra laboratory since the mid 1980s. The resistant strain (BF) was established in the following manner. H. armigera were collected in January 2006, as eggs, from both Bollgard II and conventional cotton at Breeza, NSW (an area where there had been considerable survival of H. armigera on Bollgard II cotton in the 2005/2006 cotton growing season). The strain was maintained on a diet which uses heat-denatured soybean flour, in the laboratory for 12 months without selection and was then selected once on Bollgard II cotton (Sicot 289b), expressing both Cry2Ab and Cry1Ac. Neonate, first instar larvae were sealed into vented, air tight, polystyrene 5 cm diameter petri dishes (Falcon) (4 larvae per petri dish), and allowed to feed on the cotton leaf discs (5 cm diameter) for 3 days (25 °C). Surviving larvae were transferred to artificial diet. Mortality continued to occur up to 10 days after exposure to Bt cotton. In all, there was approximately 90% larval mortality. Survivors were reared to form the ‘BF’ strain, which was maintained in the laboratory without further selection. 2.2. Rearing methods Helicoverpa armigera larvae were reared, on a diet modified from that of Shorey and Hale [17]. The diet was altered by substitution of heat-denatured soybean flour for pinto beans and the addition of wheat germ and propionic acid. The artificial diet comprised (A) soybean flour (Allied Mills) (450 g), roasted in a microwave oven on full power for 5 min, wheat germ (Allied Mills) (120 g), brewers yeast (Phytofoods) (105 g), ascorbic acid (Phytofoods) (10.1 g) and nipagen (3.3 g), sorbic acid (3.3 g), thiabendazole (0.8 g) and streptomycin sulfate (0.2 g) (all obtained from Sigma); (B) agar grade J3 (Gelita) (45 g) and H2O (1200 ml), (C) formaldehyde (40%) (BDH) (6 ml) and H20 (1500 ml); (D) propionic acid mix (propionic acid (42%)/4% phosphoric acid (BDH) (8 ml)). Ingredients (A), (C) and (D) were blended together, the agar and cold water of (B) were mixed and brought to the boil, cooled to 700 °C and then blended with the other ingredients till smooth. The diet was poured into as hallow tray and allowed to set. Helicoverpa armigera were reared in an insectary at 25 °C, 70% relative humidity in natural light. Adults were kept in tall plastic cages with shredded paper at the base and cloth lids (nappy liners), on which the eggs were collected. The moths were fed on a honey solution (5%). The eggs were surface sterilised in a sodium hypochlorite (Coles Supermarkets) solution (0.1%) and allowed to dry. Eggs were sealed into plastic, ventilated containers with a small amount of rearing diet, allowed to hatch and develop to 2nd instar. Second instar larvae were transferred to a block of diet (2 g) in 32-well trays (CD International), sealed with vented, adhesive lids (CD International) and reared to pupation. Pupae were removed from the diet, sterilised in sodium hypochlorite solution (0.1%), dried and transferred to moth cages. 2.3. Toxin Cry1Ac was obtained from the commercial formulation of B. thuringiensis kurstaki MVPIIÒ (Mycogen) (20% ai, Cell Cap (formula-
3. Results Neonate and third instar H. armigera larvae from the susceptible and resistant strains were bioassayed to examine the effects of heat denatured/raw soybean flour diets on toxicity of Cry1Ac. Results of neonate bioassays are shown in Table 1. Compared to the susceptible strain, larvae from the Bollgard selected BF strain bioassayed on diet with heat-denatured soybean flour, had Cry1Ac resistance factors of 7980 and 16,901 at the LC50 and LC99.9 levels, respectively. Resistance, however, could not be detected in neonate larvae bioassayed on raw soybean flour diet where LC50 and LC99.9 levels in the resistant and susceptible strains were not significantly different (P = 0.05). It was notable that raw soybean flour diet
57
R.V. Gunning, G.D. Moores / Pesticide Biochemistry and Physiology 97 (2010) 55–59 Table 1 Effects of heat denatured/raw soybean flour diet on the response of neonate, first instar Cry1Ac-susceptible and resistant (BF) Helicoverpa armigera larvae to Cry1Ac toxin.
a
Strain
Diet
Slope
v2
LC50 (lg/cm2) (95% fiducial limits)
RFa LC50
LC99.9 (lg/cm2) (95% fiducial limits)
RFa LC99.9
Susceptible BF Susceptible BF
Heat denatured Heat denatured Raw Raw
4.16 2.9 2.7 2.5
0.039 1.6 0.70 1.04
0.0000771 (0.000057–0.000104) 0.615 (0.48–0.75) 0.14 (0.096–0.18) 0.18 (0.12–0.26)
1 7980 1 1.2
0.000426 (0.00015–0.00123) 7.2 (3.75–13.5) 1.5 (0.75–4.35) 3.0 (0.56–16.5)
— 16,900 — 2
RF = resistance factor (calculated as the ratio of resistant LC50,99.9/susceptible LC50,99.9).
bioassays were less sensitive than heat-denatured soybean flour diet bioassays, requiring higher doses of Cry1Ac toxin to confer mortality. In the susceptible strain, LC50 and LC99.9 levels using the denatured soybean flour diet were 1816 and 3521 times lower than with the raw soybean flour diet. Third instar bioassays (Table 2) on larvae reared and bioassayed on heat-denatured soybean flour diet, also revealed Cry1Ac resistance in the BF strain. Resistance factors were 322 and 21,190 at the LC50 and LC99.9 levels, respectively. The slope of the log dose probit line for the BF strain was low (1.2), which may have exaggerated the LC99.9. Resistance in the third instar was again not detected on raw soybean flour diet in larvae from the BF strain (BF and susceptible strain LC50 and LC99.9 were not significantly different (P = 0.05). Again, the susceptible strain was more sensitive when bioassayed with heat-denatured soybean flour diet. Doses of Cry1Ac required to kill being 121- (at LC50) and 255-fold (at LC99.9) less than with raw soybean flour diet. Results of first instar H. armigera cotton leaf feeding assays are shown in Fig. 1. There was no significant mortality of resistant or susceptible larvae fed on non-Bt cotton (P = 0.05). On Bollgard II, however, there was 100% mortality of susceptible larvae (susceptible larvae died within 48 h). There was 32% mortality of resistant larvae on Bollgard II cotton.
