Investigation and characterisation of diamide insecticide resistance conferred by target-site mutations in the Ryanodine Receptor of lepidopteran pests

Lepidopteran crop pests such as Tuta absoluta, Chilo suppressalis, Spodoptera frugiperda and Plutella xylostella are becoming increasingly problematic, as populations colonise new regions of the globe. The diamide insecticides are a relatively new class that is especially effective, and important, for controlling lepidopteran pests. However, over the past 10 years, resistance to diamides has emerged amongst various lepidopteran species, and is a major threat to crop
protection in some cases. Resistance is thought to be caused by alterations to the Ryanodine Receptor, which is the target-site of Diamide insecticides, and previous studies have identified a variety of alterations, in field-populations of multiple lepidopteran species, which may be implicated in diamide resistance. This thesis aims to characterise RyR alterations in terms of their impact on the control-efficacy of diamide insecticides. A previously cloned RyR sequence from the moth, Plutella xylostella, was altered to reflect resistance-alterations present in the field. Alteration of positions 4946 and 4790 was found to reduce activation by diamides when expressed in cell lines. These same altered moth RyR sequences were inserted into Drosophila, by way of in-vivo confirmation. Flies expressing the resistant RyR genotypes survived diamide challenge more readily than those expressing the wild-type genotype of moth RyR. However, the resistant genotypes also climbed and crawled more slowly, suggesting a potential drawback of resistance. The alterations to residues 4946 and 4790 suggested a location of diamide interaction within the voltage-sensor-like domain of the RyR. In order to further define the interaction site, further alterations were made within the same region. Preliminary investigations indicated that these alterations strongly reduce diamide efficacy, when in combination, but exhibit somewhat minor reductions of efficacy as individual changes. It was concluded that the diamide interaction lies proximal to the Voltage-Sensor-like-Domain of the RyR.