Comparison of DNA extraction methods for DNA barcoding of non-biting midges

The insect group Chironomidae is used as an environmental indicator for freshwater ecosystems because of its wide distribution and species richness. The morphological identification of chironomids requires expert skill; to this end, the application of DNA barcoding for species identification is a powerful tool. We applied DNA barcoding to chironomid specimens using different preservative conditions and several protocols for extraction and sequencing of DNA and found that the success of sequencing was affected by the condition of specimens and protocols applied. Purification using a silica-membrane filter yielded significantly high DNA quality and was deemed suitable for both old and valuable specimens. On the other hand, rough extraction without any purification and extraction without membrane filters both achieved sufficient DNA quality and sequencing success rates to be applied to wellpreserved and large specimens. For old or air-dried samples, however, rough extraction requires great care because it significantly reduces the efficiency of sequencing. DNA extracted from old or poorly preserved specimens tended to be fragmented and was not successfully sequenced, but using alternative PCR primers targeting a shorter region can improve the success rate. Dried wing specimens on prepared slides and pupal exuviae collected from the water surface were also subjected to DNA extraction and subsequent sequencing, which achieved 18.0% and 41.7% success rates, respectively. Such success rates may be insufficient for some purposes of DNA barcoding, but match particular objectives, for example, a field study with less work by collecting exuviae.