4. Discussion Bioassay data indicated that resistance to Cry1Ac in Australian H. armigera was only detectable when larvae were bioassayed and reared on an artificial diet in which the soybean flour was heat denatured. The levels of Cry1Ac resistance detected in the Bollgard II cotton selected BF strain were high, 7980 and 16,901 at the LC50 and LC99.9 and 322- and 21,900-fold at the LC 50 and LC99 levels for neonates and third instars, respectively. Soybean and chickpea seeds are protected against herbivores, including insects, by anti-nutritional defence proteins, including serine protease inhibitors. Levels of these protease inhibitors are very high in soybean and chick pea seeds, comprising up to 5% [22,23]. Examples of such inhibitors are Kunitz soybean trypsin inhibitor (STI) and Kunitz chickpea trypsin inhibitor [24,25]. It is possible that it is these protease inhibitors that are responsible for masking the effects of CRY1Ac resistance in raw diet. Mid-gut serine proteases, against which these inhibitors work, are found in a wide variety of Lepidopteran pests including H. armigera, where trypsin is a major protease in the mid-gut [22]. The protease inhibitors are larval growth retardants and in cases of
Fig. 1. Response of Cry1Ac-susceptible (Sus) and Cry1Ac-resistant (BF) neonate, first instar H. armigera larvae to Bollgard II cotton terminal leaves and non-Bt cotton, in leaf feeding assays. Error bar represent 95% confidence intervals.
chronic exposure to the inhibitors, such as feeding on artificial diets containing raw legume seed flours, cause death through inability to digest protein [23,25–27]. To avoid chronic exposure to artificially high levels of protease inhibitors in artificial diets, it is normal to heat-treat legume components of diets to denature the inhibitors [17,28]. However, some laboratories involved in Bt resistance monitoring work use, or have used, raw soybean or raw chickpea flour in Helicoverpa spp. diets. [3–6,15,16]. Apart from toxic effects in raw flour diets, STI and other potent trypsin inhibitors are also known to have synergistic effects with delta endotoxins of B. thuringiensis. This has been shown in Lepidoptera (H. armigera, Manduca sexta, Heliothis virescens, Trichoplusia ni and H. zea), as well as Leptinotarsa decemlineata (Colorado potato beetle) [22,29–33]. It was suggested that the mechanism of synergism might be prevention of toxin degradation into non-toxic derivatives by gut serine proteases [31]. Therefore, diets with high levels of these inhibitors, not found in cotton, could prevent the detection of Bt resistance. Helicoverpa armigera are naturally exposed to protease inhibitors in crops. It could be argued, therefore, that resistance detected here on heat-denatured soybean flour diet would not be present on a Bt transgenic cotton diet. However, cotton is not reported to express unusually potent protease inhibitors and concentrations of
Table 2 Effects of heat denatured/raw soybean flour diet on the response of third instar Cry1Ac-susceptible and -resistant (BF) Helicoverpa armigera larvae to Cry1Ac toxin.
a
Strain
Diet
Slope
v2
LC50 (lg/cm2) (95% fiducial limits)
RFa LC50
LC99.9 (lg/cm2) (95% fiducial limits)
RFa LC99.9
Susceptible BF Susceptible BF
Heat denatured Heat denatured Raw Raw
3.0 1.2 2.2 2.5
2.5 9.6 5.8 0.38
0.27 (0.23–0.36) 87 (55–267) 32.7 (22.5–45.0) 43.7 (27–67.5)
1 332 1 1.3
3.15 (1.65–5.85) 66750 (6750–675150) 806 (245–2655) 795 (216–2917)
1 21190 1 0.99
RF = resistance factor (calculated as the ratio of resistant LC50,
99.9/susceptible
LC50,
99.9).
58
R.V. Gunning, G.D. Moores / Pesticide Biochemistry and Physiology 97 (2010) 55–59
protease inhibitors in raw legume flour are vastly greater than those present in plant tissue of crops. Furthermore, there was some 68% survival of resistant H. armigera larvae on Bollgard II leaf feeding assays. Survival on Bollgard II indicates that there could be considerable field significance, in the observed levels of Cry1Ac resistance detected in diet bioassays. Survival on Bollgard II cotton also indicates resistance to Cry2Ab in the BF strain. The significance of larvae surviving on leaf bioassays and the potential of metabolic resistance to both Cry1Ac and Cry2Ab should form the subject of further study into the impact of raw flour diets on resistance detection. STI and other trypsin inhibitors are well known to have inhibitory effects on mid-gut enzymes in H. armigera [25–27]. Therefore the inability to detect Cry1Ac resistance in the BF strain on a raw soybean flour diet may be due to the inhibition of an enzymatic metabolic resistance mechanism by protease inhibitors such as STI. Insecticide synergists act by inhibiting metabolic enzymes and STI and other protease inhibitors are known to be synergists of Bt toxins, in species including H. zea and H. armigera [22,29– 33]. Thus in this case, it may be that STI or similar protease inhibitor is also acting synergistically with Cry1Ac to suppress resistance in the BF strain. The ability of soybean serine protease inhibitors to potentiate Bt toxins has led to an interest in engineering unrelated crop plants without potent inhibitors, such as Bt cotton, to express high levels of inhibitors such as STI as a measure to prevent or ameliorate resistance to Bt toxins [21,31,32]. Non-specific esterases, derived from cell adhesion proteins, are serine hydrolases with an ability to sequester, Cry1Ac pro-toxin and activated toxin in Australian H. armigera [7]. This resistance to Cry1Ac, can be synergised by esterase inhibitors [8] and such synergism of Cry1Ac by an esterase inhibitor has also been reported in Cry1Ac-resistant diamondback moth [34]. It is possible that these non-specific esterases, found in the H. armigera midgut, are inhibited by dietary protease inhibitors found in a raw soybean flour diet. The other major effect of using a raw soybean flour diet was that the doses of Cry1Ac required for mortality in the susceptible strain were much higher than using a heat-denatured soybean flour diet, indicating that the Cry1Ac bioassay using heat-denatured soybean flour diet is more sensitive. The neonate LC50 on raw soybean flour diet was some 1816 times higher than on heat-denatured soybean flour diet and in third instars, the LC50 on raw soybean flour diet was 121 times higher than on heat-denatured soybean flour diet. The susceptible neonate LC99.9 on raw soybean flour diet (1.5 lg/cm2) was comparable with the Cry1Ac discriminating dose (susceptible LC96 of 0.25 lg/cm2) used by current Australian H. armigera Cry1Ac resistance monitoring programmes (which use raw flour diets) [5]. The greater sensitivity of H. armigera when using the Cry1Ac bioassay on heat-denatured soy flour diet could be due to destruction of protease inhibitors that would otherwise inhibit the proteases required to activate Cry toxins in the mid-gut, as STI is known to inhibit proteases that activate Cry pro-toxins in H. armigera, resulting in much-reduced levels of activated toxin [29]. This study has indicated that rearing and bioassay of H. armigera on diets that give chronic exposure to abnormally high levels of active legume protease inhibitors may be unsuitable for detection of a metabolic-based resistance to Cry1Ac in cotton populations of H. armigera in Australia. Bird and Ackhurst have previously suggested that greater susceptibility of cotton populations of H. armigera to Cry1Ac, compared to Helicoverpa punctigera, in Australia, might have been due to dietary STI inhibition of detoxificative enzymes in H. armigera [4]. This work may have important implications for the monitoring of H. armigera resistance to Bt toxins in Australia. This study has also generated some important avenues for continuing research,
as of course more data are needed to establish a causal relationship between xenobiotics in raw legume flour, mid-gut enzymes and Bt resistance in Australian H. armigera.
