Evaluation of Chilo partellus and Busseola fusca susceptibility to δ-endotoxins in Bt maize

Crop Protection 29 (2010) 115–120

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Evaluation of Chilo partellus and Busseola fusca susceptibility to d-endotoxins in Bt maize Regina M. Tende a, Stephen N. Mugo b, *, John H. Nderitu c, Florence M. Olubayo c, Josephine M. Songa d, David J. Bergvinson e a

Kenya Agricultural Research Institute (KARI), Katumani Research Center, Machakos, Kenya International Maize and Wheat Improvement Center (CIMMYT), Village Market, Nairobi 00621, Kenya Department of Plant Science and Crop Protection, University of Nairobi, Nairobi, Kenya d Alliance for a Green Revolution in Africa (AGRA), Nairobi, Kenya e Bill & Melinda Gates Foundation (BMGF), Seattle, Washington, USA b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 October 2007 Received in revised form 3 November 2009 Accepted 8 November 2009

Susceptibility of Chilo partellus (Lepidoptera, Crambidae) and Busseola fusca (Lepidoptera, Noctuidae) populations to Cry proteins from the bacterium, Bacillus thuringiensis (Bt), the d-endotoxins Cry1Ab and Cry1Ba in Bt-maize, were evaluated under biosafety greenhouse conditions. Larval feeding on Bt-maize was adjusted to deliver sub-lethal doses of d-endotoxins from the two events; survivors were reared on artificial diet to obtain successive generations. Eight generations of three C. partellus populations and five generations of a B. fusca population were screened for susceptibility on each event. Mean proportion of surviving larvae from Bt-maize plants, and the corresponding pupal weights of survivors for each population, were lower for individuals exposed to d-endotoxins. Both Bt Cry proteins expressed in maize leaves controlled C. partellus and showed stability in control, with no indication of a change in susceptibility among generations. Neither toxin, however, provided complete control of B. fusca, but no changes in susceptibility were observed after five generations of selection. Implications for development of future transgenic Bt maize events, and research for East Africa are discussed. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Bt susceptibility Bt-maize d-endotoxins Stalk borers Chilo partellus Busseola fusca

1. Introduction Stalk borers are a major limiting factor to maize (Zea mays L.) production worldwide (Pingali, 2001; James, 2003). In Kenya, the spotted stalk borer, Chilo partellus Swinhoe (Lepidoptera, Crambidae), accounts for more than 90% of the borer complex in the Lowland Tropics, Mid-altitude and the Moist Transitional areas. However, this species is almost absent in the Highland Tropic regions of Kenya (Overholt et al., 2001). Although C. partellus is more important in lowland tropical regions (Ofomata et al., 2000), it is increasingly expanding its range at higher altitudes of Kenya (Kfir, 1997). It is also the most widely distributed stalk borer species in the maize growing zones in Kenya (Kfir, 1997; Overholt et al., 2001). The African stalk borer, Busseola fusca Fuller (Lepidoptera, Noctuidae), causes economic loss to maize production in the high yielding Mid-altitude Transitional and the highlands Tropics zones

* Corresponding author. Tel.:þ254 20 722 4600; fax: þ254 20 7224601. E-mail address: [email protected] (S.N. Mugo). 0261-2194/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2009.11.008

in Kenya (Mulaa, 1995; Songa et al., 2001; De Groote, 2002). Subsequently, B. fusca accounts for 82% of all maize losses associated with stalk borers in Kenya, and can have more than one generation within the same growing season (Overholt et al., 2001). Control options for managing maize stalk borers include chemical, biological, cultural, and host plant resistance, through conventional breeding and genetic engineering. Chemical control methods are most effective; however, they are expensive to the smallholder farmer and pose risks to humans, livestock and the environment. Developing host plant resistance using conventional means is difficult due to the quantitative nature of inheritance, and the fact that the breeding procedures are expensive, involving the maintenance of two organisms, the pest and the host. Genetically engineered host plant resistance using codon optimized genes from the bacterium, Bacillus thuringiensis (Bt), has become a commercial success in many parts of the world, particularly for stalk borer control (e.g., Burkness et al., 2002; Shelton et al., 2002). Bt-maize technology is also being examined for implementation in Kenyan farming systems (Mugo et al., 2005; James, 2008). Bt d-endotoxin proteins are highly specific in their mode of action so they can be used to control a narrow range of target pests

