Trace DNA from insect skins: a comparison of five extraction protocols and direct PCR on chironomid pupal exuviae

Abstract

Insect skins (exuviae) are of extracellular origin and shed during moulting. The skins do not contain cells or DNA themselves, but epithelial cells and other cell-based structures might accidentally attach as they are shed. This source of trace DNA can be sufficient for PCR amplification and sequencing of target genes and aid in species identification through DNA barcoding or association of unknown life stages. Species identification is essential for biomonitoring programs, as species vary in sensitivities to environmental factors. However, it requires a DNA isolation protocol that optimizes the output of target DNA. Here, we compare the relative effectiveness of five different DNA extraction protocols and direct PCR in isolation of DNA from chironomid pupal exuviae. Chironomidae (Diptera) is a species-rich group of aquatic macroinvertebrates widely distributed in freshwater environments and considered a valuable bioindicator of water quality. Genomic DNA was extracted from 61.2% of 570 sampled pupal exuviae. There were significant differences in the methods with regard to cost, handling time, DNA quantity, PCR success, sequence success and the ability to sequence target taxa. The NucleoSpin(®) Tissue XS Kit, DNeasy(®) Blood and Tissue kit, and QuickExtract(™) DNA Extraction Solution provided the best results in isolating DNA from single pupal exuviae. Direct PCR and DTAB/CTAB methods gave poor results. While the observed differences in DNA isolation methods on trace DNA will be relevant to research that focuses on aquatic macroinvertebrate ecology, taxonomy and systematics, they should also be of interest for studies using environmental barcoding and metabarcoding of aquatic environments.

A Comprehensive Comparison of DNA Extraction Using Fresh and Stored Bloods in Molecular Hematology Diagnostic Study

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Conochironomus (Diptera: Chironomidae) in Asia: new and redescribed species and vouchering issues

“…The latter stage has high value in systematics and descriptive taxonomy (Brundin 1966, Krosch & Cranston 2012) and biomonitoring (Raunio et al 2007), and it can provide barcode DNA (Krosch & Cranston 2012, Kranzfelder et al 2015. Fortunately, it is likely that the development of quick extraction methods (Kranzfelder et al 2016), Direct PCR (Wong et al 2014), and cheap NGS barcoding (Meier et al 2015) will rapidly increase the number of species with matched life history stages.…”

Using DNA metabarcoding for assessing chironomid diversity and community change in mosquito controlled temporary wetlands

“…Only seven of those species can be determined based on larval morphology (Table 1). Even though various determination keys for larval and adult chironomids exist, not all taxa can be determined to species level even by experts (Kranzfelder et al 2016). Especially larvae and female midges are almost impossible to determine morphologically, since often male genitals are necessary to distinguish species.…”