Evaluation of the synergistic activities of Bacillus thuringiensis Cry proteins against Helicoverpa armigera (Lepidoptera: Noctuidae).

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Evaluation of the synergistic activities of Bacillus thuringiensis Cry proteins against Helicoverpa armigera (Lepidoptera: Noctuidae)

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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 17 April 2014 Accepted 13 June 2014 Available online xxxx Keywords: Helicoverpa armigera Bacillus thuringiensis Cry proteins Larvicidal activity Interaction Synergism

a b s t r a c t With the aim of identifying Cry proteins that would be useful in the management of the economically important lepidopteran pest Helicoverpa armigera, the larvicidal activities of binary combinations (1:1 ratios) of six Cry proteins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, Cry2Aa and Cry9Aa) were evaluated against H. armigera neonate larvae using droplet feeding bioassays. Determination of the LD50 values of individual Cry proteins and mixtures of Cry proteins enabled assessment of the nature of the interactions between Cry proteins in H. armigera. There was a more than 6000-fold difference between the LD50 values of the Cry protein mixture with the lowest larvicidal activity and the mixture with the highest larvicidal activity. Cry1Ac and Cry2Aa mixtures and Cry1Ac and Cry1Ca mixtures had the highest larvicidal activity against H. armigera, with Cry1Ac and Cry1Ca interacting synergistically. Differences in the magnitudes of the antagonistic interactions observed for different binary mixtures of Cry1A-class proteins are consistent with a model of more than one binding site for some Cry1A-class proteins in H. armigera. Binary combinations of Cry1A-class and Cry9Aa proteins showed additive interactions in neonate larvae of H. armigera, whereas combinations of Cry1Ca and Cry9Aa were statistically synergistic. The results suggest that products containing mixtures of Cry1Ac and Cry2Aa or Cry1Ac and Cry1Ca may be useful components of H. armigera pest management programs. Ó 2014 Published by Elsevier Inc.

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1. Introduction

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Bacillus thuringiensis (Bt) is a gram-positive, rod-shaped, sporeforming bacterium that produces crystal (Cry) proteins that are toxic to insects (Höfte and Whiteley, 1989). Bt-based biopesticides have been used for several decades worldwide due to their larvicidal activity against target pests and environmentally friendly characteristics. The lethality of Bt towards a wide range of insects, including lepidopteran, dipteran and coleopteran larvae, is attributed largely to the Cry proteins produced during the Bt growth cycle (Crickmore et al., 1998; Feitelson et al., 1992; Höfte and Whiteley, 1989). The Cry proteins are produced as protoxins which can be converted to active toxins upon ingestion by a susceptible insect (Feitelson et al., 1992). Synergistic interactions between Bt insecticidal proteins were first reported when it was found that the mosquitocidal activity of mixtures of diverse Cry proteins of Bt subsp. israelensis (Bti) was greater than that of the isolated proteins (Angsuthanasombat et al., 1992; Chilcott and Ellar, 1988; Poncet et al., 1995). The synergistic interaction between Bti Cyt1Aa, from the Cyt family of Bt

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⇑ Corresponding author. Fax: +27 11 717 6337. E-mail address: [email protected] (G. Bouwer).

