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New Zealand Plant Protection 66 (2013): 29-33

A biochemical viability assay is compatible with molecular methods for species identification

N.K. Richards, L.M. Winder, I.I. Iline, M.A. Novoselov, M.R. McNeill and C.B. Phillips

ABSTRACT

A biochemical viability assay was recently developed to quickly and easily assess the viability of small immobile arthropods, including eggs, intercepted on plant products. On finding a viable specimen, species identification often becomes the next hurdle. This paper demonstrates that amplifiable DNA is present in a used biochemical viability assay solution, and can be used for making taxonomic identifications. Cryptically labelled heattreated and untreated eggs of three weevil species (Listronotus bonariensis, Sitona lepidus and S. discoideus) were first tested for viability, then a 1135 bp fragment of the cytochrome c oxidase I gene was amplified from each viability assay solution in the presence of a fluorescent dye, SYBR Green. Melt curve analysis of the amplicons (n=36) revealed three distinct melt profiles that correctly corresponded to each of the three weevil species. This shows that the biochemical viability assay is compatible with the application of subsequent molecular identification methods, which will facilitate the appropriate management response.

Keywords: immobile arthropods, species identification, melt curve analysis.

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