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(Dutton et al., 2004). Depending on the promoter and specific transformation events, Bt-maize produce d-endotoxins at varying levels in selected tissues and over time, which must deliver a lethal dose in tissues where stalk borer larvae feed. This technology has been introduced into Kenya through the Insect Resistant Maize for Africa (IRMA) project (Mugo et al., 2005). The biological activity of Bt-maize events against African stalk borers has been previously reported, with the two most active events being used in the current study (Mugo et al., 2005). Frequent use of pesticides often results in loss of susceptibility through development of resistant populations within the target pest to the particular insecticide. While climatic variations play a major role in the distribution of pest species, the genetic makeup of the individuals may affect the reaction of pest populations to different control methods. Farming systems also differ from one ecological zone to another, with changing patterns of chemical usage over the years (Mulaa et al., 2005). Differentially treated insect populations may develop mechanisms to overcome effects of toxins (Oppernoorth, 1976). The cry proteins in Bt-maize Event 10 and Event 223 were found not to effectively control B. fusca (Mugo et al., 2005), and, therefore, the pest is known to already have some level of inherent tolerance to d-endotoxins tested thus far (Mugo et al., 2004, 2005). The objectives of this study were to: 1) Quantify mortality rates of three C. partellus populations to two Cry proteins expressed in experimental Bt-maize varieties, 2) Monitor survival rates and pupal weights of C. partellus populations over several generations of selection using sub-lethal doses of Cry proteins, and 3) Evaluate changes in the level of susceptibility to Bt-maize d-endotoxins by B. fusca over five generations of selection. 2. Materials and methods 2.1. Biosafety facilities The experiments were carried out at the Biosafety Level 2 Greenhouse Complex (BL2GH) located at the National Agricultural Research Laboratories (NARL) of the Kenyan Agricultural Research Institute (KARI) in Nairobi, Kenya. The facility was approved by the Kenya Standing Technical Committee on Imports and Exports (KSTCIE) for research on transgenic plants in 2004 (Mugo et al., 2005). 2.2. Bt-maize plants and d-endotoxins Two Bt transgenic maize inbred lines; Event 223 cry1Ab::Ubiquitin and Event 10 cry1Ba::Ubiquitin were used. Previous studies had shown that these two events were effective in controlling most of the stalk borer species in Kenya (Mugo et al., unpublished data). The events were developed using a maize constitutive promoter that enables the production of d-endotoxins in almost all parts of the plant. Both events were developed through backcrossing to CML216, a subtropical white maize line developed in Africa. The non-transformed version of the same maize inbred line CML216 was used as the control. Toxin expression levels for event 223 were 0.35 ug/g and the toxin expression levels of event 10 were 0.19 ug/g of fresh weight. Bt-maize seeds were sown into pots (7.5 cm  7.5 cm  9 cm) with growing media containing equal volumes of sand, loam soil and coconut peat (1:1:1 ratio). Management practices included daily watering, with fertilizer applied three times using 17N:17P:17K. Maize planting was synchronized with the pupal stage so that the emergence of neonates coincided with the 4–6 leaf stage of plant development at the time of infestation. A randomized complete block design (RCBD) was used with four replicates per treatment.