toxins, and Cry4Ba has been attributed to Cyt1Aa serving as a specific functional receptor for Cry4Ba (Cantón et al., 2011). Synergistic effects have been reported between Bt spores and Cry proteins against Plodia interpunctella (Lepidoptera: Pyralidae) (Johnson et al., 1998), between Bt subsp. japonensis Buibui strain and entomopathogenic nematodes against Cyclocephala hirta (Coleoptera: Scarabaeidae) and Cyclocephala pasadenae (Koppenhöfer and Kaya, 1997), and between Beauveria bassiana- and Bt subsp. tenebrionisbased biopesticides against Colorado potato beetle larvae (Wraight and Ramos, 2005). Antagonism, another key interaction effect, is characterized by a significantly reduced observed toxicity compared to the expected toxicity of the mixture, and has been observed between Bt Cry proteins in several lepidopteran species (Gao et al., 2010; Ibargutxi et al., 2008; Lee et al., 1996; Sauka et al., 2007). For example, Lee et al. (1996) found that Cry1Aa and Cry1Ab exhibited an antagonistic effect on fourth instar larvae of Lymantria dispar (Lepidoptera: Lymantriidae), and Sauka et al. (2007) reported antagonistic interactions between Cry1A-class proteins in neonate larvae of Epinotia aporema (Lepidoptera: Tortricidae). Antagonism has been observed also between Cry1Ac1 and Cyt1A1 in vitro and in vivo against Trichoplusia ni (Lepidoptera: Noctuidae) (del Rincón-Castro et al., 1999).

http://dx.doi.org/10.1016/j.jip.2014.06.005 0022-2011/Ó 2014 Published by Elsevier Inc.

Please cite this article in press as: Li, H., Bouwer, G. Evaluation of the synergistic activities of Bacillus thuringiensis Cry proteins against Helicoverpa armigera (Lepidoptera: Noctuidae). J. Invertebr. Pathol. (2014), http://dx.doi.org/10.1016/j.jip.2014.06.005

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The interaction between different Cry proteins can differ markedly between insect species, with major differences found between species in the same study using the same toxin preparations (Gao et al., 2010; Ibargutxi et al., 2008). In a study that evaluated the interactions among Cry protoxins, Gao et al. (2010) reported that all Cry protein mixtures (1:1 ratio) evaluated (Cry1Aa–Cry1Ab, Cry1Aa–Cry1Ca, Cry1Ac–Cry1Ca, Cry1Ac–Cry1Ba, Cry1Ab–Cry1Ac, Cry1Ab–Cry1Ba, and Cry1Ab–Cry1Ca) showed significant synergistic activity against Chilo suppressalis, but of these toxin mixtures only Cry1Aa–Cry1Ab showed synergistic activity against Sesamia inferens with the other mixtures displaying significant antagonistic interactions. Such differences in the interaction effects show the importance of evaluating the interaction effects in different insect species. The African bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), is a polyphagous pest whose larvae feed on a wide range of plants (Vassal et al., 2008). In South Africa, H. armigera is a major pest and attacks a wide range of cultivated crops and wild hosts (Moore et al., 2004; van Jaarsveld et al., 1998). Several studies have evaluated the toxicity interactions of Cry proteins against H. armigera; e.g., synergistic interactions have been observed in H. armigera between Cry1Aa and Cry1C (Xue et al., 2005) and between Cry1Ac and Cry2Ab (Ibargutxi et al., 2008), whereas Cry1Ac and Cry1Fa appeared to interact antagonistically (Ibargutxi et al., 2008). Knowledge of the nature of the interaction between Cry proteins is important in the development of highly larvicidal genetically engineered Bt strains or transgenic crops. Only a limited number of Cry protein combinations have been evaluated in toxicity interaction studies using H. armigera; e.g., Cry1Ac and Cry2Ab (Ibargutxi et al., 2008), Cry1Ac and Cry2Aa (Liao et al., 2002), Cry1Ac and Cry1Fa (Ibargutxi et al., 2008), and Cry1Aa and Cry1C (Xue et al., 2005). The limited data set of Cry protein interactions in H. armigera hampers the identification of Cry proteins that may be useful to use in combination in biopesticides or transgenic crops. In this study, we evaluated the interaction effects of all possible binary combinations of six different Cry proteins against H. armigera larvae using droplet feeding bioassays.