Acknowledgments The authors would like to acknowledge the support of the New South Wales Department of Primary Industries. Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.
References [1] R.T. Roush, B.E. Tabashnik (Eds.), Pesticide Resistance in Arthropods, Chapman and Hall, New York, 1990. [2] K.M. Olsen, J.C. Daly, E.J. Finnegan, R.J. Mahon, Changes in Cry1Ac Bt transgenic cotton in response to two environmental factors: temperature and insect damage, J. Econ. Entomol. 98 (2005) 1382–1390. [3] N.W. Forrester, L.J. Bird, Bt resistance assays for Australian Helicoverpa armigera. In: M.P. Zalucki, R.A.I. Drew, G.W. White (Eds.), Proceedings of the Sixth Australasian Applied Entomological Research Conference, Brisbane, 1998, pp. 145–148. [4] L.J. Bird, R.J. Akhurst, Variation in susceptibility of Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) (Lepidoptera; Noctuidae) to two Bacillus thuringiensis toxins, J. Invert. Path. 94 (2007) 84–94. [5] R.J. Mahon, K.M. Olsen, S. Downs, S. Addison, Frequencies of alleles conferring resistance to the Bt toxins Cry1Ac and Cry2Ab in Australian populations of Helicoverpa armigera (Lepidoptera: Noctuidae), J. Econ. Entomol. 100 (2007) 844–1853. [6] R.J. Akhurst, W. James, L.J. Bird, C. Beard, Resistance to the Cry1Ac d-endotoxin of Bacillus thuringiensis in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae), J. Econ. Entomol. 96 (2003) 1290–1298. [7] R.V. Gunning, H.T. Dang, F.C. Kemp, I.C. Nicholson, G.D. Moores, New resistance mechanism in Helicoverpa armigera threatens transgenic crops expressing Bacillus thuringiensis Cry1Ac toxin, Appl. Environ. Microbiol. 71 (2005) 2558– 2563. [8] R.V. Gunning, I.C. Nicholson, F.C. Kemp, V. Borzatta, E.L.A. Cottage, L.M. Field, G.D. Moores, Piperonyl butoxide, restores the efficacy of Bacillus thuringiensis toxin in transgenic cotton against resistant Helicoverpa armigera, Biopestic. Int. 2 (2006) 39–136. [9] F. Liu, Z. Xu, J. Chang, J. Chen, F. Meng, Y.C. Zhu, J. Shen, Resistance allele frequency to Bt cotton in field populations of Helicoverpa armigera (Lepidoptera: Noctuidae) in China, J. Econ. Entomol. 101 (2008) 933–943. [10] B.E. Tabashnik, A.J. Gassmann, D.W. Crowder, Y. Carrière, Insect resistance to Bt crops: evidence versus theory, Nat. Biotechnol. 26 (2008) 199–202. [11] R.V. Gunning, C.S. Easton, M.E. Balfe, I.G. Ferris, Pyrethroid resistance mechanisms in Australian Helicoverpa armigera, Pestic. Sci. 33 (1991) 473–490. [12] R.V. Gunning, G.D. Moores, A.L. Devonshire, Esterases and fenvalerate resistance in Australian Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), Pestic. Biochem. Physiol. 54 (1996) 12–23. [13] R.V. Gunning, G.D. Moores, A.L. Devonshire, Insensitive acetylcholine esterase and resistance to thiodicarb in Australian Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), Pestic. Biochem. Physiol. 55 (1996) 21–28. [14] G. Ma, H. Roberts, M. Sarjan, N. Featherstone, J. Lahnstein, R. Akhurst, O. Schmidt, Is the mature endotoxin Cry1Ac from Bacillus thuringiensis inactivated by a coagulation reaction in the gut lumen of resistant Helicoverpa armigera larvae?, Insect Biochem Mol. Biol. 35 (2005) 729–739. [15] N.W. Forrester, M. Cahill, L.J. Bird, J.K. Layland, Management of pyrethroid and endosulfan resistance in Helicoverpa armigera (Lepidoptera: Noctuidae) in Australia, Bull. Ent. Res. Suppl. Ser. (Suppl.1) (1993) 1–132. [16] K.M. Olsen, J.C. Daly, Plant-toxin interactions in transgenic Bt cotton and their effect on mortality of Helicoverpa armigera (Lepidoptera: Noctuidae), J. Econ. Entomol. 93 (2000) 1293–1299. [17] H.H. Shorey, R.L. Hale, Mass rearing of larvae of nine noctuid species on a simple artificial medium, J. Econ. Entomol. 58 (1965) 522–524. [18] F.A. Gould, A. Anderson, L. Reynolds, L. Bumgardner, W. Moar, Selection and genetic analysis of a Heliothis virescens (Lepidoptera: Noctuidae) strain with a high level of resistance to Bacillus thuringiensis toxins, J. Econ. Entomol. 88 (1995) 1545–1559. [19] K.A. Anilkumar, W.J. Moar, Difference between rate of resistance development to Bt Cry1Ac toxin in cotton bollworm, Helicoverpa zea (Boddie) when selected using MVP II and activated toxin, in: Proceedings of 2006 Beltwide Cotton Conferences, San Antonio, Texas, January 3–6, 2006, pp 1478–1482. [20] P.C. Marçon, I.J. Young, K.L. Steffey, B.D. Siegfried, Baseline susceptibility of European corn borer (Lepidoptera: Crambidae) to Bacillus thuringiensis toxins, J. Econ. Entomol. 92 (1999) 279–285. [21] D.J. Finney, Probit Analysis, Cambridge University Press, London, 1971. [22] S.C. Macintosh, G.M. Kishore, F.J. Perlak, P.G. Marrone, T.B. Stone, S.R. Sims, R.L. Fuchs, Potentiation of Bacillus thuringiensis insecticidal activity by serine protease inhibitors, J. Agric. Food Chem. 38 (1990) 1145–1152.
