Codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae) is a major pest of apples and pears throughout the temperate regions of the world (Barnes 1991; Boncheva et al. 2006; Soleno et al. 2008). Codling moth has been reared on a large scale at Stellenbosch, Western Cape, South Africa since 2002 for use in an area-wide integrated pest management (AW-IPM) program that includes a sterile insect technique (SIT) component (Addison 2005). Economical production and efficient rearing of large numbers of insects are a prerequisite in a program that uses sequential releases of sterile insects. In many rearing facilities selection is directed towards traits which result in high productivity such as high fertility and fecundity and a short life cycle (Sørensen et al. 2012), which sometimes has negative consequence in terms of low performance in the field (Simmons et al. 2010). An artificial larval diet, described by Guennelon et al. (1981), has been used in the facility for rearing codling moth and it includes maize meal as a major ingredient, making up 59% of the diet. Almost 30 years have passed since the first publication of the Guennelon diet but the availability, safety and quality of the original diet ingredients have changed.

The use of genetically modified (GM) maize that contains genetic material from Bacillus thuringiensis (Bt) targets stemboring Lepidoptera feeding on this crop. Bt maize is an important component in integrated management of maize pests in South Africa (Gore et al. 2002; Van Rensburg 2007; Kruger et al. 2012). Bacillus thuringiensis is a Gram positive bacterium which produces crystalline inclusions during sporulation (De Maagd et al. 1999; Boncheva et al. 2006; Konecka et al. 2007). These inclusions consist of delta-endotoxins or Cry proteins with insecticidal properties (De Maagd et al. 1999; Boncheva et al. 2006; Konecka et al. 2007). The Bt maize plant produces a protoxin that destroys cells in the insect's midgut. The toxin gains entry only if the insect ingests Bt-contaminated food causing direct mortality, indirect effects such as cessation of feeding (Fast & Regniere 1984; Harris et al. 2006) and increased dispersal of those larvae that recovered from the toxic effects (Halcomb et al. 2000; Gore et al. 2002; Harris et al. 2006).

In 2011, 72% of the total area planted with maize in South Africa was GM maize of which 45.2% contained single Bt gene events, 40.4% contained the combination of herbicide and Bt genes and 14.4% was herbicide tolerant (Anonymous 2012). South African milling companies do not distinguish between Bt and non-Bt varieties of maize and therefore, availability of commercial non-Bt maize meal is limited. Bacillus thuringiensis is known to be toxic to codling moth and Bt-crystal formulations are registered for the control of codling moth in the field (Andermatt et al. 1988; Falcon & Huber 1991; Boncheva et al. 2006). It is therefore hypothesized that the use of local maize meal as an ingredient of an artificial diet for mass-rearing codling moth represents a risk in terms of production volumes needed for SIT programs.

From 2005 to 2007, production in the SIT facility suffered from high larval mortality and fluctuations in numbers of moths produced (D. Stenekamp, personal observation). The presence of a virus infection was ruled out but various Bt concentrations were found in maize meal batches, which was thought to explain the fluctuations in the number of insects produced at the rearing facility. Therefore, the hypothesis that Bt toxins in the maize meal used in the codling moth diet was causing larval mortality was tested.

The purpose of this study was to evaluate the effect of Bt maize and commercially available organically produced maize meal on production parameters, i.e., larval mortality, development and larval dispersal, during the rearing of codling moth and to quantify toxicity of various Bt concentrations in maize meal diets.

Material and Methods

QUANTIFICATION OF CRY1AB PROTEIN IN MAIZE MEAL

The concentrations of Cry1Ab protein in the maize meal was determined using a commercially available ELISA test kit (Agdia Inc., USA) and assayed according to the manufacturers' protocol. The positive control, which had a concentration of 40 µg/mL of Bt toxin, was suitably diluted to generate a standard curve. Maize meal samples were prepared by adding 1 mL sample buffer to 1 g of maize meal and allowing the Bt toxin to dissolve. Aliquots of 100 µL were used in the Bt ELISA test. The concentration of Bt toxin in the maize meal was calculated using the absorbance values obtained by reading the unknown values of the standard curve.

