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Toxicity of Bacillus thuringiensis Cry proteins to Helicoverpa armigera (Lepidoptera: Noctuidae) in South Africa
Journal of Invertebrate Pathology 109 (2012) 110–116
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Toxicity of Bacillus thuringiensis Cry proteins to Helicoverpa armigera (Lepidoptera: Noctuidae) in South Africa Hua Li, Gustav Bouwer ⇑ School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, Wits 2050, Johannesburg, South Africa
a r t i c l e
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Article history: Received 29 June 2011 Accepted 10 October 2011 Available online 15 October 2011 Keywords: Helicoverpa armigera Bacillus thuringiensis Cry proteins Escherichia coli expression Larvicidal activity Larval growth inhibition
a b s t r a c t The susceptibility of one of the most important pests in southern Africa, Helicoverpa armigera (Lepidoptera: Noctuidae), to Bacillus thuringiensis Cry proteins was evaluated by bioassay. Cry proteins were produced in Escherichia coli BL21 cells that were transformed with plasmids containing one of six cry genes. The toxicity of each Cry protein to H. armigera larvae was determined by the diet contamination method for second instar larvae and the droplet feeding method for neonate larvae. For each of the proteins, dose– mortality and dose–growth inhibition responses were analyzed and the median lethal dose (LD50) and median inhibitory dose (ID50) determined. Second instar larvae were consistently less susceptible to the evaluated Cry proteins than neonate larvae. The relative toxicity of Cry proteins ranked differently between neonate larvae and second instar larvae. On the basis of the LD50 and ID50 values, Cry1Ab, Cry1Ac, and Cry2Aa were the most toxic of the evaluated proteins to H. armigera larvae. The study provides an initial benchmark of the toxicity of individual Cry proteins to H. armigera in South Africa. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Bacillus thuringiensis (Bt) is a gram-positive, rod shaped, sporeforming bacterium that produces one or more proteinaceous parasporal crystals during sporulation (Höfte and Whiteley, 1989). The Bt crystals may consist of either Cyt proteins or Cry proteins (Höfte and Whiteley, 1989; Schnepf et al., 1998). As a group, Cry proteins are toxic to certain species in the orders Lepidoptera, Diptera, Coleoptera, Hymenoptera and Hemiptera, and also to other invertebrates such as nematodes and mites (Crickmore et al., 2011; Schnepf et al., 1998; van Frankenhuyzen, 2009). Although order-specificity was initially considered important in the classification of Cry proteins (Höfte and Whiteley, 1989), the widely-adopted Cry nomenclature of Crickmore et al. (1998) is based on amino acid sequence divergence rather than functional aspects of the proteins, such as the specificity of toxicity. However, several Cry proteins displaying cross-order activity have been identified; for example, the primarily lepidopteran-active Cry1Ba protein was reported to be toxic to Coleoptera (Naimov et al., 2001; Zhong et al., 2000) and Diptera (Heath et al., 2004; Zhong et al., 2000), and the primarily lepidopteran-active Cry1Ia protein exhibited toxicity to coleopteran insects (Martins et al., 2008; Naimov et al., 2001; Ruiz et al., 2006). Different lepidopteran species may have different levels of susceptibility to a specific Cry protein. For example, Cry1Ac shows ⇑ Corresponding author. Fax: +27 11 717 6337. E-mail address: [email protected] (G. Bouwer). 0022-2011/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2011.10.005
high larvicidal activity against most lepidopteran insects, but very low larvicidal activity against Actebia fennica (Lepidoptera: Noctuidae) (van Frankenhuyzen et al., 1993), Spodoptera frugiperda (Lepidoptera: Noctuidae) (Luo et al., 1999), or Spodoptera littoralis (Lepidoptera: Noctuidae) (Escriche et al., 1998). The relative toxicity of Cry proteins to a susceptible lepidopteran species may also differ. For example, Cry1Ac shows significantly higher larvicidal activity than Cry2Aa against Plutella xylostella (Lepidoptera: Plutellidae) (Monnerat et al., 1999). The African bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), is a polyphagous pest whose larvae feed on a wide range of plants including many important cultivated crops (Vassal et al., 2008). In South Africa, it achieves major pest status in cotton, tomato, and maize and causes serious losses throughout its range (van Jaarsveld et al., 1998). The susceptibility of H. armigera to various Cry proteins has been evaluated in many countries over the past decades. In Australia, it was reported that only Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab killed H. armigera at doses that could be considered acceptable, whereas Cry1B, Cry1C and Cry1E were considered non-toxic (Liao et al., 2002). In India, Cry1Ac was the most toxic Cry protein to H. armigera followed by Cry1Ab and Cry1Aa (Chandrashekar et al., 2005). Although Cry1Ac was also the most toxic of the Cry proteins evaluated in another Indian study (Chakrabarti et al., 1998), the authors found that Cry1Aa was more toxic than either Cry2Aa or Cry1Ab. Studies in different countries have reported significant differences in relative toxicities for the same Cry proteins, e.g. the Cry2Aa/Cry1Ac median lethal concentration (LC50) ratio was 35.5 in India (Chakrabarti et al.,
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1998) and 1.3 in Australia (Liao et al., 2002). It is thus apparent that H. armigera larvae in different countries or from geographically distinct source populations can differ in susceptibility to the same Cry proteins. To our knowledge, no studies have determined the susceptibility of South African H. armigera populations to different Cry proteins. The assessment of the toxicity of individual Cry proteins would provide an important step in the determination of their potential for use in H. armigera control programmes in South Africa. In this study, we evaluated the toxicity of six Cry proteins to a South African population of H. armigera. 2. Materials and methods 2.1. Transformation of cry genes into Escherichia coli BL21 The recombinant E. coli strains used in this study were originally obtained from the Bacillus Genetic Stock Centre: ECE52 (cry1Aa cloned in plasmid pKK223-3 in E. coli JM103), ECE54 (cry1Ab cloned in plasmid pKK223-3 in E. coli JM103), ECE53 (cry1Ac cloned in plasmid pKK223-3 in E. coli JM103), ECE125 (cry1Ca cloned in plasmid pTZ19R in E. coli DH5a), ECE126 (cry2Aa cloned in plasmid pTZ19R in E. coli DH5a), and ECE130 (cry9Aa cloned in plasmid pSB1402 in E. coli DH5a). Plasmids containing one of the six cry genes were isolated from the relevant E. coli strain and transformed into E. coli BL21 (salt inducible strain; Invitrogen, Carlsbad, USA) to standardize the host background for Cry protein production. The plasmid isolation was performed according to the manufacturer’s instructions for the QIAprep Spin Miniprep Kit (Qiagen, Valencia, USA) and BL21 was transformed with plasmid DNA as described by Chung et al. (1989). Transformed BL21 cells were grown, selected and stored on LAON (Luria–Bertani agar with NaCl omitted, Invitrogen) plates supplemented with 100 lg/ml ampicillin using standard methods as per the manufacturer’s instructions for BL21 (Invitrogen). PCR amplification was executed according to the manufacturer’s instructions (GoTaq DNA Polymerase Kit, Promega, Madison, USA) to confirm the presence of the cry genes in the resultant BL21 transformants by using specific cry gene primers (Table 1) and the plasmids isolated from BL21 transformants as the templates. The primers were designed with cry genes in NCBI GenBank serving as the target sequences (Table 1). Throughout this study, a BL21 transformant expressing one of the Cry proteins was designated as BL21[Cry protein], for example BL21[Cry1Aa].
2.2. Production and quantitation of Cry proteins The expression of the six Cry proteins in the BL21 transformants was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) (data not shown). The SDS–PAGE results showed a prominent protein band at a molecular weight of approximately 130 kDa in BL21[Cry1Aa], BL21[Cry1Ab], BL21[Cry1Ac], and BL21[Cry9Aa]. A 65 kDa band and 140 kDa band was observed in BL21[Cry2Aa] and BL21[Cry1Ca], respectively. Since there were no distinct bands of the above mentioned sizes in the BL21 control, the presence of the expected Cry proteins in the corresponding BL21 transformants was confirmed. The molecular weights of the Cry proteins in the relevant transformants matched the published molecular weights of the Cry proteins (Gamel and Piot, 1992; Kalman et al., 1995). The six different Cry proteins were produced as protoxin inclusion bodies (crystals) in BL21 transformants. Although the expression of cry1Aa, cry1Ab and cry1Ac did not require induction since the pKK223-3 cloning vector does not utilize the T7 promoter, salt induction was performed for all BL21 transformants so as to standardize conditions and thus minimize any potential differences in protein expression profiles. NaCl induction of protein expression was performed according to the method of Donahue and Bebee (1999). After induction, all cultures were incubated at 37 °C with constant shaking (200 rpm) until the formation of crystals was confirmed (using optical microscopy) and almost all of the cells had lysed. Centrifugation of the cultures was performed for 15 min at 9000g at 4 °C and the pellets were suspended in sterile double-distilled water and the washing step was repeated twice more. The resuspended pellets were stored at 4 °C. The concentration of Cry proteins was determined by a novel quantitation strategy, hereafter referred to as the Bradford/ Pro260 method. The Bradford method (Bradford, 1976), using the Coomassie Plus Protein Assay Kit (Thermo Fisher Scientific, Rockford, USA) and bovine serum albumin (BSA) as the standard, was used to evaluate the total protein concentration of each Cry protein sample. Subsequently, the concentration of the Cry protein in the sample was calculated by the formula: Cry protein concentration (lg/ml) = (lg/ml total protein) (proportion of Cry protein to total protein), with the proportion of Cry protein to total protein being determined using the Experion Pro260 automatic electrophoresis system (Biorad, Hercules, USA). The Bradford/Pro260 method showed better accuracy and reproducibility than either densitometry analysis of SDS–PAGE gels or the Experion Pro260 system
Table 1 The sequences and characteristics of primers used to detect cry genes in E. coli BL21 transformants.