2.3. Stalk borer populations New insect cultures were established from field populations for both C. partellus and B. fusca, to reflect current genetic diversity for each species. Third to sixth instar larvae were collected from farms in various maize growing agro-ecological zones of Kenya. The C. partellus HCLT-population was collected from the Humid Coastal Lowland Tropics at Mavueni, Chonyi, Vipingo and Kikambala, along the Kenyan coast. The C. partellus DMA-population was collected from the Dry Mid-altitude areas of Gatuanyaga in Thika district in the Central Province, and from Kwa Vonza, Iveti, and Kaiti in Kitui District, Machakos and Makueni districts in Eastern Kenya. The C. partellus mixed-population was formed by random mating among populations from Coastal, Eastern and Central provinces and constituted the check-population. The B. fusca colony was obtained from Kitale within the highland tropics (HT) zone of the Rift-valley province. The original populations of each colony consisted of 300–500 larvae, established during the July–October 2004 period. Stalk borers were reared using artificial diets made from six-week old maize leaf powder and bean seed powder (Onyango and OchiengOdero, 1994; Songa et al., 2004). The diet was found to produce similar results as natural diet and perform equally well with both species. The insects were confined within an insectary, in large cages for oviposition at KARI-Katumani (37 140 E; 1 350 2400 N) at room temperature (28.0  2.0  C), and relative humidity (70  10%), under L12:D12 photoperiodism as described by Odindo and Onyango (1998) and Songa et al. (2001). 2.4. Insect bioassays for evaluation At the 4-leaf stage, 20 neonate larvae of C. partellus were placed into the whorl of each transgenic maize plant. Since not all transformed plants were expressing the cry proteins, the larvae were left to feed on the plant tissues. The plants that would lead to complete mortality of the larvae were used for the evaluation studies because toxin expression was confirmed prior to using a given plant in the selection experiment. Upon confirming the expression of the cry toxin, the three upper most leaves of the plants were cut and transferred to 1-L plastic jars for infestation. Four replicates (one plant per replicate) were used for each treatment (control, cry1Ab and cry1Ba), for each insect population. Each jar was infested with 300 neonates using a camel-hair brush. Neonate larvae of C. partellus were allowed to feed on the plants for 3–4 h then removed from the plant tissues. For B. fusca, larvae were allowed to feed for 48 h in order to ingest a higher dose of each protein, due to their reduced sensitivity to the cry proteins in these two events. After each feeding period with each toxin, larvae of both species were transferred to artificial diet following to complete their development in the absence of Bt toxin. Death of larvae occurred slowly within the artificial diet, not immediately after exposure. The d-endotoxins are released after digestion of the leaf tissues, which leads to between 84 and 98% mortality. 2.5. Rearing surviving insects Ten days after being transferred to artificial diet, surviving larvae were counted and transferred to fresh diet, and allowed to develop to the pupal stage. Pupae were harvested and weighed, then transferred to cages laid with oviposition paper where adults emerged, mated and oviposited. The butter paper media was changed every three days to synchronize the emergence of neonate larvae for the subsequent cycle of selection. Each C. partellus population was kept separate. The eggs were disinfected with 10% folmaldehyde for 15 minutes and incubated at ambient conditions

R.M. Tende et al. / Crop Protection 29 (2010) 115–120

(26–30  C, 60–80% Relative Humidity). The neonates emerging were used for infestation in the subsequent experiment. The same protocol was applied for all the subsequent generations up to the eighth cycle of selection for each of the three C. partellus colonies, and up to the fifth cycle of selection for B. fusca colony. Fewer generations of B. fusca were due to a longer life cycle lasting between 60 and 70 days, while the C. partellus life cycle ranged from 25 to50 days, at 26–30  C and 60–80% Relative Humidity. 2.6. Data analysis Insect mortality and insect counts data were subjected to arcsine square root (x) and logarithmic Ln (x þ 1) transformations respectively before analysis. Correction for control mortality was done using Abbott’s formula (Abbott, 1925):

Pt ¼ ðPo  Pc=ð100  PcÞÞ100;

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Table 2 Mean mortality (%) of eight successive generations of C. partellus populations exposed to Bt-maize Event223, Cry1Ab. Generations

Populations HCLTa

DMAb

Mixed

1 2 3 4 5 6 7 8

94.5 a 95.3 a 94.6 a 95.3 a 95.3 a 94.5 a 94.4 a 95.1 a

85.0 83.5 87.0 92.1 93.1 96.0 94.8 95.8

88.4 98.2 88.0 96.1 92.4 93.5 93.8 93.8

Mean

94.9 A

90.9 A

bc bc bc b b a a a

b b b a b b b b

93.0 A

Means within columns bearing the same lower case letter are not significantly different. Means within rows bearing the same upper case letter are not significantly different; HSD-Test (P ¼ 0.05). a Humid Coastal Lowland Tropics. b Dry Mid-Altitude.

where;