The Hoerl function (Daniel & Wood 1980):

was used to describe the relationship between the percent Bt maize meal (x) and the amount of Bt in µg of Bt/g maize meal (y). The regression constants are a, b and c and e is the base of the natural logarithm.

INSECT BIOASSAY AND RESPONSES TO BT

Adult emergence started around day 30 and lasted for up to 2 weeks (day 44) at 27 °C, a long-day photoperiod 16:8 h L:D and 75% RH (Bloem et al. 1997). In the SIT rearing facility, larval trays were moved from the larval rearing room to the adult emergence room on day 28 when adult emergence was about to start and were kept until day 44 when most of the adults had emerged. Peak emergence occurred around day 35 (Bloem et al. 1997; Bloem et al. 2000).

Experiments were repeated twice on different dates using the 5 Bt maize meal concentrations, and the control. Because of the time interval between the 2 dates, different codling moth cohorts were used and seasonal differences in temperature and humidity were apparent: in experiment 1 larvae were kept in a rearing room at 27.0 ± 2 °C, 58 ± 10% RH and in experiment 2 at 28.8 ± 2 °C and 69 ± 10% RH. The photoperiod was 18:6 h L:D. Temperature and humidity were monitored continuously at 10 min intervals using a HOBO® H8 data logger. Observations were made until day 44 when adult emergence was completed (Bloem et al. 1997).

Previous observations showed that the presence of Bt maize meal in the larval diet increased larval mortality, and/or delayed larval development and/or larvae migrating out of the diet prematurely. Therefore, on day 44 the following responses were recorded for each of codling moth larvae: (i) larval mortality, (ii) larvae not reaching adulthood by day 44 (delayed development), (iii) larvae leaving the diet, or (iv) total of the above 3 responses (total response). The concentrations causing 50% (LC₅₀) and 95% response (LC₉₅) and the 95% fiducial limits were determined by probit analysis using PoloPC (LeOra software 1987). To estimate the fiducial limits the heterogeneity factor was taken into account. This is given by

where t is the Student's t-distribution value for the appropriate confidence level (95% in this case), b is the slope of the probit regression line and Sxx is the weighted corrected sum of squares of the independent variable (concentration of Bt in this case) (Finney 1971). For g > 1 the fiducial limits run from −∞ to +∞ (Finney 1971) and cannot be estimated. Probit lines from the 2 experiments were tested for being co-incidental. If they were not co-incidental, they were tested for parallelism (homogeneity of slopes). If they were co-incidental, the data were combined (Finney 1971).

Results

Discussion

Our data clearly indicate that a Bt maize meal-based larval diet used in a codling moth production facility can adversely affect the number of moths produced. Of the 3 responses that can adversely affect colony production (larval mortality, delayed larval development and larvae leaving the diet), delayed larval development appeared to be the most important based on the slopes of the probit regression analysis. The slope for delayed larval development was higher (although not significant at P = 0.05) than the slopes for the other 2 responses (Fig. 1). In addition, the probit regression lines for delayed larval development more closely resembled the lines for total response than did those for larval mortality and larvae leaving the diet (Fig. 1). The lower slope of the regression line for larvae leaving the diet, suggests that an increase in the Bt concentration in the diet resulted in a limited increase in the response.

This report quantifies the effects of Bt maize meal in an artificial diet on codling moth and it shows that larvae were negatively affected by even small amounts of Bt in maize meal, particularly in terms of larval development. This negative effect of Bt maize meal on codling moth productivity confirms earlier results on the toxicity of different δ-endotoxins and Cry1Ab to codling moth (Andermatt et al. 1988; Pasquier et al. 1997; Rang et al. 2000; Boncheva et al. 2006). Rang et al. (2000) determined the toxicity of Cry1Ab against codling moth with a LC50 of 2.92 × 10^-1 µg/µL and Boncheva et al. (2006) at 0.078 µg/g of diet, which is less than in the present study, despite the mortality being determined after only 4 d. They used solubilized protoxins of Cry1 δ-endotoxins in a mixture with an artificial diet which could account for the difference in observed toxicity.