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2. Materials and methods

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2.1. Transformation of cry genes into Escherichia coli BL21

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Recombinant E. coli strains 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) were obtained from the Bacillus Genetic Stock Centre, USA. Plasmids containing one of the six cry genes were isolated from the relevant E. coli strains according to the manufacturer’s instructions for the QIAprep Spin Miniprep Kit (Qiagen). The plasmids were transformed into E. coli BL21 (saltinducible strain BL21-SI; Invitrogen, Carlsbad, USA) as described by Chung et al. (1989). Osmolarity is used to regulate T7 promoter-based protein expression in BL21-SI (Donahue and Bebee, 1999). 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 and as per the manufacturer’s instructions for BL21 (Invitrogen). In order to confirm the presence of the cry genes in the resultant BL21 transformants, PCR amplification was performed according to the manufacturer’s instructions for the GoTaq DNA Polymerase Kit (Promega, Madison, USA) using plasmids isolated from BL21

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transformants as templates and specific cry gene internal primers (Li and Bouwer, 2012b). Although van Frankenhuyzen (2009) concluded that there is no evidence that expression host is a major determinant of crystal toxicity or affects the toxicity ranking of Cry proteins, we used the same host strain (E. coli BL21) for the production of each of the six Cry proteins in order to standardize the host background for Cry protein production and remove any potential confounding effects that E. coli host strain may have on larvicidal activity. A BL21 transformant expressing one of the Cry proteins is designated as BL21[Cry protein], e.g. BL21[Cry1Aa].

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2.2. Production of Cry proteins in E. coli BL21

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After confirmation of the presence of the Bt cry genes in the BL21 transformants by PCR (data not shown), the expression of the six Cry proteins in the BL21 transformants was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS– PAGE). The six different Cry proteins were produced as protoxin inclusion bodies (crystals) in BL21 transformants. Because the pKK223-3 cloning vector does not utilize the T7 promoter, the expression of cry1Aa, cry1Ab and cry1Ac did not require induction. However, to standardize conditions and minimize any potential differences in protein expression profiles, osmotic induction (0.3 M NaCl) was performed for all BL21 transformants as described previously (Donahue and Bebee, 1999; Li and Bouwer, 2012a). After salt induction, cultures were incubated at 37 °C on an orbital shaker (200 rpm) until the formation of crystals was confirmed through 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 extracts of BL21 transformants were stored at 4 °C. 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 (data not shown). Since there was no distinct band of the above mentioned sizes in the BL21 (untransformed) control, the SDS–PAGE confirmed the presence of the expected Cry proteins in the corresponding BL21 transformants. 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).

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2.3. Preparation of Cry inclusion bodies

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The semi-purification of Cry protoxin inclusion bodies from BL21 transformants was performed using the method of Sayyed et al. (2005), as implemented by Li and Bouwer (2012a). The semi-purified protoxin inclusions were suspended in sterile double-distilled water and stored at 4 °C.

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2.4. Cry protein quantitation

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The concentration of Cry proteins in the semi-purified protoxin inclusions was estimated by the Bradford/Pro260 method described by Li and Bouwer (2012b). In this method, the total protein in the sample is determined by the Bradford method (Bradford, 1976), and the concentration of the Cry protein is 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). For each sample used in bioassays, the

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Cry protein concentration was the average of six separate estimates.

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2.5. Insects

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H. armigera eggs were obtained from a culture maintained at the Agricultural Research Council (Pretoria, South Africa). H. armigera larvae were reared in our laboratory on wheat-germ based artificial diet (Bot, 1966). During oviposition, H. armigera adults were provided with 5% (w/v) sucrose solution. The cultures were maintained in a growth chamber at 28 ± 1 °C and 70% relative humidity, and a photoperiod of 12:12 h (L:D).

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2.6. Dose–mortality bioassays

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The larvicidal activity of Cry proteins against H. armigera neonate larva was assessed by dose–mortality bioassays using the droplet feeding method (Hughes and Wood, 1981), as modified by Bouwer and Avyidi (2006). The neonate larvae were fed with protoxin inclusions which contained one of the six Cry proteins to determine the median lethal dose (LD50) of individual Cry proteins or a mixture (1:1 ratio) of two different Cry proteins. 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. Insects were incubated under the above described conditions. Three replicates of 20 larvae per replicate (n = 60) were used for each of five doses of a Cry treatment and for each of the controls (sterile double-distilled water). Mortality was scored 4 days after treatment. Any larva that failed to move when prodded repeatedly was considered dead.