a b c
Gene detected
Accession No.a
Plasmidb
Primer name
Sequence
Product size (bp)
cry1Aa
M11250
pKK223-3
C1Aa-d-F C1-consv-Rc
50 -TTATACTTGGTTCAGGCCC-30 50 -CTTGTGACACTTCTGCTTCC-30
1950
cry1Ab
M12661
pKK223-3
C1Ab-d-F C1-consv-Rc
50 -CAATCGGAAAATGTGCC-30 50 -CTTGTGACACTTCTGCTTCC-30
609
cry1Ac
M11608
pKK223-3
C1Ac-d-F C1-consv-Rc
50 -CAAAGACATTAATAGGCAACC-30 50 -CTTGTGACACTTCTGCTTCC-30
992
cry1Ca
AF362020
pTZ19R
C1Ca-d-F C1-consv-Rc
50 -GATCTGGAACACCTTTTTTAAC-30 50 -CTTGTGACACTTCTGCCTCC-30
1773
cry2Aa
M23723
pTZ19R
C2Aa-d-F C2Aa-d-R
50 -CCTTGCTCGTGTAAATGC-30 50 -CTAGCATATAAATTAGCGCCAG-30
507
cry9Aa
X58120
pSB1402
C9Aa-d-F C9Aa-d-R
50 -GGTTCACTTACATTGCCGGTTAG-30 50 -CGCTTTTTTTGCTGCTTCTAG-30
954
Accession number of the relevant cry gene in NCBI GenBank. Plasmid that the cry gene is cloned in. Reverse primer binds to conserved region of cry1A-class genes.
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(unpublished data). The concentrations of Cry proteins estimated by the Bradford/Pro260 method were on average 1.05 times higher than those estimated by densitometry analysis of SDS–PAGE gels (unpublished data). For each sample used in bioassays, the Cry protein concentration was the average of six separate estimates. 2.3. Insects H. armigera eggs were obtained from a culture maintained at the Agricultural Research Council (Pretoria, South Africa). This culture was established with specimens collected from the Springbok Flats (29°400 S 17°530 E) in 1974 and supplemented in 2006 with specimens collected from cotton plants near Groblersdal (25°150 S 29°250 E) in Mpumalanga, South Africa (Mawela et al., 2010). Larvae were reared in our laboratory on a wheat-germ based artificial diet (Bot, 1966). During oviposition, H. armigera adults were provided with 5% (w/v) sucrose solution. The culture was maintained in a growth chamber at 28 ± 1 °C, 70% relative humidity, and a photoperiod of 12:12 h (L:D). 2.4. Dose–mortality bioassays The toxicity of Cry proteins to H. armigera larvae was evaluated by dose–mortality bioassays. Bioassays of neonate larvae were performed using the droplet feeding method (Hughes and Wood, 1981), as adapted for H. armigera by Bouwer and Avyidi (2006). Larvae that imbibed the mixture of feeding solution and Cry protein were placed individually in wells (24-well tissue culture plate; Nalge Nunc International, Rochester, USA) containing the artificial diet. Trays were sealed with Parafilm (Sigma, St. Louis, USA), which was perforated to provide aeration, and a plastic lid. Bioassays were performed with second instar larvae (6 h after moulting) using a modification of the diet contamination method of Ferré et al. (1991). Freshly prepared diet disks (4 mm diameter and 1 mm thick) were dispensed into the wells of a 24-well tissue culture plate. Dilutions of the Cry proteins were applied uniformly over the food surface and allowed to dry. One second instar larva was placed into each well and the multi-well plates were sealed with perforated Parafilm and a plastic lid. Larvae that consumed the entire contaminated diet disk within 2 days were transferred to new individual wells containing untreated artificial diet. Four replicates of 24 larvae per replicate (n = 96) were used for each dose of a Cry protein and for the control (water only). A range of seven doses and six doses were used for bioassays of neonate and second instar larvae, respectively. For bioassays of neonate larvae, mortality was scored 4 days after treatment. For bioassays of second instar larvae, mortality was scored 4 days after the treated larvae were transferred to untreated diet. 2.5. Dose–weight bioassays The effect of sublethal doses of Cry proteins on the development and growth of H. armigera larvae was evaluated using dose–weight bioassays, with the ID50 (median inhibitory dose; the dose that caused a 50% lower larval weight compared to the controls) being used to compare the relative toxicity of the different Cry proteins. The diet contamination method and the droplet feeding method were applied to H. armigera second instar and neonate larvae, respectively, as described above. Five doses of Cry proteins were used for both second instar larvae and freshly hatched neonate larvae. For each bioassay method, a total of 40–55 larvae that survived treatment were weighed (wet weight) for each dose. Water controls were also included in the assays. The weights of neonate larvae were recorded 7 days after treatment, whereas the weights of second instar larvae were recorded 7 days after the treated larvae were transferred to uncontaminated diet.
2.6. Data analysis Dose–mortality data were subjected to probit regression analysis (Finney, 1971), and the median lethal dose (LD50) was estimated for each Cry protein. GraphPad Prism 5 (GraphPad Software Inc, San Diego, USA) was used to evaluate the dose–weight bioassay data. The ID50 estimates were derived using nonlinear regression fitting of a sigmoidal dose–response curve to Cry-treated larval weight data. The function used to calculate the ID50 was log dose versus response-variable slope (four parameters). 3. Results 3.1. Larvicidal activity of Cry proteins against neonate larvae The results of the probit analyses of the neonate dose–mortality bioassays are shown in Table 2. On the basis of LD50 values, the larvicidal activity of Cry1Ac was significantly (statistically) higher than any of the other Cry proteins evaluated and Cry2Aa was the next most toxic Cry protein evaluated. The larvicidal activity of Cry2Aa was significantly higher than that of Cry1Ab, which was in turn significantly higher than that of Cry1Aa. Cry1Ca and Cry9Aa showed the lowest larvicidal activity, with the LD50 of Cry1Ac being approximately 20,510 and 24,970 times lower than that of Cry1Ca and Cry9Aa, respectively (Table 2). 3.2. Larvicidal activity of Cry proteins against second instar larvae In bioassays against second instar larvae, the LD50 of Cry1Ac was significantly lower than the LD50 of any one of the other Cry protein evaluated (Table 3). In contrast to the results obtained for neonate larvae, Cry1Ab was significantly more toxic than Cry2Aa. The larvicidal activities of Cry1Aa, Cry9Aa, and Cry1Ca did not differ significantly from each other and the LD50 values of these proteins are at least 4000-fold higher than that of Cry1Ac (Table 3). 3.3. Dose–growth inhibition responses of neonate larvae to Cry proteins Neonate larvae that survived after treatment with Cry1Ac, Cry2Aa, Cry1Ab, Cry1Aa, or Cry9Aa showed significant stunting in their growth compared to the control larvae. On the basis of ID50 values, Cry1Ac exhibited higher larval growth inhibition activity than any of the other Cry proteins (Table 4). There was no statistical difference between the ID50 values of Cry1Ab and Cry2Aa. Cry1Aa caused statistically more growth inhibition than Cry9Aa (Table 4). Due to Cry1Ca’s poor growth inhibition activity at the evaluated doses, no ID50 could be estimated for this protein against H. armigera neonate larvae. 3.4. Dose–growth inhibition responses of second instar larvae to Cry proteins Cry1Ac exhibited significantly higher growth inhibition activity against second instar larvae than any of the other Cry proteins, whereas the weight inhibition activity of Cry1Ca was so low that no ID50 could be estimated for this protein (Table 5). The ID50 values of Cry1Ab, Cry2Aa and Cry1Aa were not significantly different. Cry9Aa had the lowest growth inhibition activity. 4. Discussion Since Cry proteins are usually found as combinations in natural Bt strains (Agaisse and Lereclus, 1995), the determination of the
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H. Li, G. Bouwer / Journal of Invertebrate Pathology 109 (2012) 110–116 Table 2 Dose–mortality responses of H. armigera neonate larvae to Cry proteins as determined by droplet feeding bioassays.
A B C
Cry proteins
LD50 (ng)A
Cry1Ac Cry2Aa Cry1Ab Cry1Aa Cry1Ca Cry9Aa
3.14 102a 1.39 101b 6.10 101c 1.02 101d 6.44 102e 7.84 102e
95% Fiducial limits Lower
Upper
2.05 102 1.07 101 5.20 101 6.48 100 3.38 102 5.33 102
4.67 102 1.80 101 7.30 101 1.57 101 1.48 103 1.27 103
Slope ± SEB
v2 (df)C
0.66 ± 0.05 1.08 ± 0.08 1.46 ± 0.13 0.68 ± 0.05 0.41 ± 0.05 0.71 ± 0.08
5.65 7.76 6.84 8.88 7.69 0.90
Slope ± SEB
v2 (df)C
0.54 ± 0.05 0.44 ± 0.04 0.39 ± 0.05 0.13 ± 0.03 0.46 ± 0.05 0.14 ± 0.03
1.9 5.7 2.8 3.0 3.9 3.2
(4) (4) (3) (4) (4) (4)
Values followed by the same letters are not significantly different (overlapping 95% fiducial limits). Slope ± standard error. Values in brackets show degrees of freedom (df).
