Epsilon Archive for Student Projects

Abstract

The escalating usage of pyrethroids has resulted in an increased awareness about resistance towards pyrethroids in insects. Pyrethroids inhibit voltage-sensitive sodium channels (VSSC) in nerve cell membranes and are composed of synthetic molecules based on pyrethrins present in pyrethrum extracts from Chrysanthemum species. VSSC are transmembrane proteins that are important for electric signalling over the membrane in insects. Mutations in the gene encoding the sodium channel have proved to be a common reason for resistance against pyrethroids. Pyrethroid resistance among pollen beetles is spread all over Sweden and also abroad and is increasing. Mapping of the gene encoding the VSSC and search for mutations known to provide resistance in insects is necessary to know if that is the reason for resistance in pollen beetles. The VSSC protein consists of four domains, where the first three domains already have been sequenced so what is left to do is to obtain the sequence encoding the fourth domain. Polymerase Chain Reaction (PCR) was used to amplify the DNA fragment out of the pollen beetle genome. The following step was to use ligation and transformation to verify the DNA fragment by sequencing of obtained clones. The experimental part with domain IV was successful and the sequence can be seen in appendix 2. However, the work to amplify the whole gene encoding the VSSC was difficult. Only one of all amplifications gave a DNA fragment of 6,000 bp, so no further experimental work was possible. Pyrethroid resistance can also be due to increased metabolism by cytochrome P450 (CYP). The experimental part with CYP aimed to sequence gene fragments encoding different CYP. Ligation and transformation of PCR products was repeated many times without any positive results. Amplified samples purified from agarose gels were sent for sequencing instead and the sequences can be seen in appendix 2.