Methods for retaining well-preserved DNA with dried specimens of insects

Abstract

Dried specimens of insects are increasingly seen as genetic resources. However, genetic analysis of dried specimens of insects is hampered by the deterioration of the DNA. In this study, we developed methods for preparing dried specimens of insects with well-preserved DNA, mainly for PCR-based genetic analysis. First, we compared the effects of either exposure to ethyl acetate vapour for from 10 min to 6 h or by freezing on the fragmentation of DNA in order to determine optimal length of time needed for killing insects using the above methods. Second, we compared the fragmentation of DNA after preservation by drying or immersion of legs in 99.5% ethanol or 99% propylene glycol in 0.2-ml tubes. We assessed degrees of fragmentation of DNA by determining polymerase chain reaction (PCR) success rates with primers for 313-, 710- and 1555-bp fragments using DNA that was collected immediately, and at one, six and 12 months after preparing the specimens. Differing times taken to kill insects did not affect the fragmentation of DNA. In dried specimens, DNA was seriously fragmented after one month, whereas that in legs prepared by immersion in 99.5% ethanol or 99% propylene glycol contained long fragments of DNA (1555 bp~) after 12 months. Propylene glycol was more suitable for preservation than ethanol, because the latter evaporates. Thus, to preserve insect DNA we suggest inserting the pin on which an insect is impaled into the hinged lid of a 0.2-ml tube containing 99% propylene glycol so that when the lid is closed the legs of the insect are preserved in the solution.