Table 3 Dose–mortality responses of H. armigera second instar larvae to Cry proteins as determined by diet contamination bioassays.
A B C
Cry proteins
LD50 (ng)A
Cry1Ac Cry1Ab Cry2Aa Cry1Aa Cry9Aa Cry1Ca
5.05 101a 5.30 100b 5.26 101c 2.16 103d 4.09 103d 2.70 104d
95% Fiducial limits Lower
Upper
2.58 101 2.50 100 2.49 101 2.72 102 2.11 103 2.04 103
9.18 101 1.03 101 1.20 102 1.70 105 8.35 103 1.85 107
(4) (4) (3) (4) (3) (4)
Values followed by the same letters are not significantly different (overlapping 95% fiducial limits). Slope ± standard error. Values in brackets show degrees of freedom (df).
Table 4 Dose–growth inhibition responses of H. armigera neonate larvae to Cry proteins as determined by droplet feeding bioassays. Cry proteins
ID50 (ng)A
Cry1Ac Cry1Ab Cry2Aa Cry1Aa Cry9Aa Cry1Ca
2.00 104a 1.00 103b 2.00 103b 1.20 101c 2.95 100 d NCC
95% Confidence intervals Lower
Upper
2.00 104 6.00 104 9.00 104 3.00 102 1.60 100
3.00 104 3.00 103 3.00 103 4.20 101 5.45 100
Slope ± SEB
1.15 ± 0.11 0.40 ± 0.05 0.44 ± 0.04 0.37 ± 0.06 0.76 ± 0.14
A Values followed by the same letters are not significantly different (overlapping 95% confidence intervals). B Slope ± standard error. Hill slope is reported. C NC: not calculated, ID50 substantially higher than the highest dose evaluated.
Table 5 Dose–growth inhibition responses of H. armigera second instar larvae to Cry proteins as determined by diet contamination bioassays. Cry proteins
ID50 (ng)A
Cry1Ac Cry1Ab Cry2Aa Cry1Aa Cry9Aa Cry1Ca
3.00 103a 2.00 102b 1.00 101bc 1.67 101bc 2.28 102c NCC
95% Confidence intervals Lower
Upper
2.00 103 7.00 103 3.00 102 3.00 102 7.53 100
4.00 103 4.00 102 3.90 103 8.19 103 6.90 103
Slope ± SEB
0.69 ± 0.07 0.46 ± 0.07 0.23 ± 0.05 0.26 ± 0.10 0.35 ± 0.08
A Values followed by the same letters are not significantly different (overlapping 95% confidence intervals). B Slope ± standard error. Hill slope is reported. C NC: not calculated, ID50 substantially higher than the highest dose evaluated.
relative toxicity of single Cry proteins using natural Bt strains is often not feasible. However, some studies have used Bt strains expressing single Cry proteins (Ibargutxi et al., 2006). We evaluated the toxicity of Cry proteins produced in E. coli, an approach
used by several studies that evaluated the toxicity of Cry proteins to H. armigera (Chakrabarti et al., 1998; Chandrashekar et al., 2005; Lee et al., 1996). By using E. coli as an expression host for cry genes, any potential synergistic interactions between Bt spores and crystals may be avoided (Johnson et al., 1998). A review of the larvicidal activity of Bt toxins to various insects showed that there is no evidence that expression host is a major determinant of crystal toxicity or affects the toxicity ranking of crystals (van Frankenhuyzen, 2009). The majority of published bioassay data for various lepidopteran species used lethal concentration (LC) and inhibitory concentration (IC) as the units of estimates to describe the toxicity of Cry proteins (van Frankenhuyzen, 2009). However, lethal dose (LD) and inhibitory dose (ID) may be preferable to LC and EC in evaluating the susceptibility of an insect to a specific toxin, because the LD and ID estimates are based on the actual dose ingested by the larvae rather than the concentration to which the insect was exposed to (Hughes and Wood, 1981). Different bioassay methods can be used to obtain LD or ED estimates, including the diet contamination method (Ignoffo, 1965) and the droplet feeding method (Hughes and Wood, 1981). However, the diet contamination method is not suitable for neonate larvae as the larvae are unlikely to fully consume even a very small block of food. The droplet feeding method is, thus, a valuable bioassay method for the estimation of LD or ED values for neonate larvae. The droplet feeding method is not suitable for post-first instar larvae because it is often practically impossible to identify the larvae which imbibed the colored feeding solution by the presence of the dye in their foreguts and midguts. From the second instar onwards, the diet contamination method may be used because these larvae are able to fully consume a small food block. To enable LD and ED estimation in this study, the toxicity of each Cry protein to H. armigera larvae was determined by the diet contamination method for second instar larvae and the droplet feeding method for neonate larvae. Bt biopesticides are usually applied when early instar larvae are present as older larvae are more tolerant (Sanahuja et al., 2011). We thus focused on the susceptibility of the first and second larval
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stages to the Cry proteins. Although several studies have compared the susceptibility of neonate and third instar larvae of H. armigera to Bt products or Cry proteins (Bird and Akhurst, 2007; Cooper, 1984; Liao et al., 2002), a consistent pattern in susceptibility is not apparent. Liao et al. (2002) demonstrated that there was no difference in LC50 values between H. armigera third instar and neonate larvae for commercial Bt products. In contrast, Bird and Akhurst (2007) reported that H. armigera neonate larvae were less susceptible to Cry1Ac than third instar larvae based on LC50 and median inhibitory concentration (IC50) values, respectively, but more susceptible to Cry2Ab than third instar larvae. In our study, neonate larvae exhibited consistently lower LD50 and ID50 values than second instar larvae. This decreased susceptibility for second instar larvae was in agreement with other studies that showed that later instar larvae of Helicoverpa zea (Lepidoptera: Noctuidae) and P. xylostella were less susceptible to Cry proteins than early instar larvae (Ali and Young, 1996; Asano et al., 1993). In this study, a steeper slope was found in most dose–mortality and dose–weight regression lines of Cry proteins in H. armigera neonate larvae compared to those of second instar larvae, suggesting a more homogenous population with regard to susceptibility to Cry proteins. A similar trend was reported by Bird and Akhurst (2007), where the slope value of concentration–mortality regression lines of Cry1Ac decreased when H. armigera neonate larvae developed to third instar larvae. There are marked differences between the absolute values of LC50 estimates for the same Cry protein in different studies (Avilla et al., 2005; Bird and Akhurst, 2007; Chakrabarti et al., 1998; Ibargutxi et al., 2008; Liao et al., 2002). The following factors are likely to contribute to the observed differences: the bioassay method used (e.g., diet incorporation or surface treatment), the insect diet used, the level of processing of the Cry proteins used in the bioassays (e.g., protoxins or activated toxin), the protein quantitation method used, and the temperature at which bioassays were performed (Avilla et al., 2005; Bird and Akhurst, 2007). Although LD bioassays have some advantages over LC bioassays, we are not aware of any studies that have determined the LD50 values for Cry proteins assayed against H. armigera. Since we estimated LD50 values rather than LC50 values and we used the droplet feeding assay method for neonate larvae, it is not possible to directly compare our absolute values of LD50 estimates with estimates in other studies that evaluated Cry proteins against H. armigera (Avilla et al., 2005; Bird and Akhurst, 2007; Chakrabarti et al., 1998; Ibargutxi et al., 2008; Liao et al., 2002). Comparisons of the toxicity data produced in different studies should, thus, focus on the relative toxicity or toxicity ranking of the Cry proteins rather than the absolute values of toxicity estimates. However, several factors can cause differences in the relative toxicity of Cry proteins to the same insect species, including: different levels of precision resulting from different bioassay methods, the use of test colonies derived from geographically distinct source populations, and evaluation of different developmental stages of a given test species (Avilla et al., 2005; van Frankenhuyzen, 2009). When reviewing the toxicity ranking reported in other studies, we believe it is important to consider whether the authors based their ranking of Cry proteins purely on numerical differences in LC50 values or on significant differences, e.g. non-overlapping 95% fiducial limits. We obtained the following ranking of larvicidal activity (LD50 values) against neonate larvae: Cry1Ac < Cry2Aa < Cry1Ab < Cry1Aa < (Cry1Ca = Cry9Aa), where < means significantly lower (non-overlapping 95% fiducial limits) and = means not significantly different. Cry1Ac and Cry2Aa thus had the highest larvicidal activities of the tested Cry proteins against H. armigera neonate larvae, which is consistent with the result obtained by other researchers (Avilla et al., 2005; Chakrabarti et al., 1998; Chandrashekar et al., 2005; Liao et al., 2002). The Cry2Aa/Cry1Ac LD50 ratio was 4.4,