R.V. Gunning, G.D. Moores / Pesticide Biochemistry and Physiology 97 (2010) 55–59 [23] K.A. Jonhston, J.A. Gatehouse, J.H. Anstee, Effects of soybean protease inhibitors on the growth and development of larval Helicoverpa armigera, J. Insect Physiol. 39 (1993) .657–664. [24] M. Kunitz, Crystallization of a trypsin inhibitor from soybean, Science 101 (1945) 668–669. [25] A. Srinivasan, A.P. Giri, A.M. Harsulkar, J.A. Gatehouse, V.S. Gupta, A kunitz trypsin inhibitor from chickpea (Cier arietinum L.) that exerts anti-metabolic effect on podborer (Helicoverpa armigera) larvae, Plant Mol. Biol. 57 (2005) 359–374. [26] C.Z. Wang, X.F. Xiang, S.F. Zhang, J.D. Qin, Effect of soybean trypsin inhibitor on the growth and digestive physiology of Helicoverpa armigera larvae, Acta Entomol. Sin. 29 (1995) 35–39. [27] R.M. Broadway, S.S. Duffey, Plant proteinase inhibitors: mechanism of action and effect on larval growth and digestive physiology of larval Heliothis zea and Spodoptera exiqua, J. Insect Physiol. 32 (1986) 827– 833. [28] R.E. Teakle, J.M. Jensen, Heliothis punctiger, in: R. Singh, R.F. Moore (Eds.), Handbook of Insect Rearing, vol. 2, Elsevier, Amsterdam, 1985, pp. 312–322.
59
[29] Z. Shao, Y. Cui, X. Liu, H. Yi, J. Ji, Z. Yu, Processing of d-endotoxin of Bacillus thuringiensis subsp. Kurstaki HD-1 in Heliothis armigera midgut juice and the effects of protease inhibitors, J. Invert. Path. 72 (1998) 3–81. [30] J. Zhang, C. Wang, J. Qin, The interactions between soybean trypsin inhibitor and d-endotoxin of Bacillus thuringiensis in Helicoverpa armigera larva, J. Invert. Path. 75 (2000) 259–266. [31] Y.C. Zhu, C.A. Abel, M.S. Chen, Interaction of Cry1Ac toxin (Bacillus thuringiensis) and proteinase inhibitors on the growth, development, and midgut proteinase activities of the bollworm Helicoverpa zea, Pestic. Biochem. Physiol. 87 (2007) 39–46. [32] J.T. Christtellier, W.A. Laing, N.P. Markwick, E.J.P. Burgess, Midgut protease activities in 12 phytophagous Lepidopteran larvae: dietary and protease inhibitor interactions, Insect Biochem. Mol. Biol. 22 (1992) 735–746. [33] J. Hubert, M. Nesvorna, R. Zemek, J. Stara, V. Stejskal, Effects of metabolic inhibitors on activity of Cry1Ab toxin to inhibit growth of Ephestia kuehniella larvae, Pest Manag. Sci. 64 (2008) 1063–1068. [34] A.H. Sayyed, G.D. Moores, N. Crickmore, D.J. Wright, Cross-resistance between a Bacillus thuringiensis Cry toxin and non-Bt insecticides in the diamondback moth, Pest. Manag. Sci. 64 (2008) 813–819.
Contents lists available at ScienceDirect
Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
The effects of diet on the detection of resistance to Cry1Ac toxin in Australian Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) Robin V. Gunning a,*, Graham D. Moores b a b
Tamworth Agricultural Institute, New South Wales Department of Industry and Investment, 4 Marsden Park Road, Calala, NSW 2340, Australia Rothamsted Research, Harpenden, Herts AL5 2JQ, UK
a r t i c l e
i n f o
Article history: Received 29 December 2008 Accepted 17 December 2009 Available online 24 December 2009 Keywords: Helicoverpa armigera Cry1Ac Resistance Bollgard II cotton
a b s t r a c t The effects of raw or heat-denatured soybean flour in an artificial diet on the detection of Cry1Ac resistance in Helicoverpa armigera were examined. Resistant neonate larvae reared on denatured soybean flour diet showed resistance factors of 7980 and 16,901 at the LC50 and LC99.9 levels, respectively. By comparison, resistance could not be detected in neonate larvae reared on raw flour diet. Third instar larvae reared on denatured flour diet showed resistance factors of 322 and 21,190 at the LC50 and LC99.9 levels. Resistance was not detected in third instar larvae reared on raw flour diet. There was 68% survival of resistant neonate larvae on Bollgard II cotton leaf feeding assays, compared to 100% mortality in a susceptible strain. We conclude that detection of CRY1Ac resistance in H. armigera from Australia can be masked, if an artificial diet gives chronic exposure to potent, protease inhibitors present in raw soy flour. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction During the last 50 years, world-wide use of synthetic insecticides to control insect pests has led to both insecticide resistance and environmental problems [1]. The introduction of genetically engineered crops incorporating the use of insecticidal proteins of Bacillus thuringiensis (Bt), has provided growers with the opportunity to significantly reduce the use of synthetic insecticides whilst managing key Lepidopteran pests. These transgenic crops, such as cotton, are now grown in many parts of the world. In Australia, China, the Indian sub-continent and Africa, transgenic cottons are largely targeted against the cotton bollworm, Helicoverpa armigera (Hübner). Within Australia, transgenic cotton varieties encoding for the expression of the single Bt delta-endotoxin Cry1Ac (INGARDÒ) for control of Helicoverpa spp. were introduced in 1996. In recognising the potential threat of resistance developing, through the continued exposure of a single endotoxin, INGARD was replaced with a two-gene variety (Bollgard IIÒ), encoded for expression of both Cry1Ac and Cry2Ab toxins. In Australia, Bollgard II was commercially released in 2003. Even with the introduction of two-gene technology the continuous expression of Bt toxins in transgenic cotton presents an enduring threat of resistance selection, a risk increased by variable Cry1Ac expression in Australia [2]. The key to managing any resistance threat is the capacity to detect any change in resistance frequency. Since the introduction of Bt cotton in Australia, there have been various Bt resistance * Corresponding author. Fax: +61 2 6763 1222. E-mail address: [email protected] (R.V. Gunning). 0048-3575/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2009.12.004
monitoring programmes for Helicoverpa spp. on Bt cotton [3–5] and H.T. Dang (unpublished). Laboratory selection has demonstrated the capacity of H. armigera in Australia to develop resistance against Cry1Ac [6] and Cry1Ac resistance has also been detected in a field derived strain [7,8]. Significant H. armigera field resistance to Cry1Ac in transgenic cotton was recently reported from China [9] and field resistance to Cry1Ac in some USA cotton populations of Helicoverpa zea has also been reported [10]. Throughout its history, Australian H. armigera has been shown to evolve a range of mechanisms to confer resistance to insecticides [11–13]. Therefore it is vital that any resistance monitoring programme developed for transgenic crops must not only be able to detect resistance at low frequencies but also be able to detect resistance resulting from any number of potential mechanisms: target-site resistance, through changes in the receptor, a resistance mechanism against toxins based on a systemic immune-induction and metabolic resistance (through increased titres of non-specific esterase in the mid-gut) have been reported for Cry1Ac resistance in Australian H. armigera [6–8,14]. As Bt toxins are ingested by Helicoverpa spp. larvae, feeding bioassays are necessary. In the Australian Bt resistance monitoring programmes for cotton populations of H. armigera, Cry1Ac toxin is administered to larvae in an artificial diet based on either soybean or chickpea flour. However, there is a dichotomy of procedures at this point; some laboratories heat-treat legume components of diets, [7,8] whilst other laboratories involved in Bt resistance monitoring work use or have used, raw soybean or raw chickpea flour in Helicoverpa spp. diets. [3–6,15,16].
