The use of PCR to amplify DNA sequences for esterases putatively associated with pesticide resistance in the diamondback moth (Plutella xylostella L.)

Plutella xylostella L. is a global pest of cruciferous crops that has demonstrated resistance to almost every class of pesticide. Resistance to pesticides such as organophosphates, carbamates and pyrethroids involves target insensitivities, and metabolic mechanisms in insect pests. Some of these metabolic mechanisms involve the activity of esterases. The polymerase chain reaction was used to amplify DNA sequences that potentially encode for esterases in P. xylostella. Primers designed from esterase sequences of other resistant species, and degenerate primers designed from conserved regions of esterase sequences were used to amplify gene fragments from genomic DNA. DNA sequences of PCR products using nested primers specific to resistant aphid species contained many ambiguous nucleotides and had no matches in a nucleotide BLAST search of the GenBank online database. A 550bp DNA sequence was obtained from PCR products using Nilaparvata lugens specific esterase primers, but a search of GenBank yielded no matches with known esterase sequences. No PCR products were obtained using primers designed from the DNA sequence of a putative P. xylostella esterase in GenBank. A 306bp DNA sequence, however, was obtained by direct sequencing of a PCR product amplified from primers manually designed from conserved regions of a Myzus persicae esterase sequence, and an apparently homologous Drosophila sequence. The nucleotide sequence and its protein translation had many matches to the esterase sequences of a variety of resistant insect species, and may contain a partial esterase active site (oxyanion hole). The 306bp sequence may provide a marker for developing a probe for field monitoring of P. xylostella resistance.