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2.7. Data analysis

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Dose–mortality data were subjected to probit regression analysis (Finney, 1971), and the median lethal dose (LD50) was estimated for Cry proteins administered independently and in mixtures. The LD50s of Cry proteins (single or mixtures) for which the 95% fiducial limits did not overlap were considered to be significantly different. The interactions between Cry proteins were evaluated using the method described by Tabashnik (1992). The expected LD50 of a mixture was calculated with the following mathematical equation (Tabashnik, 1992):

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LD50ðmÞ ¼

ra rb þ LD50ðaÞ LD50ðbÞ

1

where LD50(m) in the equation is the expected LD50 of the mixture of toxins a and b, LD50(a) is the observed LD50 for toxin a alone, LD50(b) is the observed LD50 for toxin b alone and ra and rb represent the relative proportions of toxin a and toxin b in the mixture, respectively. In determining the nature of the interaction between the Cry proteins, we compared the expected LD50(m) to the fiducial limits of the observed LD50(m) as described previously (Liao et al., 2002; Tabashnik, 1992): (1) if the expected LD50(m) is within the range of the 95% fiducial limits of the observed LD50(m), the interaction is considered additive; (2) if the expected LD50(m) is greater than the upper limit of the 95% fiducial limits of the observed LD50(m), the interaction is considered synergistic; and (3) if the expected LD50(m) is lower than the lower limit of the 95% fiducial limits of the observed LD50(m), the interaction is considered antagonistic. We report the synergism factor (SF) of mixtures to assist in evaluating both the type of interaction and the magnitude of the interaction, with the SF for a

mixture being calculated by dividing the expected LD50(m) and the observed LD50(m). Although classification of an interaction was based on significant (statistical) difference, i.e. comparison of the expected LD50(m) to the 95% fiducial limits of the observed LD50(m), statistically additive interactions with SF values >1.5 were classified as weakly synergistic.

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3. Results

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3.1. Larvicidal activity of individual Cry proteins

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On the basis of LD50s, Cry1Ac had significantly (statistically) higher larvicidal activity against H. armigera neonate larvae than any of the other Cry proteins assayed (Table 1). It was followed, in ascending order of LD50 values, by Cry2Aa, Cry1Ab, and Cry1Aa. Cry1Ca and Cry9Aa showed the lowest, and statistically similar, larvicidal activity against H. armigera neonate larva (Table 1). There was an approximately 18,200-fold difference between the LD50s of the Cry protein with the lowest LD50 (Cry1Ac) and the Cry protein with the highest LD50 (Cry9Aa).

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3.2. Larvicidal activity of binary mixtures of Cry proteins

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In H. armigera neonate larvae, a significant synergistic interaction was observed between Cry1Ab and Cry1Ca in 1:1 mixtures of these Cry proteins (Table 2). In addition, weak synergistic interactions occurred between Cry1Aa and Cry1Ca and between Cry1Ac and Cry1Ca (Table 2). In contrast, combinations of Cry1Aa and Cry1Ab or Cry1Aa and Cry1Ac exhibited significant antagonistic interactions. When combined, Cry1Ab and Cry1Ac showed a statistically additive interaction (Table 2). Additive effects were observed for eight different binary combinations of either Cry2Aa and Cry1-class proteins or Cry9Aa and Cry1-class proteins, but Cry1Ca and Cry9Aa interacted synergistically (Table 3). Although the observed LD50 of the mixture of Cry1Ca and Cry9Aa was approximately two-fold less than that of either of the individual proteins in the combination, the LD50 (309.06 ng) of this mixture was the highest of all the tested combinations (Table 3). The combinations with the highest observed larvicidal activity were (in order of ascending LD50 values): Cry1Ac–Cry1Ca (LD50 = 0.05 ng); Cry1Ac–Cry2Aa (LD50 = 0.06 ng); Cry1Ac–Cry9Aa (LD50 = 0.07 ng); and Cry1Ab–Cry1Ac (LD50 = 0.09 ng). Although the LD50 values of these mixtures were all significantly lower than that of Cry1Ab on its own (LD50 = 0.64 ng), none of these highly-toxic mixtures had an LD50 that was significantly different to either Cry1Ac (LD50 = 0.04 ng) or Cry2Aa (LD50 = 0.13 ng). Although it is noteworthy that the mixtures with the highest larvicidal activity all contained Cry1Ac as one of the Cry proteins, one of the three Cry mixtures that