56
R.V. Gunning, G.D. Moores / Pesticide Biochemistry and Physiology 97 (2010) 55–59
This work investigates whether denaturing soybean flour or using a raw soybean flour in an artificial diet results in changes to the observed mortality in a CRY1Ac feeding bioassay.
tion). MVP contains a modified Cry1Ac protein produced by a recombinant strain of the bacterium Psuedomonas fluorescens. MVP contains only Cry1Ac toxin in an inert formulation [18,19].
2. Experimental methods
2.4. Diet bioassays
2.1. Insects
For Cry1Ac bioassays, two forms of the artificial diet were prepared. The standard diet (described above) incorporating heatdenatured soybean flour and a diet using raw soybean flour. Feeding bioassays with Cry1Ac, using neonate and third instar larvae were performed using both diets. Cry1Ac was overlayed onto the diet by surface treatment. Neonate and third instar (30–40 mg) larval bioassays were performed in 128-well trays (CD International, USA). Blocks of diet (0.5 g) (made from denatured or non-denatured soybean proteins), were placed into each well and compressed to fit. The resulting surface was approximately 1 cm2. Six concentrations of toxin were used with dilutions made in Triton-X detergent (Roche) (0.1%), to obtain uniform spreading onto the diet [20]. Each well was treated with solution (30 ll). Diet in control wells was treated with Triton-X (0.1%) only. Neonate or third instar larvae were transferred singly into wells, which were then covered with vented adhesive lids (CD International, USA). Thirty larvae were used for each replicate at each dose. The trays were held at 25 °C at 70% RH in natural light. For neonates, mortality was recorded at 7 days. In accordance with previous MVP bioassay methods on Helicoverpa spp. third instar larvae were given fresh, untreated food after 4 days (N.W. Forrester, L.J. Bird, unpublished) and transferred to 32-well trays to allow sufficient space for the larva to grow. Mortality was recorded at 10 days. Larvae were considered to be alive if growth had occurred and they were actively feeding. Data were analysed by probit analysis [21]. In order to relate artificial diet bioassay data to survival on Bt Cry proteins expressed by transgenic cotton, feeding bioassays were carried out on Bollgard II cotton. Neonate resistant and susceptible larvae (not exposed to artificial diet) were placed on 5 cm diameter cotton leaf discs cut from leaves of irrigated, field grown non-Bt cotton (Delta18RF) and irrigated, field grown Bollgard II (Delta 12BRF) expressing both Cry1Ac and Cry2Ab. (Bollgard II was expressing Cry1Ac toxin at a rate of 8–12 ppm and Cry2Ab 55–88 ppm, respectively (EnvirologiX QuantiPlate™ Kits). Thirty-two neonate, first instar larvae were sealed into vented, air tight polystyrene 5 cm diameter petri dishes (Falcon) (4 larvae per petri dish), and allowed to feed on the cotton leaf discs (5 cm diameter) for 4 days (25 °C). Each experiment was replicated 21 times. Initial mortality was assessed at this time. Larvae were considered alive, if actively feeding and growing. Surviving larvae were retained on the standard artificial diet (heat-denatured soybean flour), and further mortality (if any) was assessed 10 days after first exposure to the cotton leaf material. Data were analysed by Prism 5 Software (GraphPad, USA), means and 95% confidence limits were calculated.
A Bt susceptible strain of H. armigera (GR), originally obtained from CSIRO Entomology was used as the susceptible strain for these studies. This strain has been in culture in CSIRO’s Canberra laboratory since the mid 1980s. The resistant strain (BF) was established in the following manner. H. armigera were collected in January 2006, as eggs, from both Bollgard II and conventional cotton at Breeza, NSW (an area where there had been considerable survival of H. armigera on Bollgard II cotton in the 2005/2006 cotton growing season). The strain was maintained on a diet which uses heat-denatured soybean flour, in the laboratory for 12 months without selection and was then selected once on Bollgard II cotton (Sicot 289b), expressing both Cry2Ab and Cry1Ac. Neonate, first instar larvae were sealed into vented, air tight, polystyrene 5 cm diameter petri dishes (Falcon) (4 larvae per petri dish), and allowed to feed on the cotton leaf discs (5 cm diameter) for 3 days (25 °C). Surviving larvae were transferred to artificial diet. Mortality continued to occur up to 10 days after exposure to Bt cotton. In all, there was approximately 90% larval mortality. Survivors were reared to form the ‘BF’ strain, which was maintained in the laboratory without further selection. 2.2. Rearing methods Helicoverpa armigera larvae were reared, on a diet modified from that of Shorey and Hale [17]. The diet was altered by substitution of heat-denatured soybean flour for pinto beans and the addition of wheat germ and propionic acid. The artificial diet comprised (A) soybean flour (Allied Mills) (450 g), roasted in a microwave oven on full power for 5 min, wheat germ (Allied Mills) (120 g), brewers yeast (Phytofoods) (105 g), ascorbic acid (Phytofoods) (10.1 g) and nipagen (3.3 g), sorbic acid (3.3 g), thiabendazole (0.8 g) and streptomycin sulfate (0.2 g) (all obtained from Sigma); (B) agar grade J3 (Gelita) (45 g) and H2O (1200 ml), (C) formaldehyde (40%) (BDH) (6 ml) and H20 (1500 ml); (D) propionic acid mix (propionic acid (42%)/4% phosphoric acid (BDH) (8 ml)). Ingredients (A), (C) and (D) were blended together, the agar and cold water of (B) were mixed and brought to the boil, cooled to 700 °C and then blended with the other ingredients till smooth. The diet was poured into as hallow tray and allowed to set. Helicoverpa armigera were reared in an insectary at 25 °C, 70% relative humidity in natural light. Adults were kept in tall plastic cages with shredded paper at the base and cloth lids (nappy liners), on which the eggs were collected. The moths were fed on a honey solution (5%). The eggs were surface sterilised in a sodium hypochlorite (Coles Supermarkets) solution (0.1%) and allowed to dry. Eggs were sealed into plastic, ventilated containers with a small amount of rearing diet, allowed to hatch and develop to 2nd instar. Second instar larvae were transferred to a block of diet (2 g) in 32-well trays (CD International), sealed with vented, adhesive lids (CD International) and reared to pupation. Pupae were removed from the diet, sterilised in sodium hypochlorite solution (0.1%), dried and transferred to moth cages. 2.3. Toxin Cry1Ac was obtained from the commercial formulation of B. thuringiensis kurstaki MVPIIÒ (Mycogen) (20% ai, Cell Cap (formula-
3. Results Neonate and third instar H. armigera larvae from the susceptible and resistant strains were bioassayed to examine the effects of heat denatured/raw soybean flour diets on toxicity of Cry1Ac. Results of neonate bioassays are shown in Table 1. Compared to the susceptible strain, larvae from the Bollgard selected BF strain bioassayed on diet with heat-denatured soybean flour, had Cry1Ac resistance factors of 7980 and 16,901 at the LC50 and LC99.9 levels, respectively. Resistance, however, could not be detected in neonate larvae bioassayed on raw soybean flour diet where LC50 and LC99.9 levels in the resistant and susceptible strains were not significantly different (P = 0.05). It was notable that raw soybean flour diet
57
R.V. Gunning, G.D. Moores / Pesticide Biochemistry and Physiology 97 (2010) 55–59 Table 1 Effects of heat denatured/raw soybean flour diet on the response of neonate, first instar Cry1Ac-susceptible and resistant (BF) Helicoverpa armigera larvae to Cry1Ac toxin.