Pt ¼ corrected mortality ð%Þ; Po ¼ observed mortality ð%Þ; and Pc ¼ control mortality ð%Þ: Pupal weight ratios were obtained and subjected to arc-sin (square root (x)) transformation. Natural logarithm for count data, Ln(x þ 1) were used to avoid infinite results. The data were then subjected to analysis of variance (ANOVA), and the means were separated using Tukey’s Studentized Range (HSD) Test (SAS, 2000), at P ¼ 0.05. 3. Results 3.1. Response of C. partellus populations to cry proteins in Bt-maize High mortality rate means for Event 10 (97.2%) and Event 223 (97.8%) were observed for the Bt-maize fed larvae, during the first few days after transfer to artificial diet. A mean of only 35.1% mortality was observed within the control (data not shown). Larval death, therefore, occurred a few days after infestation in the artificial diet. Survival of C. partellus was significantly lower in Bt-maize (2.1–3.3%) for all populations and events, compared to the control (30–42%) after being transferred into artificial diet (Table 1). Larval mortality of C. partellus was greatest for Event 223 (90.9–94.9%; Table 2) compared to a slightly lower mortality rate for larvae exposed to Event 10 (86.8–93.5%; Table 3). Similar levels of efficacy against the C. partellus HCLT population were observed for eight generations of cry1Ab and cry1Ba exposure. Table 1 Composition (%) of stem borer conspecific populations of C. partellus that reached adult stage after feeding on Bt maize events and a non-Bt maize check plants (CML216). Treatments

CML216 Event 10 Event 223

Populations HCLTa

DMAb

Mixed

Means

42.0aA 2.6bA 2.1bA

33.3aAB 3.3bA 2.4bA

30.0abB 2.5bA 2.2bA

35.1aAB 2.8bA 2.2bA

Means within columns bearing the same lower case letter are not significantly different. Means within rows bearing the same upper case letter are not significantly different; HSD-Test (P ¼ 0.05). a Humid Coastal Lowland Tropics. b Dry Mid-Altitude.

There was a marked reduction in pupal weights of the insects that were exposed to the Cry proteins, with the control experiments maintaining high pupal weights. Pupal weights of the insects exposed to the cry1Ab proteins ranged from 27.3 to 39.7 mg, while pupal weights of the CML216 were 83.3–108.1 mg in all C. partellus populations (Table 4). This represented a weight reduction of 62.8–66.6% between the Bt fed insects and the control populations. Pupal weights of the insects exposed to Cry1Ba proteins ranged from 29.2 to 44.5 mg for C. partellus populations (Table 5). These results indicate weight reductions of 60.2–66.7%. 13.2. Monitoring changes in tolerance to Cry1Ab and Cry1Ba d-endotoxins by B. fusca 3.2.1. Mortality of larvae and pupal weights for B. fusca Mortality of B. fusca larvae was lower than for C. partellus from the two Bt-maize events, at 34.8% and 49.6%, for Event 10 and Event 223, respectively (Table 6), compared to 92.9% for C. partellus from Event 223 (Table 2) and 90.4% from Event 10 (Table 3). Mortality for B. fusca remained fairly stable from the first to the fifth generation for each of the cry proteins with no significant differences observed. However, only low levels of control were observed for B. fusca by the Cry proteins, compared to C. partellus, where over 90% control was achieved. There was no observed mortality in the CML 216 control. The CML216 control had high pupal weights ranging from 189.9 to 208.7 mg (Table 7). Bt events resulted in reduced pupal weights Table 3 Mean mortality (%) of eight successive generations of C. partellus populations exposed to Bt-maize Event10, Cry1Ba. Generations

Populations HCLTa

DMAb

Mixed

1 2 3 4 5 6 7 8

91.2 93.6 92.9 94.6 94.6 94.5 93.4 93.4

55.6 80.0 88.3 92.1 93.1 95.4 94.8 95.2

85.4 88.9 86.7 92.8 93.4 93.0 93.3 93.0

Mean

93.5 A

b a ab a a a a a

c b ab a a a a a

86.8 B

b ab ab ab a ab a ab

90.8 AB

Means within columns bearing the same lower case letter are not significantly different. Means within rows bearing the same upper case letter are not significantly different; HSD-Test (P ¼ 0.05). a Humid Coastal Lowland Tropics. b Dry Mid-Altitude.