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Table 1 Dose–mortality responses of H. armigera neonate larvae to Cry proteins as determined by droplet feeding bioassays. Cry proteins

LD50 (ng)A

Cry1Ac Cry2Aa Cry1Ab Cry1Aa Cry1Ca Cry9Aa

0.04a 0.13b 0.64c 11.99d 690.94e 727.84e

95% Fiducial limits Lower

Upper

0.02 0.08 0.51 8.90 419.07 430.45

0.06 0.18 0.80 16.50 1420.01 1556.39

Slope ± SEB

0.52 ± 0.05 0.63 ± 0.05 0.85 ± 0.07 0.64 ± 0.04 0.52 ± 0.06 0.46 ± 0.06

A Values followed by the same letters are not significantly different (overlapping 95% fiducial limits). B Slope ± standard error.


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Table 2 Larvicidal activity of binary mixtures of Cry1-class proteins against H. armigera neonate larvae as determined by droplet feeding bioassays. Cry proteinsA

Observed

Expected

SFD

Slope ± SEE

1.60*w 0.84 4.13* 0.23D 0.63D 1.72*w

0.48 ± 0.06 0.64 ± 0.09 0.48 ± 0.07 0.65 ± 0.08 0.95 ± 0.10 0.70 ± 0.07

95% Fiducial limits LD50 (ng)B Cry1Ac–Cry1Ca Cry1Ab–Cry1Ac Cry1Ab–Cry1Ca Cry1Aa–Cry1Ac Cry1Aa–Cry1Ab Cry1Aa–Cry1Ca A B C D E *w *

D

a

0.05 0.09ab 0.31b 0.34b 1.92c 13.70d

Lower

Upper

LD50 (ng)C

0.02 0.04 0.12 0.17 1.24 7.52

0.11 0.18 0.72 0.61 2.96 24.33

0.08 0.08 1.28 0.08 1.22 23.57

Cry protein were assayed at a ratio of 1:1. Values followed by the same letters are not significantly different (overlapping 95% fiducial limits). Expected LD50(m), calculated by the method of Tabashnik (1992). SF (synergism factor) is the expected LD50(m) divided by the observed LD50(m). Slope ± standard error. Weak synergistic interaction; statistically an additive interaction, but SF > 1.5. Statistically significant synergistic interaction. Statistically significant antagonistic interaction.

Table 3 Larvicidal activity of binary mixtures of Cry1-class, Cry2Aa and Cry9Aa proteins against H. armigera neonate larvae as determined by droplet feeding bioassays. Cry proteinsA

Observed

Expected

SFD

Slope ± SEE

1.02 1.14 1.37 1.18 0.75 0.89 0.96 0.82 2.29*

0.72 ± 0.08 0.45 ± 0.08 0.66 ± 0.09 0.73 ± 0.09 1.21 ± 0.13 0.52 ± 0.08 0.72 ± 0.13 0.78 ± 0.15 0.77 ± 0.13