a
Strain
Diet
Slope
v2
LC50 (lg/cm2) (95% fiducial limits)
RFa LC50
LC99.9 (lg/cm2) (95% fiducial limits)
RFa LC99.9
Susceptible BF Susceptible BF
Heat denatured Heat denatured Raw Raw
4.16 2.9 2.7 2.5
0.039 1.6 0.70 1.04
0.0000771 (0.000057–0.000104) 0.615 (0.48–0.75) 0.14 (0.096–0.18) 0.18 (0.12–0.26)
1 7980 1 1.2
0.000426 (0.00015–0.00123) 7.2 (3.75–13.5) 1.5 (0.75–4.35) 3.0 (0.56–16.5)
— 16,900 — 2
RF = resistance factor (calculated as the ratio of resistant LC50,99.9/susceptible LC50,99.9).
bioassays were less sensitive than heat-denatured soybean flour diet bioassays, requiring higher doses of Cry1Ac toxin to confer mortality. In the susceptible strain, LC50 and LC99.9 levels using the denatured soybean flour diet were 1816 and 3521 times lower than with the raw soybean flour diet. Third instar bioassays (Table 2) on larvae reared and bioassayed on heat-denatured soybean flour diet, also revealed Cry1Ac resistance in the BF strain. Resistance factors were 322 and 21,190 at the LC50 and LC99.9 levels, respectively. The slope of the log dose probit line for the BF strain was low (1.2), which may have exaggerated the LC99.9. Resistance in the third instar was again not detected on raw soybean flour diet in larvae from the BF strain (BF and susceptible strain LC50 and LC99.9 were not significantly different (P = 0.05). Again, the susceptible strain was more sensitive when bioassayed with heat-denatured soybean flour diet. Doses of Cry1Ac required to kill being 121- (at LC50) and 255-fold (at LC99.9) less than with raw soybean flour diet. Results of first instar H. armigera cotton leaf feeding assays are shown in Fig. 1. There was no significant mortality of resistant or susceptible larvae fed on non-Bt cotton (P = 0.05). On Bollgard II, however, there was 100% mortality of susceptible larvae (susceptible larvae died within 48 h). There was 32% mortality of resistant larvae on Bollgard II cotton.
4. Discussion Bioassay data indicated that resistance to Cry1Ac in Australian H. armigera was only detectable when larvae were bioassayed and reared on an artificial diet in which the soybean flour was heat denatured. The levels of Cry1Ac resistance detected in the Bollgard II cotton selected BF strain were high, 7980 and 16,901 at the LC50 and LC99.9 and 322- and 21,900-fold at the LC 50 and LC99 levels for neonates and third instars, respectively. Soybean and chickpea seeds are protected against herbivores, including insects, by anti-nutritional defence proteins, including serine protease inhibitors. Levels of these protease inhibitors are very high in soybean and chick pea seeds, comprising up to 5% [22,23]. Examples of such inhibitors are Kunitz soybean trypsin inhibitor (STI) and Kunitz chickpea trypsin inhibitor [24,25]. It is possible that it is these protease inhibitors that are responsible for masking the effects of CRY1Ac resistance in raw diet. Mid-gut serine proteases, against which these inhibitors work, are found in a wide variety of Lepidopteran pests including H. armigera, where trypsin is a major protease in the mid-gut [22]. The protease inhibitors are larval growth retardants and in cases of
Fig. 1. Response of Cry1Ac-susceptible (Sus) and Cry1Ac-resistant (BF) neonate, first instar H. armigera larvae to Bollgard II cotton terminal leaves and non-Bt cotton, in leaf feeding assays. Error bar represent 95% confidence intervals.
chronic exposure to the inhibitors, such as feeding on artificial diets containing raw legume seed flours, cause death through inability to digest protein [23,25–27]. To avoid chronic exposure to artificially high levels of protease inhibitors in artificial diets, it is normal to heat-treat legume components of diets to denature the inhibitors [17,28]. However, some laboratories involved in Bt resistance monitoring work use, or have used, raw soybean or raw chickpea flour in Helicoverpa spp. diets. [3–6,15,16]. Apart from toxic effects in raw flour diets, STI and other potent trypsin inhibitors are also known to have synergistic effects with delta endotoxins of B. thuringiensis. This has been shown in Lepidoptera (H. armigera, Manduca sexta, Heliothis virescens, Trichoplusia ni and H. zea), as well as Leptinotarsa decemlineata (Colorado potato beetle) [22,29–33]. It was suggested that the mechanism of synergism might be prevention of toxin degradation into non-toxic derivatives by gut serine proteases [31]. Therefore, diets with high levels of these inhibitors, not found in cotton, could prevent the detection of Bt resistance. Helicoverpa armigera are naturally exposed to protease inhibitors in crops. It could be argued, therefore, that resistance detected here on heat-denatured soybean flour diet would not be present on a Bt transgenic cotton diet. However, cotton is not reported to express unusually potent protease inhibitors and concentrations of
Table 2 Effects of heat denatured/raw soybean flour diet on the response of third instar Cry1Ac-susceptible and -resistant (BF) Helicoverpa armigera larvae to Cry1Ac toxin.
a
Strain
Diet
Slope
v2
LC50 (lg/cm2) (95% fiducial limits)
RFa LC50
LC99.9 (lg/cm2) (95% fiducial limits)
RFa LC99.9
Susceptible BF Susceptible BF
Heat denatured Heat denatured Raw Raw
3.0 1.2 2.2 2.5
2.5 9.6 5.8 0.38
0.27 (0.23–0.36) 87 (55–267) 32.7 (22.5–45.0) 43.7 (27–67.5)
1 332 1 1.3
3.15 (1.65–5.85) 66750 (6750–675150) 806 (245–2655) 795 (216–2917)
1 21190 1 0.99
RF = resistance factor (calculated as the ratio of resistant LC50,
99.9/susceptible
LC50,
99.9).
