Non-destructive DNA extraction from Chironomidae, including of fragile pupal exuviae, extends analysable collections and enhances vouchering

It is well established that the traditional taxonomy and nomenclature of Chironomidae relies on adult males whose usually characteristic genitalia provide evidence of species distinction. In the early days some names were based on female adults of variable distinctiveness – but females are difficult to identify (Ekrem et al. 2010) and many of these names remain dubious. In Russia especially, a system based on larval morphology grew in parallel to the conventional adult-based system. The systems became reconciled with the studies that underlay the production of the Holarctic generic keys to Chironomidae, commencing notably with the larval volume (Wiederholm, 1983). Ever since Thienemann’s pioneering studies, it has been evident that the pupa, notably the cast skins (exuviae) provide a wealth of features that can aid in identification (e.g. Wiederholm, 1986). Furthermore, the pupae can be readily associated with name-bearing adults when a pharate (‘cloaked’) adult stage is visible within the pupa. Association of larvae with the name-bearing later stages has been much more difficult, time-consuming and fraught with risk of failure. Yet it is identification of the larval stage that is needed by most applied researchers due to the value of the immature stages of the family in aquatic monitoring for water quality, although the pupal stage also has advocates (reviewed by Sinclair & Gresens, 2008). Few use the adult stage for such purposes as their provenance and association with the water body can be verified only by emergence trapping, and sampling of adults lies outside regular aquatic monitoring protocols.

A note on sampling chironomids for RNA-based studies of natural populations that retains critical morphological vouchers

The rapid uptake of transcriptomic approaches in freshwater ecology has seen a wealth of data produced concerning the ways in which organisms interact with their environment on a molecular level. Typically, such studies focus either at the community level and so don’t require species identifications, or on laboratory strains of known species identity or natural populations of large, easily identifiable taxa. For chironomids, impediments still exist for applying these technologies to natural populations because they are small-bodied and often require time-consuming secondary sorting of stream material and morphological voucher preparation to confirm species diagnosis. These procedures limit the ability to maintain RNA quantity and quality in such organisms because RNA degrades rapidly and gene expression can be altered rapidly in organisms; thereby limiting the inclusion of such taxa in transcriptomic studies. Here, we demonstrate that these limitations can be overcome and outline an optimised protocol for collecting, sorting and preserving chironomid larvae that enables retention of both morphological vouchers and RNA for subsequent transcriptomics purposes. By ensuring that sorting and voucher preparation are completed within <4 hours after collection and that samples are kept cold at all times, we successfully retained both RNA and morphological vouchers from all specimens. Although not prescriptive in specific methodology, we anticipate that this paper will assist in promoting transcriptomic investigations of the sublethal impact on chironomid gene expression of changes to aquatic environments.