which is within the wide Cry2Aa/Cry1Ac LC50 ratio range obtained in other studies, for example: 35.5 (Chakrabarti et al., 1998), 10.2 (Ibargutxi et al., 2008), 6.6 (Babu et al., 2002), 1.8 (Avilla et al., 2005), and 1.3 (Liao et al., 2002). Although most H. armigera studies have also found Cry1Ac and Cry2Aa to be the most toxic to neonate larvae, there are differences between the toxicity rankings of Cry1Ab, Cry1Aa, Cry1Ca, and Cry9Aa in our study and other studies (e.g. Chakrabarti et al., 1998). In agreement with our results, other studies have reported that Cry9Aa has very low toxicity to H. armigera neonate larvae (Chakrabarti et al., 1998; Liao et al., 2002). Sublethal effects (feeding inhibition and growth inhibition) are considered more sensitive indicators of Cry protein toxicity than lethal effects (van Frankenhuyzen, 2009). To further characterize the toxicity of the Cry proteins to neonate larvae, we estimated ID50 values. We obtained the following ranking of ID50 values against neonate larvae: Cry1Ac < (Cry1Ab = Cry2Aa) < Cry1Aa < Cry9Aa. Due to Cry1Ca’s poor growth inhibition activity at the evaluated doses, no ID50 could be estimated for Cry1Ca. Although the ranking is very similar to that obtained for LD50 values, there are differences, e.g. Cry2Aa < Cry1Ab based on LD50 values, but Cry2Aa = Cry1Ab based on ID50 values. When considering both the LD50 and ID50 values obtained for neonate larvae, it is apparent that the most toxic Cry proteins were Cry1Ac, Cry1Ab, and Cry2Aa. We obtained the following ranking of larvicidal activity (LD50 values) against second instar larvae: Cry1Ac < Cry1Ab < Cry2Aa < (Cry1Aa = Cry9Aa = Cry1Ca). Cry1Ac was again the most toxic of the evaluated proteins, but in contrast to the results for neonate larvae Cry1Ab was more toxic than Cry2Aa, and Cry1Aa clustered with Cry9Aa and Cry1Ca. To our knowledge, no other published studies have evaluated the toxicity of Cry proteins against second instar H. armigera larvae, and we can thus not directly compare our results to other studies. On the basis of ID50 values, Cry1Ac was the most toxic Cry protein to second instar larvae, with the ranking as follows: Cry1Ac < (Cry1Ab = Cry2Aa = Cry1Aa). Due to its low sublethal activity, no ID50 could be estimated for Cry1Ca. Low toxicity of Cry1C proteins has been reported for other H. armigera populations (Chakrabarti et al., 1998; Liao et al., 2002). The South African H. armigera population was generally susceptible to most of the tested Cry proteins, and Cry1Ac was consistently more toxic than any of the other evaluated Cry proteins to both neonate and second instar larvae. Although not as toxic as Cry1Ac, Cry1Ab and Cry2Aa could be considered as good candidates for the control of H. armigera in South Africa. In bioassays conducted over three seasons, Bird and Akhurst (2007) found significant differences in susceptibility to Cry1Ac and Cry2Ab spore-crystal mixes in Australian strains of H. armigera. Although inter-strain differences in susceptibility to a Cry protein is not unexpected, a critical issue is whether these differences in susceptibility affect the ranking of the toxicity of the Cry proteins in the different strains. To address this issue, we analyzed the LC50 data for those H. armigera strains for which Bird and Akhurst (2007) reported bioassay data for both Cry1Ac and Cry2Ab (n = 15). This analysis of the strain-specific LC50 values showed that Cry1Ac was more toxic than Cry2Ab in all the tested strains, with a mean Cry2Ab/Cry1Ac LC50 ratio of 3.0 ± 0.3 (SE). The Cry2Ab/ Cry1Ac LC50 ratio for their H. armigera laboratory strain, which had been cultured in the laboratory for approximately 4 years, was 2.2. Our analysis of the data presented by Bird and Akhurst (2007) suggests that, in the absence of significant field-developed resistance to specific Cry proteins, inter-strain variation in susceptibility to Cry proteins may not be large enough to cause different H. armigera strains to have different Cry toxicity rankings. Nonetheless, evaluation of the susceptibility of different South African H. armigera populations or strains to the Cry proteins used in our study may provide information that will be useful in pest control programmes.
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The choice of Cry proteins to pyramid in transgenic plants or recombinant Bt strains should consider which combinations of Cry proteins are more likely to prevent or delay the development of resistance in insects. From a resistance management standpoint, Cry1Ac and Cry2Aa would be a good combination for the control of H. armigera because these two proteins utilize different receptor binding sites (Estela et al., 2004; Gahan et al., 2005). The rationale for combining Cry1Ac and Cry2Aa is supported by studies that looked at Cry-resistant strains: a Cry1Ac-resistant strain of H. armigera was not resistant to Cry2Aa or Cry2Ab (Akhurst et al., 2003), and a Cry2Ab-resistant strain of H. armigera, which was crossresistant to Cry2Aa, was susceptible to Cry1Ac (Mahon et al., 2007). In conclusion, this is the first report of the toxicity of Cry proteins to a South African H. armigera population. Because we estimated LD50 and ID50 values in both neonate and second instar larvae, we believe that this study presents a more comprehensive evaluation of Cry toxicity than other studies that evaluated the toxicity of Cry proteins to H. armigera. The study provides an initial benchmark of the toxicity of individual Cry proteins to H. armigera in South Africa. In order to gain insight into the variation of H. armigera susceptibility in South Africa, future studies will evaluate the susceptibility of multiple, geographically distinct H. armigera populations to a range of Cry proteins. Acknowledgments We thank Michelle Grant for assistance with the maintenance of the H. armigera cultures. This project was partially funded by a LIFELab Biotechnology Regional Innovation Centre research grant awarded to Gustav Bouwer. We thank anonymous reviewers for comments that improved this manuscript. References Agaisse, H., Lereclus, D., 1995. How does Bacillus thuringiensis produce so much insecticidal crystal protein? J. Bacteriol. 177, 6027–6032. Akhurst, R.J., James, W., Bird, L.J., Beard, C., 2003. Resistance to the Cry1Ac dendotoxin of Bacillus thuringiensis in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae). J. Econ. Entomol. 96, 1290–1299. Ali, A., Young, S.Y., 1996. Activity of Bacillus thuringiensis Berliner against different ages and stages of Helicoverpa zea (Lepidoptera: Noctuidae) on cotton. J. Entomol. Sci. 31, 1–8. Asano, S., Maruyama, T., Iwasa, T., Seki, A., Takahashi, M., Soares Jnr, G.G., 1993. Evaluation of biological activity of Bacillus thuringiensis test samples using a diet incorporation method with diamondback moth, Plutella xylostella (Linnaeus) (Lepidoptera: Yponomeutidae). Appl. Entomol. Zool. 28, 513–524. Avilla, C., Vargas-Osuna, E., González-Cabrera, J., Ferré, J., González-Zamora, J.E., 2005. Toxicity of several delta-endotoxins of Bacillus thuringiensis against Helicoverpa armigera (Lepidoptera: Noctuidae) from Spain. J. Invertebr. Pathol. 90, 51–54. Babu, B.G., Udayasuriyan, V., Mariam, M.A., Sivakumar, N.C., Bharathi, M., Balasubramanian, G., 2002. Comparative toxicity of Cry1Ac and Cry2Aa dendotoxins of Bacillus thuringiensis against Helicoverpa armigera (H.). Crop Protect. 21, 817–822. Bird, L.J., Akhurst, R.J., 2007. Variation in susceptibility of Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) (Lepidoptera: Noctuidae) in Australia to two Bacillus thuringiensis toxins. J. Invertebr. Pathol. 94, 84–94. Bot, J., 1966. Rearing Heliothis armigera Hübner and Prodemia litura F. on an artificial diet. South Afr. J. Agric. Sci. 9, 535–538. Bouwer, G., Avyidi, D., 2006. Application of the droplet feeding assay method to two economically important African lepidopteran pests. Afr. Entomol. 14, 195–198. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Chakrabarti, S.K., Mandaokar, A., Kumar, P.A., Sharma, R.P., 1998. Efficacy of Lepidopteran specific d-endotoxins of Bacillus thuringiensis against Helicoverpa armigera. J. Invertebr. Pathol. 72, 336–337. Chandrashekar, K., Kumari, A., Kalia, V., Gujar, G.T., 2005. Baseline susceptibility of the American bollworm, Helicoverpa armigera (Hübner) to Bacillus thuringiensis Berl var. kurstaki and its endotoxins in India. Curr. Sci. 88, 167–175. Chung, C.T., Niemela, S.L., Miller, R.H., 1989. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc. Nat. Acad. Sci. USA 86, 2172–2175. Cooper, D.J., 1984. The application of a model to achieve predicted mortality in a field trial using Bacillus thuringiensis to control Heliothis punctiger. Entomol. Exp. Appl. 36, 253–259.
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Crickmore, N., Zeigler, D.R., Feitelson, J., Schnepf, E., Van Rie, J., Lereclus, D., Baum, J., Dean, D.H., 1998. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 807–813. Crickmore, N., Zeigler, D.R., Schnepf, E., Van Rie, J., Lereclus, D., Baum, J., Bravo, A., Dean, D.H., 2011. Bacillus thuringiensis toxin nomenclature.
