A PCR detection method for mutations in receptor–protein genes from Busseola fusca potentially involved in Bt–resistance
Genetically modified (GM) crops attracted interest globally when use of these crops resulted in significant increases in yield and production. These increases were due to protection of crops from pests, weeds and diseases. However, evolution of resistance by pests threatens the continued efficacy of GM crops. One such example is the resistance to Cry1Ac toxin in Helicoverpa armigera (Lepidoptera: Noctuidae). Resistance in this pest was due to a mutation in the aminopeptidase N1 (APN) Cry receptor gene, encoding the receptor for Cry1Ac. Laboratory studies have indicated that species in families Noctuidae, Pyralidae and Plutellidae can develop resistance to Bttoxins. To date, field-evolved resistance has only been reported in Busseola fusca (Fuller) (Lepidoptera: Noctuidae) in South Africa, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) in the south-eastern United States, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) in Puerto Rico, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) in India, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in northern China and Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae) in The Philippines and Hawaii. Resistance development in lepidopteran species is thus a common phenomenon. The stem borer B. fusca is a major insect pest to Bt-maize in the Vaalharts irrigation scheme (South Africa). The first official report of B. fusca resistance to Cry1Ab toxin was recorded in 2007, although farmers observed increased damage to Bt-maize from stem borers as early as 2004. A second report of resistance in an area nearby followed in 2009. No study has yet been done to determine the molecular mechanism of B. fusca resistance to Cry1Ab. As mentioned, a mutation in the APN receptor gene is responsible for H. armigera resistance to Cry1Ac. Although B. fusca has developed resistance to the B. thuringiensis Cry1Ab toxin, the binding-patterns and -sites of Cry1Ac and Cry1Ab are similar. Thus a similar mutation may be responsible for B. fusca resistance to Cry1Ab. Aminopeptidase, cadherin and alkaline phosphatase are the major Cry toxin receptors that have been identified in lepidopteran species. The present study was concerned with the investigation of mutations in these receptor genes. However, in order to study mutations, sequence data of receptor genes are essential. Degenerate primers were designed based on conserved regions observed in multiple protein sequence alignments of aminopeptidase N (isogenes 1 to 6), cadherin and alkaline phosphatase of several lepidopteran species. Primers were degenerate to take into consideration the variant regions in receptor gene sequences among lepidopteran species. These primers were used to amplify genomic DNA (gDNA) from susceptible and resistant larvae by using PCR. Sequences of PCR amplicons were determined through Sanger sequencing reactions and subjected to BLAST searches. Results of the BLAST searches showed some similarities to the respective receptor genes. These sequences were also used in phylogenetic analysis. This analysis intended to determine the phylogenetic relationship of the respective receptor genes between B. fusca and other lepidopteran species. Mutations could not be identified in the present study, due to a lack in receptor gene sequence data for B. fusca. Thus a goal of the present study was to generate sequence data for B. fusca. In addition to the proposed objectives, cytochrome b gene sequences of B. fusca were used to determine the phylogenetic relationship between B. fusca and other lepidopteran species. Genome sequencing of B. fusca is recommended, as this will provide a platform for genomic, transcriptomic and proteomic studies on this species. These studies will provide much needed information, which can be used to formulate strategies to prevent resistance development in and spread of resistance to other B. fusca populations in sub-Saharan Africa.