A procedure for combined genitalia dissection and DNA extraction in Lepidoptera

Abstract

Extraction of DNA from Lepidoptera is a destructive procedure and curators are often reluctant to provide museum specimens for molecular investigations. On the other hand, dissection of abdomens and genitalia is a standard procedure for description and identification of species and generally accepted even for type material. We present a method that combines the investigation of morphological traits in genitalia with the analysis of DNA sequence information by modifying the dissection protocol. Maceration of abdomens in potassium hydroxide is replaced by enzymatic digestion of soft tissue followed by DNA extraction. DNA extracted from abdomens is suitable for sequencing, as shown for the mitochondrial COI gene appropriate for species identification. Enzymatically treated abdomens proved to be sufficient for preservation of morphological traits. Recommendations are given for appropriate treatment of collected specimens and for routine use of enzymatic digestion.

Identity of the ailanthus webworm moth (Lepidoptera, Yponomeutidae), a complex of two species: evidence from DNA barcoding, morphology and ecology

“…Following the protocol of Knolke et al (2005) DNA was extracted from old type specimens during genitalia dissections for those which still had the abdomen intact, otherwise DNA was extracted from a leg. From these old DNA extracts, barcodes were assembled from six PCR amplicons (Hausman et al 2009;Rougerie et al in preparation).…”

“…However, we also view this study as an example of successful integrative taxonomic eff orts. Our conclusions were achieved through examination of Atteva type specimens, where available and involved a combined genitalia dissection and DNA extraction procedure (Knolke et al 2005) and the sequencing of mini-barcodes (Hausman et al 2009;Rougerie et al personal communication). Where type specimens have been lost, original illustrations were examined.…”

The use of Proteinase K to access genitalia morphology, vouchering and DNA extraction in minute wasps

“…We used only specimens conserved in alcohol and collected in or after 2005 to increase success in DNA extraction and amplification (Knölke et al 2005). For both KOH and PK digestion protocols, the genitalia were removed from the last metasomal segment with a sterile needle and were stored in polyethylene genitalia vials filled with glycerin.…”

“…This is true for several groups of Hymenoptera. Knölke et al (2005) first proposed extracting DNA as a by-product of genitalia preparation in Lepidoptera, avoiding morphological damage, which is usually achieved when a leg is dissected for DNA extraction. The Proteinase K (PK) treatment preserved morphological traits in Lepidoptera during DNA extraction from genitalia, but the authors did not evaluate efficacy of diaphanization and structure preservation of genitalia after the PK treatment (Knölke et al 2005, Hundsdoerfer andKitching 2010).…”

The Bupleurum (Apiaceae) feeding species of Trifurcula (Glaucolepis): new species, biology and distribution (Lepidoptera: Nepticulidae)

“…DNA extraction from larvae was usually destructive; from abdomens and some larvae the non-destructive protocol by Knölke et al (2005) was followed, allowing the preparation of the genitalia or larval skin as well. Details of methods are presented by , we provide here the COI DNA barcode for four out of the five species, only lacking Trifurcula siciliae sp.…”