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Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D.R., Dean, D.H., 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Mol. Biol. Rev. 62, 775–806. van Frankenhuyzen, K., 2009. Insecticidal activity of Bacillus thuringiensis crystal proteins. J. Invertebr. Pathol. 101, 1–16. van Frankenhuyzen, K., Gringorten, J.L., Gauthier, D., Milne, R.E., Masson, L., Peferoen, M., 1993. Toxicity of activated Cry1 proteins from Bacillus thuringiensis to six forest lepidoptera and Bombyx mori. J. Invertebr. Pathol. 62, 295–301.
van Jaarsveld, M.J., Basson, N.C.J., Marais, P., 1998. Synthetic pyrethroid resistance in field strains of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in South Africa. Afr. Plant Protect. 4, 15–18. Vassal, J.M., Brevault, T., Achaleke, J., Menozzi, P., 2008. Genetic structure of the polyphagous pest Helicoverpa armigera (Lepidoptera: Noctuidae) across the subSaharan cotton belt. Commun. Agric. Appl. Biol. Sci. 73, 433–437. Zhong, C., Ellar, D.J., Bishop, A., Johnson, C., Lin, S., Hart, E.R., 2000. Characterization of a Bacillus thuringiensis d-endotoxin which is toxic to insects in three orders. J. Invertebr. Pathol. 76, 131–139.
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Toxicity of Bacillus thuringiensis Cry proteins to Helicoverpa armigera (Lepidoptera: Noctuidae) in South Africa Hua Li, Gustav Bouwer ⇑ School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, Wits 2050, Johannesburg, South Africa
a r t i c l e
i n f o
Article history: Received 29 June 2011 Accepted 10 October 2011 Available online 15 October 2011 Keywords: Helicoverpa armigera Bacillus thuringiensis Cry proteins Escherichia coli expression Larvicidal activity Larval growth inhibition
a b s t r a c t The susceptibility of one of the most important pests in southern Africa, Helicoverpa armigera (Lepidoptera: Noctuidae), to Bacillus thuringiensis Cry proteins was evaluated by bioassay. Cry proteins were produced in Escherichia coli BL21 cells that were transformed with plasmids containing one of six cry genes. The toxicity of each Cry protein to H. armigera larvae was determined by the diet contamination method for second instar larvae and the droplet feeding method for neonate larvae. For each of the proteins, dose– mortality and dose–growth inhibition responses were analyzed and the median lethal dose (LD50) and median inhibitory dose (ID50) determined. Second instar larvae were consistently less susceptible to the evaluated Cry proteins than neonate larvae. The relative toxicity of Cry proteins ranked differently between neonate larvae and second instar larvae. On the basis of the LD50 and ID50 values, Cry1Ab, Cry1Ac, and Cry2Aa were the most toxic of the evaluated proteins to H. armigera larvae. The study provides an initial benchmark of the toxicity of individual Cry proteins to H. armigera in South Africa. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Bacillus thuringiensis (Bt) is a gram-positive, rod shaped, sporeforming bacterium that produces one or more proteinaceous parasporal crystals during sporulation (Höfte and Whiteley, 1989). The Bt crystals may consist of either Cyt proteins or Cry proteins (Höfte and Whiteley, 1989; Schnepf et al., 1998). As a group, Cry proteins are toxic to certain species in the orders Lepidoptera, Diptera, Coleoptera, Hymenoptera and Hemiptera, and also to other invertebrates such as nematodes and mites (Crickmore et al., 2011; Schnepf et al., 1998; van Frankenhuyzen, 2009). Although order-specificity was initially considered important in the classification of Cry proteins (Höfte and Whiteley, 1989), the widely-adopted Cry nomenclature of Crickmore et al. (1998) is based on amino acid sequence divergence rather than functional aspects of the proteins, such as the specificity of toxicity. However, several Cry proteins displaying cross-order activity have been identified; for example, the primarily lepidopteran-active Cry1Ba protein was reported to be toxic to Coleoptera (Naimov et al., 2001; Zhong et al., 2000) and Diptera (Heath et al., 2004; Zhong et al., 2000), and the primarily lepidopteran-active Cry1Ia protein exhibited toxicity to coleopteran insects (Martins et al., 2008; Naimov et al., 2001; Ruiz et al., 2006). Different lepidopteran species may have different levels of susceptibility to a specific Cry protein. For example, Cry1Ac shows ⇑ Corresponding author. Fax: +27 11 717 6337. E-mail address: [email protected] (G. Bouwer). 0022-2011/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2011.10.005
high larvicidal activity against most lepidopteran insects, but very low larvicidal activity against Actebia fennica (Lepidoptera: Noctuidae) (van Frankenhuyzen et al., 1993), Spodoptera frugiperda (Lepidoptera: Noctuidae) (Luo et al., 1999), or Spodoptera littoralis (Lepidoptera: Noctuidae) (Escriche et al., 1998). The relative toxicity of Cry proteins to a susceptible lepidopteran species may also differ. For example, Cry1Ac shows significantly higher larvicidal activity than Cry2Aa against Plutella xylostella (Lepidoptera: Plutellidae) (Monnerat et al., 1999). The African bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), is a polyphagous pest whose larvae feed on a wide range of plants including many important cultivated crops (Vassal et al., 2008). In South Africa, it achieves major pest status in cotton, tomato, and maize and causes serious losses throughout its range (van Jaarsveld et al., 1998). The susceptibility of H. armigera to various Cry proteins has been evaluated in many countries over the past decades. In Australia, it was reported that only Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab killed H. armigera at doses that could be considered acceptable, whereas Cry1B, Cry1C and Cry1E were considered non-toxic (Liao et al., 2002). In India, Cry1Ac was the most toxic Cry protein to H. armigera followed by Cry1Ab and Cry1Aa (Chandrashekar et al., 2005). Although Cry1Ac was also the most toxic of the Cry proteins evaluated in another Indian study (Chakrabarti et al., 1998), the authors found that Cry1Aa was more toxic than either Cry2Aa or Cry1Ab. Studies in different countries have reported significant differences in relative toxicities for the same Cry proteins, e.g. the Cry2Aa/Cry1Ac median lethal concentration (LC50) ratio was 35.5 in India (Chakrabarti et al.,
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1998) and 1.3 in Australia (Liao et al., 2002). It is thus apparent that H. armigera larvae in different countries or from geographically distinct source populations can differ in susceptibility to the same Cry proteins. To our knowledge, no studies have determined the susceptibility of South African H. armigera populations to different Cry proteins. The assessment of the toxicity of individual Cry proteins would provide an important step in the determination of their potential for use in H. armigera control programmes in South Africa. In this study, we evaluated the toxicity of six Cry proteins to a South African population of H. armigera. 2. Materials and methods 2.1. Transformation of cry genes into Escherichia coli BL21 The recombinant E. coli strains used in this study were originally obtained from the Bacillus Genetic Stock Centre: ECE52 (cry1Aa cloned in plasmid pKK223-3 in E. coli JM103), ECE54 (cry1Ab cloned in plasmid pKK223-3 in E. coli JM103), ECE53 (cry1Ac cloned in plasmid pKK223-3 in E. coli JM103), ECE125 (cry1Ca cloned in plasmid pTZ19R in E. coli DH5a), ECE126 (cry2Aa cloned in plasmid pTZ19R in E. coli DH5a), and ECE130 (cry9Aa cloned in plasmid pSB1402 in E. coli DH5a). Plasmids containing one of the six cry genes were isolated from the relevant E. coli strain and transformed into E. coli BL21 (salt inducible strain; Invitrogen, Carlsbad, USA) to standardize the host background for Cry protein production. The plasmid isolation was performed according to the manufacturer’s instructions for the QIAprep Spin Miniprep Kit (Qiagen, Valencia, USA) and BL21 was transformed with plasmid DNA as described by Chung et al. (1989). Transformed BL21 cells were grown, selected and stored on LAON (Luria–Bertani agar with NaCl omitted, Invitrogen) plates supplemented with 100 lg/ml ampicillin using standard methods as per the manufacturer’s instructions for BL21 (Invitrogen). PCR amplification was executed according to the manufacturer’s instructions (GoTaq DNA Polymerase Kit, Promega, Madison, USA) to confirm the presence of the cry genes in the resultant BL21 transformants by using specific cry gene primers (Table 1) and the plasmids isolated from BL21 transformants as the templates. The primers were designed with cry genes in NCBI GenBank serving as the target sequences (Table 1). Throughout this study, a BL21 transformant expressing one of the Cry proteins was designated as BL21[Cry protein], for example BL21[Cry1Aa].