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Table 4 Effects of Cry1Ab protein Bt maize events on pupal weights (mg) (meansem) of conspecific populations of C. partellus. Generations

Populations HCLTa

1 2 3 4 5 6 7 8

DMAb

Mixed

CML216

Event223

CML216

Event223

CML216

Event223

95.4  1.1bc 93.1  1.1c 99.1  1.2a 98.0  1.2ab 98.9  1.1ab 100.2  1.1a 101.5  1.2a 101.9  1.3a

39.7  2.0a 37.7  1.9ab 32.7  1.8bc 35.5  1.8a 32.9  1.6bc 34.1  1.7bc 35.2  2.0abc 30.5  2.1c

96.1  3.1bc 103.6  1.8ab 95.8  1.6abc 86.2  1.6c 89.3  1.4bc 108.1  0.8a 101.7  1.2abc 102.5  1.0ab

34.1  1.7a 31.3  1.3a 35.2  2.3a 33.3  1.8a 35.3  2.2a 31.7  1.8a 33.0  2.0a 31.2  1.6a

89.8  2.0bc 83.3  1.4c 104.3  2.0a 94.1  1.4abc 99.2  1.4ab 104.2  1.2a 96.6  1.3ab 92.0  1.1abc

31.5  1.9ab 31.6  1.7ab 27.3  1.9b 31.1  1.9ab 31.6  1.9ab 32.4  1.9a 34.5  1.9a 35.2  2.0a

Mean Reduction

65.0%

62.8%

66.6%

Means within columns bearing the same lower case letter are not significantly different; Tukey’s Studentized Range HSD test (P ¼ 0.05). Bt-maize; Event223 cry1Ab::Ubiquitin. a Humid Coastal Lowland Tropics. b Dry Mid-Altitude.

of 165.9–201.9 mg. Event 223 had a greater effect on the development of the B. fusca larvae, which led to reduced pupal weights compared to the control, in generations 1, 3 and 5. However, generations 2 and 4 showed no significant difference among pupal weights for CML216, Event 10 and Event 223. 4. Discussion 4.1. Number of surviving insects While the number of C. partellus and B. fusca larvae collected from Bt-maize plants was similar to that from the control plants, larval death occurred during rearing on artificial diet that contained Bt toxin. For susceptible insects, this should be expected, given the Bt d-endotoxin mode of action. Susceptible larvae are known to stop feeding within 1–2 h after ingestion of Bt maize tissue and death occurs within 1–2 dafter ingestion of d-endotoxins (Gill et al., 1992). There were significant differences in the number of surviving larvae from the transgenic Bt-maize used in the experiment compared to the CML216 control. High mortalities observed from the Bt-maize were attributed to the effects of the d-endotoxins to the target insects. The larvae counts of DMA-colony first generation from Event 10 plants were relatively higher compared to the means of insect from the rest of the cycles. Mortality for B. fusca was lower compared to those of C. partellus. These findings confirm earlier ones, which showed that the events used in this research work were not completely effective in controlling B. fusca (Mugo et al., 2004, 2005).

Exposure of the insects to the Bt-maize Cry proteins did not guarantee feeding by all individuals that were recovered and reared to maturity. Some larvae may not have fed on the maize plant, but were recovered and developed to the adult stage, thereby representing escapes. This could have led to the significant difference observed in the Event 10 surviving larvae and pupal weights of DMA population that were recorded in the generation. Observations on the response of the C. partellus populations indicate that the Bt-maize cry proteins did not affect the colonies differently. Susceptibility was maintained in the colonies after exposure to the cry proteins throughout the generations developed and tested. The three colonies showed little variation in the mean number of surviving larvae from each event. This may imply that the populations do not harbor significant differences genetically, or there is no variability in susceptibility to Cry1Ab and Cry1Ba d-endotoxins. Neither Event 10 (cry1Ba) nor Event 223 (cry1Ab) showed a significant difference in the number of surviving larvae and their corresponding pupal weights. An increase in the mean count of surviving larvae from the control (CML216) of the DMA-colony may be due to adaptation to the elevated temperatures (>30  C) within the biosafety greenhouse complex, considering that these insects were not exposed to the Cry proteins, the differences can only be attributed to other factors other than d-endotoxins per se. A possible explanation could be that the individuals from the HCLT region were well adapted to high temperatures found in the coastal region, while the population from the Dry Mid-altitude (DMA) maize growing zone required time to acclimate to the high temperatures within the biosafety greenhouse complex.