58
R.V. Gunning, G.D. Moores / Pesticide Biochemistry and Physiology 97 (2010) 55–59
protease inhibitors in raw legume flour are vastly greater than those present in plant tissue of crops. Furthermore, there was some 68% survival of resistant H. armigera larvae on Bollgard II leaf feeding assays. Survival on Bollgard II indicates that there could be considerable field significance, in the observed levels of Cry1Ac resistance detected in diet bioassays. Survival on Bollgard II cotton also indicates resistance to Cry2Ab in the BF strain. The significance of larvae surviving on leaf bioassays and the potential of metabolic resistance to both Cry1Ac and Cry2Ab should form the subject of further study into the impact of raw flour diets on resistance detection. STI and other trypsin inhibitors are well known to have inhibitory effects on mid-gut enzymes in H. armigera [25–27]. Therefore the inability to detect Cry1Ac resistance in the BF strain on a raw soybean flour diet may be due to the inhibition of an enzymatic metabolic resistance mechanism by protease inhibitors such as STI. Insecticide synergists act by inhibiting metabolic enzymes and STI and other protease inhibitors are known to be synergists of Bt toxins, in species including H. zea and H. armigera [22,29– 33]. Thus in this case, it may be that STI or similar protease inhibitor is also acting synergistically with Cry1Ac to suppress resistance in the BF strain. The ability of soybean serine protease inhibitors to potentiate Bt toxins has led to an interest in engineering unrelated crop plants without potent inhibitors, such as Bt cotton, to express high levels of inhibitors such as STI as a measure to prevent or ameliorate resistance to Bt toxins [21,31,32]. Non-specific esterases, derived from cell adhesion proteins, are serine hydrolases with an ability to sequester, Cry1Ac pro-toxin and activated toxin in Australian H. armigera [7]. This resistance to Cry1Ac, can be synergised by esterase inhibitors [8] and such synergism of Cry1Ac by an esterase inhibitor has also been reported in Cry1Ac-resistant diamondback moth [34]. It is possible that these non-specific esterases, found in the H. armigera midgut, are inhibited by dietary protease inhibitors found in a raw soybean flour diet. The other major effect of using a raw soybean flour diet was that the doses of Cry1Ac required for mortality in the susceptible strain were much higher than using a heat-denatured soybean flour diet, indicating that the Cry1Ac bioassay using heat-denatured soybean flour diet is more sensitive. The neonate LC50 on raw soybean flour diet was some 1816 times higher than on heat-denatured soybean flour diet and in third instars, the LC50 on raw soybean flour diet was 121 times higher than on heat-denatured soybean flour diet. The susceptible neonate LC99.9 on raw soybean flour diet (1.5 lg/cm2) was comparable with the Cry1Ac discriminating dose (susceptible LC96 of 0.25 lg/cm2) used by current Australian H. armigera Cry1Ac resistance monitoring programmes (which use raw flour diets) [5]. The greater sensitivity of H. armigera when using the Cry1Ac bioassay on heat-denatured soy flour diet could be due to destruction of protease inhibitors that would otherwise inhibit the proteases required to activate Cry toxins in the mid-gut, as STI is known to inhibit proteases that activate Cry pro-toxins in H. armigera, resulting in much-reduced levels of activated toxin [29]. This study has indicated that rearing and bioassay of H. armigera on diets that give chronic exposure to abnormally high levels of active legume protease inhibitors may be unsuitable for detection of a metabolic-based resistance to Cry1Ac in cotton populations of H. armigera in Australia. Bird and Ackhurst have previously suggested that greater susceptibility of cotton populations of H. armigera to Cry1Ac, compared to Helicoverpa punctigera, in Australia, might have been due to dietary STI inhibition of detoxificative enzymes in H. armigera [4]. This work may have important implications for the monitoring of H. armigera resistance to Bt toxins in Australia. This study has also generated some important avenues for continuing research,
as of course more data are needed to establish a causal relationship between xenobiotics in raw legume flour, mid-gut enzymes and Bt resistance in Australian H. armigera.
Acknowledgments The authors would like to acknowledge the support of the New South Wales Department of Primary Industries. Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.
References [1] R.T. Roush, B.E. Tabashnik (Eds.), Pesticide Resistance in Arthropods, Chapman and Hall, New York, 1990. [2] K.M. Olsen, J.C. Daly, E.J. Finnegan, R.J. Mahon, Changes in Cry1Ac Bt transgenic cotton in response to two environmental factors: temperature and insect damage, J. Econ. Entomol. 98 (2005) 1382–1390. [3] N.W. Forrester, L.J. Bird, Bt resistance assays for Australian Helicoverpa armigera. In: M.P. Zalucki, R.A.I. Drew, G.W. White (Eds.), Proceedings of the Sixth Australasian Applied Entomological Research Conference, Brisbane, 1998, pp. 145–148. [4] L.J. Bird, R.J. Akhurst, Variation in susceptibility of Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) (Lepidoptera; Noctuidae) to two Bacillus thuringiensis toxins, J. Invert. Path. 94 (2007) 84–94. [5] R.J. Mahon, K.M. Olsen, S. Downs, S. Addison, Frequencies of alleles conferring resistance to the Bt toxins Cry1Ac and Cry2Ab in Australian populations of Helicoverpa armigera (Lepidoptera: Noctuidae), J. Econ. Entomol. 100 (2007) 844–1853. [6] R.J. Akhurst, W. James, L.J. Bird, C. Beard, Resistance to the Cry1Ac d-endotoxin of Bacillus thuringiensis in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae), J. Econ. Entomol. 