2.2. Production and quantitation of Cry proteins The expression of the six Cry proteins in the BL21 transformants was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) (data not shown). The SDS–PAGE results showed a prominent protein band at a molecular weight of approximately 130 kDa in BL21[Cry1Aa], BL21[Cry1Ab], BL21[Cry1Ac], and BL21[Cry9Aa]. A 65 kDa band and 140 kDa band was observed in BL21[Cry2Aa] and BL21[Cry1Ca], respectively. Since there were no distinct bands of the above mentioned sizes in the BL21 control, the presence of the expected Cry proteins in the corresponding BL21 transformants was confirmed. The molecular weights of the Cry proteins in the relevant transformants matched the published molecular weights of the Cry proteins (Gamel and Piot, 1992; Kalman et al., 1995). The six different Cry proteins were produced as protoxin inclusion bodies (crystals) in BL21 transformants. Although the expression of cry1Aa, cry1Ab and cry1Ac did not require induction since the pKK223-3 cloning vector does not utilize the T7 promoter, salt induction was performed for all BL21 transformants so as to standardize conditions and thus minimize any potential differences in protein expression profiles. NaCl induction of protein expression was performed according to the method of Donahue and Bebee (1999). After induction, all cultures were incubated at 37 °C with constant shaking (200 rpm) until the formation of crystals was confirmed (using optical microscopy) and almost all of the cells had lysed. Centrifugation of the cultures was performed for 15 min at 9000g at 4 °C and the pellets were suspended in sterile double-distilled water and the washing step was repeated twice more. The resuspended pellets were stored at 4 °C. The concentration of Cry proteins was determined by a novel quantitation strategy, hereafter referred to as the Bradford/ Pro260 method. The Bradford method (Bradford, 1976), using the Coomassie Plus Protein Assay Kit (Thermo Fisher Scientific, Rockford, USA) and bovine serum albumin (BSA) as the standard, was used to evaluate the total protein concentration of each Cry protein sample. Subsequently, the concentration of the Cry protein in the sample was calculated by the formula: Cry protein concentration (lg/ml) = (lg/ml total protein) (proportion of Cry protein to total protein), with the proportion of Cry protein to total protein being determined using the Experion Pro260 automatic electrophoresis system (Biorad, Hercules, USA). The Bradford/Pro260 method showed better accuracy and reproducibility than either densitometry analysis of SDS–PAGE gels or the Experion Pro260 system
Table 1 The sequences and characteristics of primers used to detect cry genes in E. coli BL21 transformants.
a b c
Gene detected
Accession No.a
Plasmidb
Primer name
Sequence
Product size (bp)
cry1Aa
M11250
pKK223-3
C1Aa-d-F C1-consv-Rc
50 -TTATACTTGGTTCAGGCCC-30 50 -CTTGTGACACTTCTGCTTCC-30
1950
cry1Ab
M12661
pKK223-3
C1Ab-d-F C1-consv-Rc
50 -CAATCGGAAAATGTGCC-30 50 -CTTGTGACACTTCTGCTTCC-30
609
cry1Ac
M11608
pKK223-3
C1Ac-d-F C1-consv-Rc
50 -CAAAGACATTAATAGGCAACC-30 50 -CTTGTGACACTTCTGCTTCC-30
992
cry1Ca
AF362020
pTZ19R
C1Ca-d-F C1-consv-Rc
50 -GATCTGGAACACCTTTTTTAAC-30 50 -CTTGTGACACTTCTGCCTCC-30
1773
cry2Aa
M23723
pTZ19R
C2Aa-d-F C2Aa-d-R
50 -CCTTGCTCGTGTAAATGC-30 50 -CTAGCATATAAATTAGCGCCAG-30
507
cry9Aa
X58120
pSB1402
C9Aa-d-F C9Aa-d-R
50 -GGTTCACTTACATTGCCGGTTAG-30 50 -CGCTTTTTTTGCTGCTTCTAG-30
954
Accession number of the relevant cry gene in NCBI GenBank. Plasmid that the cry gene is cloned in. Reverse primer binds to conserved region of cry1A-class genes.
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(unpublished data). The concentrations of Cry proteins estimated by the Bradford/Pro260 method were on average 1.05 times higher than those estimated by densitometry analysis of SDS–PAGE gels (unpublished data). For each sample used in bioassays, the Cry protein concentration was the average of six separate estimates. 2.3. Insects H. armigera eggs were obtained from a culture maintained at the Agricultural Research Council (Pretoria, South Africa). This culture was established with specimens collected from the Springbok Flats (29°400 S 17°530 E) in 1974 and supplemented in 2006 with specimens collected from cotton plants near Groblersdal (25°150 S 29°250 E) in Mpumalanga, South Africa (Mawela et al., 2010). Larvae were reared in our laboratory on a wheat-germ based artificial diet (Bot, 1966). During oviposition, H. armigera adults were provided with 5% (w/v) sucrose solution. The culture was maintained in a growth chamber at 28 ± 1 °C, 70% relative humidity, and a photoperiod of 12:12 h (L:D). 2.4. Dose–mortality bioassays The toxicity of Cry proteins to H. armigera larvae was evaluated by dose–mortality bioassays. Bioassays of neonate larvae were performed using the droplet feeding method (Hughes and Wood, 1981), as adapted for H. armigera by Bouwer and Avyidi (2006). Larvae that imbibed the mixture of feeding solution and Cry protein were placed individually in wells (24-well tissue culture plate; Nalge Nunc International, Rochester, USA) containing the artificial diet. Trays were sealed with Parafilm (Sigma, St. Louis, USA), which was perforated to provide aeration, and a plastic lid. Bioassays were performed with second instar larvae (6 h after moulting) using a modification of the diet contamination method of Ferré et al. (1991). Freshly prepared diet disks (4 mm diameter and 1 mm thick) were dispensed into the wells of a 24-well tissue culture plate. Dilutions of the Cry proteins were applied uniformly over the food surface and allowed to dry. One second instar larva was placed into each well and the multi-well plates were sealed with perforated Parafilm and a plastic lid. Larvae that consumed the entire contaminated diet disk within 2 days were transferred to new individual wells containing untreated artificial diet. Four replicates of 24 larvae per replicate (n = 96) were used for each dose of a Cry protein and for the control (water only). A range of seven doses and six doses were used for bioassays of neonate and second instar larvae, respectively. For bioassays of neonate larvae, mortality was scored 4 days after treatment. For bioassays of second instar larvae, mortality was scored 4 days after the treated larvae were transferred to untreated diet. 2.5. Dose–weight bioassays The effect of sublethal doses of Cry proteins on the development and growth of H. armigera larvae was evaluated using dose–weight bioassays, with the ID50 (median inhibitory dose; the dose that caused a 50% lower larval weight compared to the controls) being used to compare the relative toxicity of the different Cry proteins. The diet contamination method and the droplet feeding method were applied to H. armigera second instar and neonate larvae, respectively, as described above. Five doses of Cry proteins were used for both second instar larvae and freshly hatched neonate larvae. For each bioassay method, a total of 40–55 larvae that survived treatment were weighed (wet weight) for each dose. Water controls were also included in the assays. The weights of neonate larvae were recorded 7 days after treatment, whereas the weights of second instar larvae were recorded 7 days after the treated larvae were transferred to uncontaminated diet.
2.6. Data analysis Dose–mortality data were subjected to probit regression analysis (Finney, 1971), and the median lethal dose (LD50) was estimated for each Cry protein. GraphPad Prism 5 (GraphPad Software Inc, San Diego, USA) was used to evaluate the dose–weight bioassay data. The ID50 estimates were derived using nonlinear regression fitting of a sigmoidal dose–response curve to Cry-treated larval weight data. The function used to calculate the ID50 was log dose versus response-variable slope (four parameters). 3. Results 3.1. Larvicidal activity of Cry proteins against neonate larvae The results of the probit analyses of the neonate dose–mortality bioassays are shown in Table 2. On the basis of LD50 values, the larvicidal activity of Cry1Ac was significantly (statistically) higher than any of the other Cry proteins evaluated and Cry2Aa was the next most toxic Cry protein evaluated. The larvicidal activity of Cry2Aa was significantly higher than that of Cry1Ab, which was in turn significantly higher than that of Cry1Aa. Cry1Ca and Cry9Aa showed the lowest larvicidal activity, with the LD50 of Cry1Ac being approximately 20,510 and 24,970 times lower than that of Cry1Ca and Cry9Aa, respectively (Table 2). 3.2. Larvicidal activity of Cry proteins against second instar larvae In bioassays against second instar larvae, the LD50 of Cry1Ac was significantly lower than the LD50 of any one of the other Cry protein evaluated (Table 3). In contrast to the results obtained for neonate larvae, Cry1Ab was significantly more toxic than Cry2Aa. The larvicidal activities of Cry1Aa, Cry9Aa, and Cry1Ca did not differ significantly from each other and the LD50 values of these proteins are at least 4000-fold higher than that of Cry1Ac (Table 3). 3.3. Dose–growth inhibition responses of neonate larvae to Cry proteins Neonate larvae that survived after treatment with Cry1Ac, Cry2Aa, Cry1Ab, Cry1Aa, or Cry9Aa showed significant stunting in their growth compared to the control larvae. On the basis of ID50 values, Cry1Ac exhibited higher larval growth inhibition activity than any of the other Cry proteins (Table 4). There was no statistical difference between the ID50 values of Cry1Ab and Cry2Aa. Cry1Aa caused statistically more growth inhibition than Cry9Aa (Table 4). Due to Cry1Ca’s poor growth inhibition activity at the evaluated doses, no ID50 could be estimated for this protein against H. armigera neonate larvae. 3.4. Dose–growth inhibition responses of second instar larvae to Cry proteins Cry1Ac exhibited significantly higher growth inhibition activity against second instar larvae than any of the other Cry proteins, whereas the weight inhibition activity of Cry1Ca was so low that no ID50 could be estimated for this protein (Table 5). The ID50 values of Cry1Ab, Cry2Aa and Cry1Aa were not significantly different. Cry9Aa had the lowest growth inhibition activity. 4. Discussion Since Cry proteins are usually found as combinations in natural Bt strains (Agaisse and Lereclus, 1995), the determination of the
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H. Li, G. Bouwer / Journal of Invertebrate Pathology 109 (2012) 110–116 Table 2 Dose–mortality responses of H. armigera neonate larvae to Cry proteins as determined by droplet feeding bioassays.
