A non-destructive enzymatic method to extract DNA from arthropod specimens: Implications for morphological and molecular studies

Abstract

There is a growing necessity to integrate morphological and genetic studies. This paper proposes a new technique that allows DNA extraction of arthropods while still keeping intact the entire morphology of the specimens. The technique uses Proteinase K to dissolve protein tissues and preserve the chitinous exoskeleton of specimens. The method is fast, cheap, non-toxic, and allows for good morphological preparations of specimens retaining much of their tridimensional structure. The methodology works fine with specimens preserved in different kinds of media, such as for dry (pinned) specimens, and specimens preserved in Ethanol. In addition, it allows the extraction of DNA from fresh specimens, as well as from specimens preserved for a long time. The technique works well for morphological studies alone, but allows the generation of an associated genomic library at an individual-scale. Among the advantages of the new technique is the possibility of extracting DNA from the entire specimen (necessary for the study of diseases transmitted by arthropod vectors), while still keeping the morphology intact for correct taxonomic identification. In addition, in comparison with methods that extract DNA from small tissue samples (e.g., from legs or wings), the method allows for the extraction of a larger amount of DNA and is better suited for small specimens.

Novel Single Hematophagous Insect RNA Detection Method Supports Its Use as Sentinels to Survey Flaviviruses Circulation

“…Agarose gel electrophoresis analysis of the total RNA extracted from 10 specimens of dipteran maintained in 80% ethanol by the nondestructive nucleic acid extraction method described before [21] showed integrity, and an approximately amount of 1 μg/mL, per specimen (Figure 1). The presence of RNA was also confirmed by RT/PCR to amplify a 464 bp corresponding to the 28S rRNA from dipteran, after treatment of RNA with DNase I (Figure 2).…”

“…The technique of RNA extraction from a single mosquito preserving their chitinous cytoskeleton is described for the first time, and its use can contribute not only to the detection of infectious agents with RNA genome but also to the evolutionary and morphological studies of Diptera since the RNA molecule is an important tool to understand the physiology and evolutionary relationships among organisms [29], and the physical structure of the insect is maintained [21]. Also, vectorial capability of arthropods to several infectious agents can be investigated using specific molecules, with conditional expression profiles, as biomarkers.…”

“…For RNA extraction, each dipteran was digested for 16 h at 56°C, inserted in 200 μL of a lysis buffer composed of 200 mM Tris-HCl (pH 7.5), 250 mM NaCl, 25 mM EDTA (pH 8.0), 0.5% of SDS, and 400 μg/mL of proteinase K as described for DNA extraction [21]. Before digestion, ethanol from insects stored in 80% ethanol was removed after washing twice with 1 mL of 1× PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , and 1.8 mM KH 2 PO 4 ).…”

“…Hematophagous insects are present in different wild natural and urban environments, being an excellent group of animal to be used as sentinel. Thus, in this work, the single-insect nucleic acid extraction method [21] was evaluated in hematophagous dipterans collected in a Brazilian municipal public park using CDC traps, in order to detect RNA from flaviviruses.…”

Description and molecular characterization of Chrysopilus kafkai sp. nov. (Diptera: Rhagionidae) from Serra da Bodoquena (Mato Grosso do Sul, Brazil)

“…We used the DNA extraction method proposed by Santos et al (2018). The specimens were clarified with Proteinase K preserving its external morphology.…”