95% Fiducial limits LD50 (ng)B Cry1Ac–Cry2Aa Cry1Ac–Cry9Aa Cry2Aa–Cry9Aa Cry1Ca–Cry2Aa Cry1Ab–Cry2Aa Cry1Aa–Cry2Aa Cry1Ab–Cry9Aa Cry1Aa–Cry9Aa Cry1Ca–Cry9Aa A B C D E *

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a

0.06 0.07abd 0.19abc 0.22bc 0.29c 0.29cd 1.33e 28.77f 309.06g

Lower

Upper

LD50 (ng)C

0.03 0.02 0.09 0.12 0.20 0.12 0.72 9.68 183.13

0.11 0.18 0.35 0.37 0.42 0.61 2.40 53.89 574.90

0.06 0.08 0.26 0.26 0.22 0.26 1.28 23.59 708.91

Cry proteins were assayed at a ratio of 1:1. Values followed by the same letters are not significantly different (overlapping 95% fiducial limits). Expected LD50(m), calculated by the method of Tabashnik (1992). SF (synergism factor) is the expected LD50(m) divided by the observed LD50(m). Slope ± standard error. Statistically significant synergistic interaction.

exhibited an antagonistic interaction (Cry1Ca–Cry9Aa, Cry1Aa– Cry1Ab; Cry1Aa–Cry1Ac) contained Cry1Ac.

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4. Discussion

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Bt biopesticides are usually applied when early instar larvae are present as older larvae are more tolerant (Sanahuja et al., 2011). We thus decided to focus on interaction effects in neonate (first instar) larvae. Because lethal dose (LD) 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), LD may be preferable to LC in evaluating the interaction effects of Cry proteins. Since the diet contamination method (Ignoffo, 1965) is not suitable for LD estimation in neonate larvae of H. armigera, as the larvae are unlikely to fully consume even a very small block of food in 24 h, we used the droplet feeding bioassay method. In the evaluation of synergistic interactions using the method of Tabashnik (1992), empirical estimation of the LD50 of each Cry protein in the mixture is a prerequisite for calculating the expected LD50 of the mixture (i.e. LD50(m)). We obtained the following ranking of larvicidal activity (LD50 values) against neonate larvae: Cry1Ac < Cry2Aa < Cry1Ab < Cry1Aa < (Cry1Ca = Cry9Aa), where 1.5 (a 50% increase in toxicity) was used to indicate a synergistic interaction. When applying the classification method of Wirth et al. (2004), the interactions between Cry1Ac and Cry1Ca (SF = 1.60) and between Cry1Aa and Cry1Ca (SF = 1.72) were synergistic. When using our classification method, both Cry1Ac–Cry1Ca and Cry1Aa–Cry1Ca mixtures were classified as weakly synergistic, whereas the Cry1Ab–Cry1Ca mixture (SF = 4.13) was classified as synergistic. Xue et al. (2005) reported synergistic activity between Cry1Aa and Cry1C (1:1 ratio) against neonate larvae of both Spodoptera exigua (SF = 4.0) and H. armigera (SF = 2.7). Although Xue et al. (2005) did not state the tertiary rank of the Cry1C used in their study, it is noteworthy that our study and that of Xue et al. (2005) showed synergistic effects for combinations of Cry1A-class and Cry1C-class proteins against geographically distinct populations of H. armigera. In light of the high SF values obtained for Cry1Ac–Cry1Ca and Cry1Aa–Cry1Ca and the significant synergistic interaction observed between Cry1Ab and Cry1Ca, we believe that a more in depth study of the interactions between Cry1A-class and Cry1Cclass proteins would contribute to our understanding of the synergistic interactions between Cry proteins in H. armigera. To the best of our knowledge, no published studies have evaluated the synergistic interaction between Cry9-class and other Cry