Table 5 Effects of Cry1Ba protein Bt maize events on pupal weights (mg) (mean  SEM) of conspecific populations of C. partellus. Generations

Populations HCLTa

1 2 3 4 5 6 7 8 Mean Reduction

DMAb

Mixed

CML 216

Event 10

CML 216

Event 10

CML 216

Event 10

95.4  1.1bc 93.1  1.1c 99.1  1.2a 98.0  1.2ab 98.9  1.1ab 100.2  1.1a 101.5  1.2a 101.9  1.3a

36.4  1.8a 38.9  2.2a 35.2  1.8a 38.1  1.6a 34.2  1.6a 35.8  1.8a 36.3  1.7a 39.5  1.6a

96.1  3.1bc 103.6  1.8ab 95.8  1.6abc 86.2  1.6c 89.3  1.4bc 108.1  0.8a 101.7  1.2abc 102.5  1.0ab

44.5  1.7a 33.5  1.4bc 33.9  2.0bc 29.2  1.4c 32.5  1.8bc 32.3  1.9bc 34.1  2.3bc 37.0  2.1b

89.8  2.0bc 83.3  1.4c 104.3  2.0a 94.1  1.4abc 99.2  1.4ab 104.2  1.2a 96.6  1.3ab 92.0  1.1abc

36.5  2.1ab 29.8  2.1c 31.5  2.1bc 32.5  1.4abc 39.8  2.5a 34.1  1.6ab 34.8  2.1ab 36.2  1.7a

66.7%

60.2

64.4%

Means within columns bearing the same lower case letter are not significantly different; Tukey’s Studentized Range HSD Test (P ¼ 0.05). Bt-maize; Event10 cry1Ba::Ubiquitin. a Humid Coastal Lowland Tropics. b Dry Mid-Altitude.

R.M. Tende et al. / Crop Protection 29 (2010) 115–120 Table 6 Mean mortality (%) of five successive generations of B. fusca larvae (mean  SEM) exposed to Bt-maize events and a control. Generations

CML216

Event 10

Event 223

1 2 3 4 5

0.0  0.0aD 0.0  0.0aD 0.0  0.0aD 0.0  0.0aD 0.0  0.0aD

34.8  0.9aBC 32.9  1.0aBC 25.6  1.0aC 35.8  0.9aBC 45.1  0.8abB

47.8  0.8abAB 44.8  0.8abB 37.8  0.9aBC 59.0  0.7bA 59.8  0.7bA

Mean

0.0  0.0D

34.8  0.9BC

49.6  0.8AB

Means within columns bearing the same lower case letter are not significantly different. Means within rows bearing the same upper case letter are not significantly different; HSD-Test (P ¼ 0.05).

4.2. Pupal weights The pupal weights from the control experiments in the two populations remained stable, thus implying that growth and development was not affected. Heinrichs et al. (1985) found that population increase and growth index of an insect can give information on antibiosis type of resistance. No negative observations were made on the control, indicating that there were no notable factors inhibited or interfered with the insects’ metabolic activities. This agrees with the findings of Panda and Khush (1995) that in the absence of toxins or inhibiting factors, developmental process goes on uninterrupted through the insects’ life cycle. There were significant differences in the number of surviving larvae and their corresponding pupal weights between the transgenic Bt-maize events used in the experiment, in comparison to the CML216 control. Slight increase in mean counts observed in the Event 10-DMA colony might not be a reflection of differences in genetic variation. The high mortalities observed from the Bt-maize were attributed to the effects of the d-endotoxins to the target insects. The pupal weight of insects that were exposed to the cry proteins was less, compared to the insects fed on non-transgenic maize. This could mean that the d-endotoxins interfered with the insects’ metabolism, either by interfering with feed intake or its assimilation within the insects’ mid-gut, which agrees with the findings of Fenemore (1984) that a pest which feeds on a resistant plant does not develop properly due to the presence of toxic substances in a plant. Heinrichs et al. (1985) indicated that toxicity of a chemical is a function of insects’ body weight. The pupal weights for C. partellus colonies exposed to cry proteins agree with these findings. The low pupal weight recorded is an indication of toxicity by the d-endotoxins. The subsequent generations of insects maintained low pupal weights for up to eight generations of exposure. This can be used as an indicator that despite exposure to sub-lethal doses of d-endotoxins, no changes in susceptibility was observed for these generations to cry proteins in Bt-maize. There was no increase in population growth and weight gain in generations of insects that Table 7 Mean pupal weights (mg) (mean  SE) of B. fusca for five generations of selection after 48 hours of larvae feeding on Bt-maize Event10 and Event223 and CML216 control. Generations