Delayed larval development was more pronounced in experiment 1 than in experiment 2 (LC₅₀ and LC₉₅ values in Table 1). This difference was most likely due to temperature related differences in potency of the toxin. Blomefield (2003) determined that the degree day units (DDU) for larval development from first to fifth instar at 25 °C (at a lower threshold temperature of 10 °C) were 321.6 °D for males and 329 °D for females. This resulted in a development time of 20–21 d. At the average temperature of 27.0 °C (experiment 1) the DDU were 357 °D at day 21 and at 28.8 °C (experiment 2), the DDU were 378 °D. Therefore, the larvae were expected to develop faster in experiment 2 than in experiment 1. By day 44, many of the larvae feeding on the Bt maize still had not reached the fifth instar stage (Table 2) in both experiments, although the DDU reached 748 °D. This was more than double the expected time of 321.6 °D for codling moth larvae (Blomefield 2003) to reach the fifth instar. Although there was a difference in temperature between the 2 experiments, the codling moth larvae in the control (0% Bt maize meal) still had a higher percentage emergence and faster development compared to the Bt maize meal-fed larvae in both experiments. The delayed larval development most likely was induced by ingestion of a sub lethal dose of Bt endotoxin which resulted in anorexia (Retnakaran et al. 1983; Harris et al. 2006). Dandekar et al. (1998) also reported a decreased rate of larval development in codling moth feeding on transgenic somatic walnuts with high levels of CryIA(c) gene expression, but low levels of CryIA(c) protein.

Larval dispersal can be associated with acute toxicity (Harris et al. 2006), and in our experiments, codling moth larvae were observed leaving the diet and it was assumed that they would die. Harris et al. (2006) found that light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae) larvae stopped feeding after ingesting food contaminated with Bt and they showed an increased tendency to disperse after the larva recovered from the toxic effects. If the dose of the toxin was not lethal, the midgut was repaired in time to resume larval development (Harris et al. 2006).

Fig. 1.

Probit response on log Bt concentration of Cydia pomonella larvae reared on a diet containing maize where the responses were: (A) larval mortality Y = 2.980 + 1.27(X); (B) delayed larval development, Y = 3.789 + 1.886(X) for experiment 1 and Y = 2.840 + 1.886(X) for experiment 2; (C) larvae leaving the diet, Y = 3.595 + 0.681(X); (D) total response or response A + B + C, Y = 4.133 + 2.344(X) for experiment 1 and Y = 3.592 + 2.344(X) for experiment 2. Y is the probit response and X is the log Bt concentration.

Table 1.

Chi-square (χ²) goodness of fit values with the degrees of freedom (df) and probability levels (P) for the co-incidental and parallel probit regression lines of 4 responses on log Bt concentration, with the LC₅₀ and LC₉₅ values and their 95% fiducial limits (FL).

Table 2.

The number of Cydia pomonella larvae and pupae recorded on day 44 after exposure to different amounts of Bacillus thuringiensis (Bt) endotoxin in the 2 experiments combined giving a total of 100 C. pomonella in each treatment.

These results demonstrate that Bt maize meal has a deleterious effect on codling moth development even at low concentrations. It has a direct toxic effect causing larval mortality as well as an indirect effect resulting in delayed development. Both effects are of major concern in mass-rearing facilities where lepidopteran insects are reared. In our experiments, ideally the non-Bt isoline of maize should have been used as the control diet. However, the Bt maize was a mixture of 2 Bt cultivars and meal of the non-Bt isoline was unavailable. The nutritional properties of the organically grown maize meal may have differed from those of the Bt-maize meal. However, our data indicate that the risk of using South African milled maize meal in an artificial diet for rearing codling moth is high. The alternative is to find a suitable replacement for commercial maize meal, which can include organic maize meal, whole wheat flour (Brinton et al. 1969) or soybean flour (Howell 1972; Hansen & Anderson 2006). However these ingredients are more expensive. Extensive testing, evaluation and GM status of these products are needed before being accepted as a suitable replacement. Optimization of insectary-rearing of codling moths may be enhanced through modification of diet ingredients, and thus the latter have important implications for pest management.