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proteins in H. armigera larvae. In our study, all 1:1 combinations of Cry1A-class and Cry9Aa proteins (Cry1Ac–Cry9Aa, Cry2Aa–Cry9Aa, Cry1Ab–Cry9Aa, Cry1Aa–Cry9Aa) showed additive interactions in neonate larvae of H. armigera. In contrast, a 1:1 ratio of Cry1Ca and Cry9Aa was statistically synergistic with a SF = 2.29. Ibargutxi et al. (2008) noted then when the LC50 value of one toxin is very high, the r/LC50 ratio used in the Tabashnik formula (1992) tends to zero, with the result that its relative contribution to the larvicidal activity of a mixture in which the other toxin has a low LC50 value is relatively insignificant. Although this may impact on the reliability of classifying interactions as synergistic using the Tabashnik formula (1992), it is noteworthy that Cry1Ca (high LD50) was found to interact synergistically not only with Cry proteins with low LD50 values (e.g. Cry1Aa or Cry1Ab) but also with a Cry protein (Cry9Aa) that has an LD50 that is only 1.05-fold larger than its own LD50. In light of the presence of Cry1Ca in several mixtures (Cry1Aa–Cry1Ca, Cry1Ab–Cry1Ca, Cry1Ac–Cry1Ca and Cry1Ca–Cry9Aa) showing evidence of synergistic interactions, we believe that further evaluation of the interaction between Cry1Ca and other Cry proteins is warranted. The four most toxic mixtures were Cry1Ac–Cry1Ca, Cry1Ac– Cry2Aa, Cry1Ac–Cry9Aa, and Cry1Ab–Cry1Ac, but not one of the 15 mixtures evaluated had an LD50 that was statistically different to that of Cry1Ac. The presence of a synergistic interaction does not mean that a mixture is per se a good mixture for the control of a pest. This is illustrated by the fact that a mixture of Cry1Ca and Cry9Aa displayed a strong synergistic interaction (SF = 2.29) and the LD50 of the mixture was approximately two-fold less than either of the individual proteins in the mixture, but the LD50 of this mixture was the highest of all the mixtures tested. Of the four most toxic mixtures, only one of these (Cry1Ac–Cry1Ca) showed activity suggestive of a synergistic activity (SF > 1.5, but not statistically different). In choosing Cry mixtures for sustained use in a pest control program, the Bt toxins that are used in combination should ideally not share a receptor in order to avoid a single mutation conferring cross-resistance to both Cry proteins. Xu et al. (2005) stated that the cross-resistance pattern and inheritance mode of Cry1Ac resistance in a strain of H. armigera was consistent with ‘‘mode 1’’ resistance, which is characterized by high resistance to at least one Cry1A protein but little or no cross-resistance to Cry1C (Tabashnik et al., 1998). Ren et al. (2013) showed that both Cry1Ca and Cry1Ac could bind to bacterially expressed truncated cadherin (rHaBtRp) from H. armigera, but Cry1Ac does not share the same binding sites with Cry1Ca in rHaBtRp. The evidence of ‘‘mode 1’’ resistance in H. armigera and the fact that Cry1Ac–Cry1Ca had the lowest LD50 of the evaluated mixtures suggest that a mixture of Cry1Ac and Cry1C would be beneficial from both a pest control and resistance management perspective. Since the Cry1Ac–Cry2Aa mixture had an LD50 statistically identical to that of Cry1Ac– Cry1Ca and Cry1Ac and Cry2Aa do not share a common binding site in H. armigera (Estela et al., 2004; Hernández-Rodríguez et al., 2008), Cry1Ac and Cry2Aa appear to be good candidates for gene stacking in genetically engineered products developed to control H. armigera. To the best of our knowledge, this study is the first to report the interaction activity between Cry1A-class proteins and Cry1Ca and between Cry1Ca and Cry9Aa against H. armigera. In evaluating the interaction effects of all 15 binary combinations of six different Cry proteins, this study has significantly expanded the database of published interactions between Cry proteins in H. armigera larvae. Evaluation of the same Cry mixtures against a range of H. armigera populations is required to determine if any of the interaction classifications of this study are generalizable to H. armigera as a species.

