Genetic hitchhiking and resistance evolution to transgenic Bt toxins: insights from the African stalk borer Busseola fusca (Noctuidae)

Abstract

Since transgenic crops expressing Bacillus thuringiensis (Bt) toxins were first released, resistance evolution leading to failure in control of pests populations has been observed in a number of species. Field resistance of the moth Busseola fusca was acknowledged 8 years after Bt maize was introduced in South Africa. Since then, field resistance of this corn borer has been observed at several locations, raising questions about the nature, distribution and dynamics of the resistance trait. Using genetic markers, our study identified four outlier loci clearly associated with resistance. In addition, genetic structure at neutral loci reflected extensive gene flow among populations. A realistically parameterised model suggests that resistance could travel in space at speed of several kilometres a year. Markers at outlier loci delineated a geographic region associated with resistance spread. This was an area of approximately 100 km radius, including the location where resistance was first reported. Controlled crosses corroborated these findings and showed significant differences of progeny survival on Bt plants depending on the origin of the resistant parent. Last, our study suggests diverse resistance mutations, which would explain the widespread occurrence of resistant larvae in Bt fields across the main area of maize production in South Africa.

Figure 1

Map of the South African localities where larvae of Busseola fusca were collected. Empty dots represent Bt fields; dark-grey dots, non-Bt field; square dots, locations sampled for the controlled crosses (Va, Be and Li). Location where resistance was initially reported is represented by a cross. Numbers are the field IDs as displayed in Table 1.

Figure 2

Isolation by distance in B. fusca populations (non-outlier loci). (a) Pairwise kinship coefficients (F_ij) as a function of distance (km). The curve corresponds to the regression of kinship as a function of log-transformed distance; dotted lines represent the 95% confidence interval of the regression slope. (b) Between-field analysis performed on Principal Coordinate (PCO) axes. Barycentre of each field sampled is denoted by its ID (number); individuals collected in the different fields correspond to grey dots; lines relate each individual to its field of origin.

Figure 3

Comparison of population structures between both the outliers and non-outlier loci, for individuals collected in Bt fields. The dendrogram (left) was obtained based on Euclidean genetic distances obtained with the data set of outlier loci. Two genetic groups are distinguished: A₁ and A₂. Comparative STRUCTURE plots (right) of the 14 Bt fields using outlier loci and non-outlier loci. The number of clusters in each analysis was set at K=2. The length of individual bars corresponds to the probability of membership to a cluster. Numbers identify the Bt field sampled.

Figure 4

Marker frequency at four outliers loci for the different locations sampled. Black-and-white pies represent Bt fields; shaded-grey pies, non-Bt fields. The darker fraction of the pies corresponds to the absence of marker (i.e., homozygote for the null allele). The shaded area delineates the region A₁, while by default A₂ is composed of any fields outside this region.

Figure 5

Survival of progeny on Bt maize after 21 days. Crosses were performed between individuals originating from a susceptible Tanzanian strain (Tz) and individuals originating from Bt fields in three different locations: Lichtenburg (Li × Tz), Bethleem (Be × Tz) and Valhaarts (Va × Tz), the latter being in the area A₁. The control consisted of the three same types of cross for which progeny was reared on non-Bt maize. M/F indicate whether the resistant parent was a male (M) or a female (F); error bars represent standard errors of the different proportions.

Figure 6

Expected resistance spread due to selection operated by Bt toxins (ω=0.6) after k=26 generations. Resistance was assumed initially confined at the location where it was first reported (centre of the circles). Dotted, dashed and solid circles correspond to distances obtained with increased axial dispersal variance (σ²=0.75, 3 and 6 km² per generation). External border of the hatched ring represents the dispersal parameter required σ²≈25 km² to encompass all resistant populations collected in this study. Empty dots correspond to sampled Bt fields; dark-grey dots, non-Bt field; square dots, locations sampled for the controlled crosses (Va, Be and Li). Numbers are the field IDs as displayed in Table 1.