Factors affecting DNA preservation from museum‐collected lepidopteran specimens

Abstract

Recent innovations in molecular genetics made DNA an intriguing molecule not only in molecular biology, but also in ecology and evolutionary and conservation biology. Despite this general interest, several discrepancies have been reported in the literature regarding the techniques for preserving insects for DNA analysis, prompting us to analyse the effects of different storage conditions on lepidopteran DNA preservation. In particular, in the present paper, adults of the cabbage moth, Mamestra brassicae (L.) (Lepidoptera: Noctuidae), were stored under various conditions in order to verify which method is the most suitable to preserve lepidopteran specimens for DNA studies. Mamestra brassicae adults were stored by rapid desiccation with silica gel, by preservation in acetone, 2‐propanol, Carnoy's or ethanol (both at 75 and 100% concentrations) solutions, and finally by storage in an ultracold freezer and liquid nitrogen. Adults preserved by each method were used to extract DNA at the aim of verifying the size of the extracted DNAs, the extraction yield and the possibility of using these samples to amplify both short and long DNA sequences by polymerase chain reaction. The results were compared with those obtained using fresh samples acting as controls. Acetone preservation appeared to be the most recommendable method for moth specimens as it proved to be a good storage medium for DNA analysis, it is cost‐effective, and it is applicable not only to field surveys, but also to obtain efficient and low‐cost storage of lepidopteran specimens in museum collections.

Analysis of tissue dependent DNA yield for optimal sampling of micro-moths in large-scale biodiversity surveys

“…The availability of molecular techniques that can obtain DNA sequence data from tiny amounts of tissue or from old museum specimens (Mandrioli et al, 2006;Meusnier et al, 2008) has led to an increased interest in museum collections which are important not only for studying species at a molecular level but also to integrate DNA data into studies previously conducted at a morphological level only (Carter et al, 1997;Quicke et al, 1999;Knölke et al, 2005;De Prins et al, 2009). However, extraction of DNA is invasive and entails the destruction of the entire specimen, or a part of it.…”

Bumblebee (Hymenoptera: Apidae) sample storage for a posteriori molecular studies: Interactions between sample storage and DNA-extraction techniques

“…The principal problems faced by researchers wishing to use DNA from stored specimens in molecular studies result from DNA shearing and inter-strand cross-linking (Lindahl, 1993;Mandrioli et al, 2006). Shearing, the degradation of DNA into smaller fragments, is caused by a variety of agents, including UV radiation, high temperatures, pH and salinity (Lindahl, 1993).…”

“…Shearing can affect both the extraction and amplification of DNA because short fragments precipitate less than long ones (Lindahl, 1993;Dessauer et al, 1996). Inter-strand crosslinking is due to chemical changes in the DNA (including the addition of alkyl groups) that inhibit denaturation and, consequently, amplification (Mandrioli et al, 2006). Proper storage conditions prevent or delay DNA degrada-tion and maintain larger DNA fragments that aid in PCR experiments (Mandrioli et al, 2006).…”

“…Inter-strand crosslinking is due to chemical changes in the DNA (including the addition of alkyl groups) that inhibit denaturation and, consequently, amplification (Mandrioli et al, 2006). Proper storage conditions prevent or delay DNA degrada-tion and maintain larger DNA fragments that aid in PCR experiments (Mandrioli et al, 2006). However, the comparative efficiencies of different storage methods for maintaining DNA integrity have received relatively little attention, with most studies using previously collected specimens for analyses which restricts their ability to conduct more detailed experiments of optimal storage (Post et al, 1993;Reiss et al, 1995;Koch et al, 1998;Fukatsu, 1999;Quicke et al, 1999;Dean & Ballard, 2001;Mandrioli et al, 2006).…”

“…Insects can also be preserved in a variety of chemical solvents. Fukatsu (1999) and Mandrioli et al (2006) have investigated the efficiency of a range of chemical storage conditions, including acetone and ethanol at various concentrations, methanol, diethyl ether, ethyl acetate, chloroform, 2-propanol, and Carnoy's solution. However, during field expeditions, entomologists predominantly use entomological boxes or ethanol to store collected specimens.…”