A B C
Cry proteins
LD50 (ng)A
Cry1Ac Cry2Aa Cry1Ab Cry1Aa Cry1Ca Cry9Aa
3.14 102a 1.39 101b 6.10 101c 1.02 101d 6.44 102e 7.84 102e
95% Fiducial limits Lower
Upper
2.05 102 1.07 101 5.20 101 6.48 100 3.38 102 5.33 102
4.67 102 1.80 101 7.30 101 1.57 101 1.48 103 1.27 103
Slope ± SEB
v2 (df)C
0.66 ± 0.05 1.08 ± 0.08 1.46 ± 0.13 0.68 ± 0.05 0.41 ± 0.05 0.71 ± 0.08
5.65 7.76 6.84 8.88 7.69 0.90
Slope ± SEB
v2 (df)C
0.54 ± 0.05 0.44 ± 0.04 0.39 ± 0.05 0.13 ± 0.03 0.46 ± 0.05 0.14 ± 0.03
1.9 5.7 2.8 3.0 3.9 3.2
(4) (4) (3) (4) (4) (4)
Values followed by the same letters are not significantly different (overlapping 95% fiducial limits). Slope ± standard error. Values in brackets show degrees of freedom (df).
Table 3 Dose–mortality responses of H. armigera second instar larvae to Cry proteins as determined by diet contamination bioassays.
A B C
Cry proteins
LD50 (ng)A
Cry1Ac Cry1Ab Cry2Aa Cry1Aa Cry9Aa Cry1Ca
5.05 101a 5.30 100b 5.26 101c 2.16 103d 4.09 103d 2.70 104d
95% Fiducial limits Lower
Upper
2.58 101 2.50 100 2.49 101 2.72 102 2.11 103 2.04 103
9.18 101 1.03 101 1.20 102 1.70 105 8.35 103 1.85 107
(4) (4) (3) (4) (3) (4)
Values followed by the same letters are not significantly different (overlapping 95% fiducial limits). Slope ± standard error. Values in brackets show degrees of freedom (df).
Table 4 Dose–growth inhibition responses of H. armigera neonate larvae to Cry proteins as determined by droplet feeding bioassays. Cry proteins
ID50 (ng)A
Cry1Ac Cry1Ab Cry2Aa Cry1Aa Cry9Aa Cry1Ca
2.00 104a 1.00 103b 2.00 103b 1.20 101c 2.95 100 d NCC
95% Confidence intervals Lower
Upper
2.00 104 6.00 104 9.00 104 3.00 102 1.60 100
3.00 104 3.00 103 3.00 103 4.20 101 5.45 100
Slope ± SEB
1.15 ± 0.11 0.40 ± 0.05 0.44 ± 0.04 0.37 ± 0.06 0.76 ± 0.14
A Values followed by the same letters are not significantly different (overlapping 95% confidence intervals). B Slope ± standard error. Hill slope is reported. C NC: not calculated, ID50 substantially higher than the highest dose evaluated.
Table 5 Dose–growth inhibition responses of H. armigera second instar larvae to Cry proteins as determined by diet contamination bioassays. Cry proteins
ID50 (ng)A
Cry1Ac Cry1Ab Cry2Aa Cry1Aa Cry9Aa Cry1Ca
3.00 103a 2.00 102b 1.00 101bc 1.67 101bc 2.28 102c NCC
95% Confidence intervals Lower
Upper
2.00 103 7.00 103 3.00 102 3.00 102 7.53 100
4.00 103 4.00 102 3.90 103 8.19 103 6.90 103
Slope ± SEB
0.69 ± 0.07 0.46 ± 0.07 0.23 ± 0.05 0.26 ± 0.10 0.35 ± 0.08
A Values followed by the same letters are not significantly different (overlapping 95% confidence intervals). B Slope ± standard error. Hill slope is reported. C NC: not calculated, ID50 substantially higher than the highest dose evaluated.
relative toxicity of single Cry proteins using natural Bt strains is often not feasible. However, some studies have used Bt strains expressing single Cry proteins (Ibargutxi et al., 2006). We evaluated the toxicity of Cry proteins produced in E. coli, an approach
used by several studies that evaluated the toxicity of Cry proteins to H. armigera (Chakrabarti et al., 1998; Chandrashekar et al., 2005; Lee et al., 1996). By using E. coli as an expression host for cry genes, any potential synergistic interactions between Bt spores and crystals may be avoided (Johnson et al., 1998). A review of the larvicidal activity of Bt toxins to various insects showed that there is no evidence that expression host is a major determinant of crystal toxicity or affects the toxicity ranking of crystals (van Frankenhuyzen, 2009). The majority of published bioassay data for various lepidopteran species used lethal concentration (LC) and inhibitory concentration (IC) as the units of estimates to describe the toxicity of Cry proteins (van Frankenhuyzen, 2009). However, lethal dose (LD) and inhibitory dose (ID) may be preferable to LC and EC in evaluating the susceptibility of an insect to a specific toxin, because the LD and ID estimates are based on the actual dose ingested by the larvae rather than the concentration to which the insect was exposed to (Hughes and Wood, 1981). Different bioassay methods can be used to obtain LD or ED estimates, including the diet contamination method (Ignoffo, 1965) and the droplet feeding method (Hughes and Wood, 1981). However, the diet contamination method is not suitable for neonate larvae as the larvae are unlikely to fully consume even a very small block of food. The droplet feeding method is, thus, a valuable bioassay method for the estimation of LD or ED values for neonate larvae. The droplet feeding method is not suitable for post-first instar larvae because it is often practically impossible to identify the larvae which imbibed the colored feeding solution by the presence of the dye in their foreguts and midguts. From the second instar onwards, the diet contamination method may be used because these larvae are able to fully consume a small food block. To enable LD and ED estimation in this study, the toxicity of each Cry protein to H. armigera larvae was determined by the diet contamination method for second instar larvae and the droplet feeding method for neonate larvae. Bt biopesticides are usually applied when early instar larvae are present as older larvae are more tolerant (Sanahuja et al., 2011). We thus focused on the susceptibility of the first and second larval
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stages to the Cry proteins. Although several studies have compared the susceptibility of neonate and third instar larvae of H. armigera to Bt products or Cry proteins (Bird and Akhurst, 2007; Cooper, 1984; Liao et al., 2002), a consistent pattern in susceptibility is not apparent. Liao et al. (2002) demonstrated that there was no difference in LC50 values between H. armigera third instar and neonate larvae for commercial Bt products. In contrast, Bird and Akhurst (2007) reported that H. armigera neonate larvae were less susceptible to Cry1Ac than third instar larvae based on LC50 and median inhibitory concentration (IC50) values, respectively, but more susceptible to Cry2Ab than third instar larvae. In our study, neonate larvae exhibited consistently lower LD50 and ID50 values than second instar larvae. This decreased susceptibility for second instar larvae was in agreement with other studies that showed that later instar larvae of Helicoverpa zea (Lepidoptera: Noctuidae) and P. xylostella were less susceptible to Cry proteins than early instar larvae (Ali and Young, 1996; Asano et al., 1993). In this study, a steeper slope was found in most dose–mortality and dose–weight regression lines of Cry proteins in H. armigera neonate larvae compared to those of second instar larvae, suggesting a more homogenous population with regard to susceptibility to Cry proteins. A similar trend was reported by Bird and Akhurst (2007), where the slope value of concentration–mortality regression lines of Cry1Ac decreased when H. armigera neonate larvae developed to third instar larvae. There are marked differences between the absolute values of LC50 estimates for the same Cry protein in different studies (Avilla et al., 2005; Bird and Akhurst, 2007; Chakrabarti et al., 1998; Ibargutxi et al., 2008; Liao et al., 2002). The following factors are likely to contribute to the observed differences: the bioassay method used (e.g., diet incorporation or surface treatment), the insect diet used, the level of processing of the Cry proteins used in the bioassays (e.g., protoxins or activated toxin), the protein quantitation method used, and the temperature at which bioassays were performed (Avilla et al., 2005; Bird and Akhurst, 2007). Although LD bioassays have some advantages over LC bioassays, we are not aware of any studies that have determined the LD50 values for Cry proteins assayed against H. armigera. Since we estimated LD50 values rather than LC50 values and we used the droplet feeding assay method for neonate larvae, it is not possible to directly compare our absolute values of LD50 estimates with estimates in other studies that evaluated Cry proteins against H. armigera (Avilla et al., 2005; Bird and Akhurst, 2007; Chakrabarti et al., 1998; Ibargutxi et al., 2008; Liao et al., 2002). Comparisons of the toxicity data produced in different studies should, thus, focus on the relative toxicity or toxicity ranking of the Cry proteins rather than the absolute values of toxicity estimates. However, several factors can cause differences in the relative toxicity of Cry proteins to the same insect species, including: different levels of precision resulting from different bioassay methods, the use of test colonies derived from geographically distinct source populations, and evaluation of different developmental stages of a given test species (Avilla et al., 2005; van Frankenhuyzen, 2009). When reviewing the toxicity ranking reported in other studies, we believe it is important to consider whether the authors based their ranking of Cry proteins purely on numerical differences in LC50 values or on significant differences, e.g. non-overlapping 95% fiducial limits. We obtained the following ranking of larvicidal activity (LD50 values) against neonate larvae: Cry1Ac < Cry2Aa < Cry1Ab < Cry1Aa < (Cry1Ca = Cry9Aa), where < means significantly lower (non-overlapping 95% fiducial limits) and = means not significantly different. Cry1Ac and Cry2Aa thus had the highest larvicidal activities of the tested Cry proteins against H. armigera neonate larvae, which is consistent with the result obtained by other researchers (Avilla et al., 2005; Chakrabarti et al., 1998; Chandrashekar et al., 2005; Liao et al., 2002). The Cry2Aa/Cry1Ac LD50 ratio was 4.4,