CML216

Event10

Event223

1 2 3 4 5

195.9  0.06abA 169.4  0.06bA‘ 208.7  0.07aA 189.9  0.06abA 210.4  0.11aA

174.6  0.06abB 169.1  0.11bA 201.9  0.08aA 191.8  0.07abA 191.4  0.06abA

172.4  0.06aB 175.4  0.11aA 165.9  0.06aB 193.8  0.08aA 170.3  0.06aB

Average

194.9  0.06A

185.8  0.06A

175.6  0.06B

Means within columns bearing the same lower case letter are not significantly different. Means within rows bearing the same upper case letter are not significantly different; HSD-Test (P ¼ 0.05).

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were exposed to the d-endotoxins. Susceptibility was maintained within the generations of these individuals. B. fusca colony showed marked difference in the pupal weights recorded from the Bt-maize with event 223 maintaining relatively low weights throughout the five cycles of selection. Lack of a clear marked difference in weights between the Bt-maize Event 10 and the control (CML216) could be interpreted to mean that the Cry1Ba had no effect on B. fusca. The cycle-to-cycle variation in weight gain may reflect a non-genetic cause such as changes in environmental conditions or management procedures, rather than indications of genetic change within the insect. Similar findings were obtained with European corn borer, Ostrinia nubilalis (Hu¨bner) (Lepidoptera: Crambidae) (Robertson et al., 1995; Marcon et al., 1999; Farinos et al., 2003). The failure of a chemical treatment in the field to control a pest is not adequate proof of the existence of genetic resistance by the insect pest (Fenemore, 1984). The fact that Bt-maize Event10::Ubiquitin and Event223 cry1Ab::Ubiquitin did not adequately control B. fusca is not proof that there were one or more resistant genes in the B. fusca populations tested in this study. However, given the Bt doses tested, and compared with the results for C. partellus, these results do suggest that B. fusca is inherently more difficult to control with the Bt toxins studied thus far. Indeed, the recent documentation of Bt resistance among field populations of B. fusca in South African maize (Van Rensburg, 2007) reinforce the need for Bt events that express a high dose of one or more toxins, and that resistance management practices should be carefully developed (e.g., Ostlie et al., 1997; Shelton et al., 2002). In summary, we did not observe significant changes in susceptibility of either pest to the Cry proteins, for either stalk borer species, or population. Our results do confirm that B. fusca is inherently less susceptible to both Bt toxins. However, this does not imply that resistant biotypes of this pest are present in the Kenyan populations, given the relatively low Bt doses used in the study. Under field conditions, each stalk borer species would likely be exposed to higher Bt dose events throughout the larval stage, via leaf and fruiting maize tissue; thus larvae would need to overcome more challenging toxin exposure. Indeed, future work with both species should, therefore, be done with higher expression levels of Bt toxins within artificial diet studies, and directly with Bt maize plants. The recent results with B. fusca in South Africa (Van Rensburg, 2007) underscore the need for both the development of Bt events that provide a high dose of toxin to target pests, and effective resistance management strategies (e.g., Ostlie et al., 1997; Shelton et al., 2002) to minimize the development of pest resistance to novel toxins.

Acknowledgement The authors acknowledge financial support from the Rockefeller Foundation and the Syngenta Foundation for Sustainable Agriculture through the KARI/CIMMYT IRMA Project, and technical assistance from A. Chavangi of CIMMYT, W. Buyse of ICRAF, D. Mutisya, and N. Kithuka of KARI-Katumani Center. Assistance given by Prof. S. Shibairo and E. Obudho from the University of Nairobi is also acknowledged.

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