Application of molecular techniques to non-lethal tissue samples of endangered butterfly populations (Parnassiuss apollo L.) in Norway for conservation management

Tissue from the wing tips of the endangered Apollo butterfly (Parnassius apollo L.) were collected at the end of a breeding season from two sites in Norway: Bandak and Gjendetunga/Bukkelægret about 250 km apart. We investigated the suitability of such non-lethal samples as a source of DNA for studying these populations. Universal mitochondrial DNA (mtDNA) primers were used to amplify DNA from the wing tip extractions. Primers spanning the 12s rRNA region produced 0.35 kb fragments that proved to be invariable in the selected samples when tested for restriction length polymorphisms (RFLPs). These sequences had high homology with analogous mtDNA from other insect sources, e.g. lepidoptera [Spodoptera litura (Fabricius)], and served to authenticate the DNA. Random amplified polymorphic DNA (RAPDs) technology was then used to screen for genetic variability. Of several primers tested, Primer-01 (Operon Kit F) produced a profile that differentiated the population with 88% (P<0.001) efficiency based on multivariate logistic regression analysis of banding profiles. RAPDs were also adapted to develop different molecular marker approaches to screen the samples quickly and cheaply. This work indicates that reliable ‘molecular tags’ can be designed to track demographic populations to study their distribution without endangering the butterfly and highlights the suitability of these markers for use in future population studies