96 (2003) 1290–1298. [7] R.V. Gunning, H.T. Dang, F.C. Kemp, I.C. Nicholson, G.D. Moores, New resistance mechanism in Helicoverpa armigera threatens transgenic crops expressing Bacillus thuringiensis Cry1Ac toxin, Appl. Environ. Microbiol. 71 (2005) 2558– 2563. [8] R.V. Gunning, I.C. Nicholson, F.C. Kemp, V. Borzatta, E.L.A. Cottage, L.M. Field, G.D. Moores, Piperonyl butoxide, restores the efficacy of Bacillus thuringiensis toxin in transgenic cotton against resistant Helicoverpa armigera, Biopestic. Int. 2 (2006) 39–136. [9] F. Liu, Z. Xu, J. Chang, J. Chen, F. Meng, Y.C. Zhu, J. Shen, Resistance allele frequency to Bt cotton in field populations of Helicoverpa armigera (Lepidoptera: Noctuidae) in China, J. Econ. Entomol. 101 (2008) 933–943. [10] B.E. Tabashnik, A.J. Gassmann, D.W. Crowder, Y. Carrière, Insect resistance to Bt crops: evidence versus theory, Nat. Biotechnol. 26 (2008) 199–202. [11] R.V. Gunning, C.S. Easton, M.E. Balfe, I.G. Ferris, Pyrethroid resistance mechanisms in Australian Helicoverpa armigera, Pestic. Sci. 33 (1991) 473–490. [12] R.V. Gunning, G.D. Moores, A.L. Devonshire, Esterases and fenvalerate resistance in Australian Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), Pestic. Biochem. Physiol. 54 (1996) 12–23. [13] R.V. Gunning, G.D. Moores, A.L. Devonshire, Insensitive acetylcholine esterase and resistance to thiodicarb in Australian Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), Pestic. Biochem. Physiol. 55 (1996) 21–28. [14] G. Ma, H. Roberts, M. Sarjan, N. Featherstone, J. Lahnstein, R. Akhurst, O. Schmidt, Is the mature endotoxin Cry1Ac from Bacillus thuringiensis inactivated by a coagulation reaction in the gut lumen of resistant Helicoverpa armigera larvae?, Insect Biochem Mol. Biol. 35 (2005) 729–739. [15] N.W. Forrester, M. Cahill, L.J. Bird, J.K. Layland, Management of pyrethroid and endosulfan resistance in Helicoverpa armigera (Lepidoptera: Noctuidae) in Australia, Bull. Ent. Res. Suppl. Ser. (Suppl.1) (1993) 1–132. [16] K.M. Olsen, J.C. Daly, Plant-toxin interactions in transgenic Bt cotton and their effect on mortality of Helicoverpa armigera (Lepidoptera: Noctuidae), J. Econ. Entomol. 93 (2000) 1293–1299. [17] H.H. Shorey, R.L. Hale, Mass rearing of larvae of nine noctuid species on a simple artificial medium, J. Econ. Entomol. 58 (1965) 522–524. [18] F.A. Gould, A. Anderson, L. Reynolds, L. Bumgardner, W. Moar, Selection and genetic analysis of a Heliothis virescens (Lepidoptera: Noctuidae) strain with a high level of resistance to Bacillus thuringiensis toxins, J. Econ. Entomol. 88 (1995) 1545–1559. [19] K.A. Anilkumar, W.J. Moar, Difference between rate of resistance development to Bt Cry1Ac toxin in cotton bollworm, Helicoverpa zea (Boddie) when selected using MVP II and activated toxin, in: Proceedings of 2006 Beltwide Cotton Conferences, San Antonio, Texas, January 3–6, 2006, pp 1478–1482. [20] P.C. Marçon, I.J. Young, K.L. Steffey, B.D. Siegfried, Baseline susceptibility of European corn borer (Lepidoptera: Crambidae) to Bacillus thuringiensis toxins, J. Econ. Entomol. 92 (1999) 279–285. [21] D.J. Finney, Probit Analysis, Cambridge University Press, London, 1971. [22] S.C. Macintosh, G.M. Kishore, F.J. Perlak, P.G. Marrone, T.B. Stone, S.R. Sims, R.L. Fuchs, Potentiation of Bacillus thuringiensis insecticidal activity by serine protease inhibitors, J. Agric. Food Chem. 38 (1990) 1145–1152.
R.V. Gunning, G.D. Moores / Pesticide Biochemistry and Physiology 97 (2010) 55–59 [23] K.A. Jonhston, J.A. Gatehouse, J.H. Anstee, Effects of soybean protease inhibitors on the growth and development of larval Helicoverpa armigera, J. Insect Physiol. 39 (1993) .657–664. [24] M. Kunitz, Crystallization of a trypsin inhibitor from soybean, Science 101 (1945) 668–669. [25] A. Srinivasan, A.P. Giri, A.M. Harsulkar, J.A. Gatehouse, V.S. Gupta, A kunitz trypsin inhibitor from chickpea (Cier arietinum L.) that exerts anti-metabolic effect on podborer (Helicoverpa armigera) larvae, Plant Mol. Biol. 57 (2005) 359–374. [26] C.Z. Wang, X.F. Xiang, S.F. Zhang, J.D. Qin, Effect of soybean trypsin inhibitor on the growth and digestive physiology of Helicoverpa armigera larvae, Acta Entomol. Sin. 29 (1995) 35–39. [27] R.M. Broadway, S.S. Duffey, Plant proteinase inhibitors: mechanism of action and effect on larval growth and digestive physiology of larval Heliothis zea and Spodoptera exiqua, J. Insect Physiol. 32 (1986) 827– 833. [28] R.E. Teakle, J.M. Jensen, Heliothis punctiger, in: R. Singh, R.F. Moore (Eds.), Handbook of Insect Rearing, vol. 2, Elsevier, Amsterdam, 1985, pp. 312–322.
59
[29] Z. Shao, Y. Cui, X. Liu, H. Yi, J. Ji, Z. Yu, Processing of d-endotoxin of Bacillus thuringiensis subsp. Kurstaki HD-1 in Heliothis armigera midgut juice and the effects of protease inhibitors, J. Invert. Path. 72 (1998) 3–81. [30] J. Zhang, C. Wang, J. Qin, The interactions between soybean trypsin inhibitor and d-endotoxin of Bacillus thuringiensis in Helicoverpa armigera larva, J. Invert. Path. 75 (2000) 259–266. [31] Y.C. Zhu, C.A. Abel, M.S. Chen, Interaction of Cry1Ac toxin (Bacillus thuringiensis) and proteinase inhibitors on the growth, development, and midgut proteinase activities of the bollworm Helicoverpa zea, Pestic. Biochem. Physiol. 87 (2007) 39–46. [32] J.T. Christtellier, W.A. Laing, N.P. Markwick, E.J.P. Burgess, Midgut protease activities in 12 phytophagous Lepidopteran larvae: dietary and protease inhibitor interactions, Insect Biochem. Mol. Biol. 22 (1992) 735–746. [33] J. Hubert, M. Nesvorna, R. Zemek, J. Stara, V. Stejskal, Effects of metabolic inhibitors on activity of Cry1Ab toxin to inhibit growth of Ephestia kuehniella larvae, Pest Manag. Sci. 64 (2008) 1063–1068. [34] A.H. Sayyed, G.D. Moores, N. Crickmore, D.J. Wright, Cross-resistance between a Bacillus thuringiensis Cry toxin and non-Bt insecticides in the diamondback moth, Pest. Manag. Sci. 64 (2008) 813–819.