which is within the wide Cry2Aa/Cry1Ac LC50 ratio range obtained in other studies, for example: 35.5 (Chakrabarti et al., 1998), 10.2 (Ibargutxi et al., 2008), 6.6 (Babu et al., 2002), 1.8 (Avilla et al., 2005), and 1.3 (Liao et al., 2002). Although most H. armigera studies have also found Cry1Ac and Cry2Aa to be the most toxic to neonate larvae, there are differences between the toxicity rankings of Cry1Ab, Cry1Aa, Cry1Ca, and Cry9Aa in our study and other studies (e.g. Chakrabarti et al., 1998). In agreement with our results, other studies have reported that Cry9Aa has very low toxicity to H. armigera neonate larvae (Chakrabarti et al., 1998; Liao et al., 2002). Sublethal effects (feeding inhibition and growth inhibition) are considered more sensitive indicators of Cry protein toxicity than lethal effects (van Frankenhuyzen, 2009). To further characterize the toxicity of the Cry proteins to neonate larvae, we estimated ID50 values. We obtained the following ranking of ID50 values against neonate larvae: Cry1Ac < (Cry1Ab = Cry2Aa) < Cry1Aa < Cry9Aa. Due to Cry1Ca’s poor growth inhibition activity at the evaluated doses, no ID50 could be estimated for Cry1Ca. Although the ranking is very similar to that obtained for LD50 values, there are differences, e.g. Cry2Aa < Cry1Ab based on LD50 values, but Cry2Aa = Cry1Ab based on ID50 values. When considering both the LD50 and ID50 values obtained for neonate larvae, it is apparent that the most toxic Cry proteins were Cry1Ac, Cry1Ab, and Cry2Aa. We obtained the following ranking of larvicidal activity (LD50 values) against second instar larvae: Cry1Ac < Cry1Ab < Cry2Aa < (Cry1Aa = Cry9Aa = Cry1Ca). Cry1Ac was again the most toxic of the evaluated proteins, but in contrast to the results for neonate larvae Cry1Ab was more toxic than Cry2Aa, and Cry1Aa clustered with Cry9Aa and Cry1Ca. To our knowledge, no other published studies have evaluated the toxicity of Cry proteins against second instar H. armigera larvae, and we can thus not directly compare our results to other studies. On the basis of ID50 values, Cry1Ac was the most toxic Cry protein to second instar larvae, with the ranking as follows: Cry1Ac < (Cry1Ab = Cry2Aa = Cry1Aa). Due to its low sublethal activity, no ID50 could be estimated for Cry1Ca. Low toxicity of Cry1C proteins has been reported for other H. armigera populations (Chakrabarti et al., 1998; Liao et al., 2002). The South African H. armigera population was generally susceptible to most of the tested Cry proteins, and Cry1Ac was consistently more toxic than any of the other evaluated Cry proteins to both neonate and second instar larvae. Although not as toxic as Cry1Ac, Cry1Ab and Cry2Aa could be considered as good candidates for the control of H. armigera in South Africa. In bioassays conducted over three seasons, Bird and Akhurst (2007) found significant differences in susceptibility to Cry1Ac and Cry2Ab spore-crystal mixes in Australian strains of H. armigera. Although inter-strain differences in susceptibility to a Cry protein is not unexpected, a critical issue is whether these differences in susceptibility affect the ranking of the toxicity of the Cry proteins in the different strains. To address this issue, we analyzed the LC50 data for those H. armigera strains for which Bird and Akhurst (2007) reported bioassay data for both Cry1Ac and Cry2Ab (n = 15). This analysis of the strain-specific LC50 values showed that Cry1Ac was more toxic than Cry2Ab in all the tested strains, with a mean Cry2Ab/Cry1Ac LC50 ratio of 3.0 ± 0.3 (SE). The Cry2Ab/ Cry1Ac LC50 ratio for their H. armigera laboratory strain, which had been cultured in the laboratory for approximately 4 years, was 2.2. Our analysis of the data presented by Bird and Akhurst (2007) suggests that, in the absence of significant field-developed resistance to specific Cry proteins, inter-strain variation in susceptibility to Cry proteins may not be large enough to cause different H. armigera strains to have different Cry toxicity rankings. Nonetheless, evaluation of the susceptibility of different South African H. armigera populations or strains to the Cry proteins used in our study may provide information that will be useful in pest control programmes.
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The choice of Cry proteins to pyramid in transgenic plants or recombinant Bt strains should consider which combinations of Cry proteins are more likely to prevent or delay the development of resistance in insects. From a resistance management standpoint, Cry1Ac and Cry2Aa would be a good combination for the control of H. armigera because these two proteins utilize different receptor binding sites (Estela et al., 2004; Gahan et al., 2005). The rationale for combining Cry1Ac and Cry2Aa is supported by studies that looked at Cry-resistant strains: a Cry1Ac-resistant strain of H. armigera was not resistant to Cry2Aa or Cry2Ab (Akhurst et al., 2003), and a Cry2Ab-resistant strain of H. armigera, which was crossresistant to Cry2Aa, was susceptible to Cry1Ac (Mahon et al., 2007). In conclusion, this is the first report of the toxicity of Cry proteins to a South African H. armigera population. Because we estimated LD50 and ID50 values in both neonate and second instar larvae, we believe that this study presents a more comprehensive evaluation of Cry toxicity than other studies that evaluated the toxicity of Cry proteins to H. armigera. The study provides an initial benchmark of the toxicity of individual Cry proteins to H. armigera in South Africa. In order to gain insight into the variation of H. armigera susceptibility in South Africa, future studies will evaluate the susceptibility of multiple, geographically distinct H. armigera populations to a range of Cry proteins. Acknowledgments We thank Michelle Grant for assistance with the maintenance of the H. armigera cultures. This project was partially funded by a LIFELab Biotechnology Regional Innovation Centre research grant awarded to Gustav Bouwer. We thank anonymous reviewers for comments that improved this manuscript. References Agaisse, H., Lereclus, D., 1995. How does Bacillus thuringiensis produce so much insecticidal crystal protein? J. Bacteriol. 177, 6027–6032. Akhurst, R.J., James, W., Bird, L.J., Beard, C., 2003. Resistance to the Cry1Ac dendotoxin of Bacillus thuringiensis in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae). J. Econ. Entomol. 96, 1290–1299. Ali, A., Young, S.Y., 1996. Activity of Bacillus thuringiensis Berliner against different ages and stages of Helicoverpa zea (Lepidoptera: Noctuidae) on cotton. J. Entomol. Sci. 31, 1–8. Asano, S., Maruyama, T., Iwasa, T., Seki, A., Takahashi, M., Soares Jnr, G.G., 1993. Evaluation of biological activity of Bacillus thuringiensis test samples using a diet incorporation method with diamondback moth, Plutella xylostella (Linnaeus) (Lepidoptera: Yponomeutidae). Appl. Entomol. Zool. 28, 513–524. Avilla, C., Vargas-Osuna, E., González-Cabrera, J., Ferré, J., González-Zamora, J.E., 2005. Toxicity of several delta-endotoxins of Bacillus thuringiensis against Helicoverpa armigera (Lepidoptera: Noctuidae) from Spain. J. Invertebr. Pathol. 90, 51–54. Babu, B.G., Udayasuriyan, V., Mariam, M.A., Sivakumar, N.C., Bharathi, M., Balasubramanian, G., 2002. Comparative toxicity of Cry1Ac and Cry2Aa dendotoxins of Bacillus thuringiensis against Helicoverpa armigera (H.). Crop Protect. 21, 817–822. Bird, L.J., Akhurst, R.J., 2007. Variation in susceptibility of Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) (Lepidoptera: Noctuidae) in Australia to two Bacillus thuringiensis toxins. J. Invertebr. Pathol. 94, 84–94. Bot, J., 1966. Rearing Heliothis armigera Hübner and Prodemia litura F. on an artificial diet. South Afr. J. Agric. Sci. 9, 535–538. Bouwer, G., Avyidi, D., 2006. Application of the droplet feeding assay method to two economically important African lepidopteran pests. Afr. Entomol. 14, 195–198. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Chakrabarti, S.K., Mandaokar, A., Kumar, P.A., Sharma, R.P., 1998. Efficacy of Lepidopteran specific d-endotoxins of Bacillus thuringiensis against Helicoverpa armigera. J. Invertebr. Pathol. 72, 336–337. Chandrashekar, K., Kumari, A., Kalia, V., Gujar, G.T., 2005. Baseline susceptibility of the American bollworm, Helicoverpa armigera (Hübner) to Bacillus thuringiensis Berl var. kurstaki and its endotoxins in India. Curr. Sci. 88, 167–175. Chung, C.T., Niemela, S.L., Miller, R.H., 1989. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc. Nat. Acad. Sci. USA 86, 2172–2175. Cooper, D.J., 1984. The application of a model to achieve predicted mortality in a field trial using Bacillus thuringiensis to control Heliothis punctiger. Entomol. Exp. Appl. 36, 253–259.
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