Isoelectric focusing electrophoresis and RFLP analysis: Two methods for immature Orius spp. identification

Abstract

Two techniques were used to discriminate between second instar Orius minutus (L) and Orius sauteri (Poppius) which occur sympatrically in eggplant fields of Japan. Isoelectric focusing (IEF) was used to discriminate between the two species utilizing the enzyme isocitrate dehydrogenase (IDH). Moreover, IEF was also shown to detect the presence of the prey Thrips palmi Karny in O. sauteri nymphs immediately after feeding by using phosphogluco-isomerase (PGI). PCR generated internal transcribed spacer 1 (ITS-1) regions for nymphs of both species were also diagnostic as the O. minutus PCR product was much larger than the O. sauteri PCR product (900 vs. 600 bp). Moreover, by comparing ITS-1 PCR product sizes and using the restriction enzyme HinfI on ITS-1 products of similar size to produce restriction fragment length polymorphisms (RFLPs), consistent differences among five Japanese Orius species were also demonstrated.