Acknowledgments

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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.

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Cas9 system.

Receptors and Lethal Effect of Bacillus thuringiensis Insecticidal Crystal Proteins to the Anticarsia gemmatalis (Lepidoptera, Noctuidae).

Bacillus thuringiensis Vip3Aa Toxin Resistance in Heliothis virescens (Lepidoptera: Noctuidae).

Mitochondrial DNA and trade data support multiple origins of Helicoverpa armigera (Lepidoptera, Noctuidae) in Brazil.

Oral Administration of TAT-PTD-Diapause Hormone Fusion Protein Interferes With Helicoverpa armigera (Lepidoptera: Noctuidae) Development.

Comparative feeding performance and digestive physiology of Helicoverpa armigera (Lepidoptera: Noctuidae) larvae-fed 11 corn hybrids.

Development, survivorship and reproduction of Helicoverpa armigera (Lepidoptera: Noctuidae) under fluctuating temperatures.

A droplet digital PCR (ddPCR) assay to detect Helicoverpa armigera (Lepidoptera: Noctuidae) in bulk trap samples.

Mitochondrial P-glycoprotein ATPase contributes to insecticide resistance in the cotton bollworm, Helicoverpa armigera (Noctuidae: Lepidoptera).

A brave new world for an old world pest: Helicoverpa armigera (Lepidoptera: Noctuidae) in Brazil.

Adaptation of Habrobracon hebetor (Hymenoptera: Braconidae) to Rearing on Ephestia kuehniella (Lepidoptera: Pyralidae) and Helicoverpa armigera (Lepidoptera: Noctuidae).

Acute, sublethal, and combination effects of azadirachtin and Bacillus thuringiensis on the cotton bollworm, Helicoverpa armigera.

Molecular Characterization and Function Analysis of the Vitellogenin Receptor from the Cotton Bollworm, Helicoverpa armigera (Hübner) (Lepidoptera, Noctuidae).

Species From the Heliothinae Complex (Lepidoptera: Noctuidae) in Tucumán, Argentina, an Update of Geographical Distribution of Helicoverpa armigera.

Characterization of lepidopteran-specific cry1 and cry2 gene harbouring native Bacillus thuringiensis isolates toxic against Helicoverpa armigera.

Identification and validation of reference genes for normalization of gene expression analysis using qRT-PCR in Helicoverpa armigera (Lepidoptera: Noctuidae).

Baseline Susceptibility of Field Populations of Helicoverpa armigera to Bacillus thuringiensis Vip3Aa Toxin and Lack of Cross-Resistance between Vip3Aa and Cry Toxins.

Antibiotics influence the toxicity of the delta endotoxins of Bacillus thuringiensis towards the cotton bollworm, Helicoverpa armigera.

Identification and Evaluation of Suitable Reference Genes for Normalization of MicroRNA Expression in Helicoverpa armigera (Lepidoptera: Noctuidae) Using Quantitative Real-Time PCR.

Isolation and Characterization of Gut Bacterial Proteases Involved in Inducing Pathogenicity of Bacillus thuringiensis Toxin in Cotton Bollworm, Helicoverpa armigera.

Coexpression of the silent cry2Ab27 together with cry1 genes in Bacillus thuringiensis subsp. aizawai SP41 leads to formation of amorphous crystal toxin and enhanced toxicity against Helicoverpa armigera.

Helicoverpa zea (Lepidoptera: Noctuidae) and Spodoptera frugiperda (Lepidoptera: Noctuidae) Responses to Sorghum bicolor (Poales: Poaceae) Tissues From Lowered Lignin Lines.

Draft Genome Sequence of Bacillus thuringiensis Serovar Tolworthi Strain Na205-3, an Isolate Toxic for Helicoverpa armigera.

Regulation of cry gene expression in Bacillus thuringiensis.