Dichlorvos exposure impedes extraction and amplification of DNA from insects in museum collections
© Espeland et al; licensee BioMed Central Ltd. 2010
Received: 10 September 2009
Accepted: 18 January 2010
Published: 18 January 2010
The insecticides dichlorvos, paradichlorobenzene and naphthalene have been commonly used to eradicate pest insects from natural history collections. However, it is not known how these chemicals affect the DNA of the specimens in the collections. We thus tested the effect of dichlorvos, paradichlorobenzene and naphthalene on DNA of insects (Musca domestica) by extracting and amplifying DNA from specimens exposed to insecticides in two different concentrations over increasing time intervals.
The results clearly show that dichlorvos impedes both extraction and amplification of mitochondrial and nuclear DNA after relatively short time, whereas paradichlorobenzene and naphthalene do not.
Collections treated with paradichlorobenzene and naphthalene, are better preserved concerning DNA, than those treated with dichlorvos. Non toxic pest control methods should, however, be preferred due to physical damage of specimens and putative health risks by chemicals.
Natural history collections are an invaluable source of biological data [1–3]. These collections record the distribution of known taxa in space and time and document both what we know and what we don't know about the world's biota . Biologists all over the world have been extracting ecological, morphological, phylogenetic, diversity and biogeographic data from museum specimens for decades, if not decennia . More recently these specimens are also in frequent use for the extraction of DNA in e.g. molecular phylogenetic, population genetic and conservation genetic studies [5–9]. It could also be expected that Natural history collections will be much more important in molecular studies in the near future owing to; 1) difficulties to collect fresh biological material from many regions and the extinction of taxa due to habitat loss, and 2) the development of new high-throughput sequencing methods  and protocols that makes it possible to use these techniques for PCR-product sequencing  and conducting extensive molecular studies based on fragmented DNA in museum collections.
Museum collections are prone to attacks by insect pests, especially beetles of the family Dermestidae (Coleoptera). If left unattended these pests can completely destroy an insect collection within a few months time. Hence a variety of methods have been developed to eradicate the pest insects e.g. fumigation or other treatments with insecticides [12, 13], traps [14–16], heating [17–19] or freezing of infested specimens [20–22] and modified atmosphere [23–28].
Many different insecticides have been used in eradication of pest insects in collections. The use is declining, but it is still utilized in many museums [29, 30]. Several studies of the effects of insecticides on the pest insects e.g. [12, 31] and their effect on different materials in museum collections [32, 33] have been performed, but there are few studies of how insecticides affect the DNA of the specimens in natural history collections. Whitten et al  found no effect of sulphuryl fluoride (Vikane) on the DNA of herbarium specimens. According to Kigawa et al.  methyl bromide, ethylene oxide, propylene oxide and methyl iodide all affected the DNA in both freeze-dried mushrooms and chicken muscle negatively, whereas sulphuryl fluoride did not. To our knowledge no studies on the effects of insect DNA have been performed.
Naphthalene, paradichlorobenzene and dichlorvos are some of the most frequently used insecticides in insect collections, but their effect on the DNA of insect specimens is not known. We therefore exposed dried insects to various concentrations of these insecticides over a period of 20 months (605 days), extracted DNA from the specimens and ran both total DNA extracts and polymerase chain reaction (PCR) products on agarose gels to investigate effects of these insecticides on the DNA of insect specimens.
The six insecticide treatments and controls in the current study.
1 High concentration
2 Low concentration
Extraction dates and length of pesticide exposure (in days) for all samples.
Pesticide exposure (days)
DNA was extracted from whole houseflies using the Qiagen DNeasy Tissue Extraction kit (Qiagen Inc., Valencia, California) which yields DNA fragments of length 50 000 kb and shorter. Twelve μl of the aliquots were run directly on 1% agarose gels in 0.5× TBE buffer for 5 hours and visualized under UV light.
Fragments of comparable length of one mitochondrial (COI, 658 bp; primers LCO-HCO ) and one nuclear gene (EF1a, 716 bp; primers M46.1-R [37, 38]) were amplified using Ready-To-Go™ PCR Beads (Amersham Pharmacia Biotech, Piscataway, New Jersey). Reaction mixtures consisting of 2 μl template, 1 μl primer (10 μm, forward and reverse) 16 μl dH20 and beads were heated to 95°C for 5 minutes, followed by 40 cycles of 30 seconds at 95°C, 30 seconds at a specific annealing temperature (52°C for EF1a and to 50°C for COI) and 50 seconds at 72°C, and then a final extension of 8 minutes at 72°. PCR products were visualized by ultraviolet light on a 0.8% agarose gel after electrophoresis.
If fragmentation is seen in both extraction and amplification then there is evidence that these insecticides cause degradation of DNA. If, on the other hand, initial gel runs on extracts exposed to insecticides are identical to controls, but amplification of genes are impossible or very difficult we have evidence that insecticides might inhibit amplification.
Effect on total DNA
Amplification of nuclear and mitochondrial DNA
The use of DNA from organisms in museum collection is increasing and it is thus important to curate the collections with this in mind. Dichlorvos clearly affects the DNA of insects negatively already after four months of exposure and the effect increases over time, whereas naphthalene and paradichlorobenzene do not seem to affect DNA, at least not over a time period of 20 months. Negative effects on DNA are observed both in total DNA extractions and amplification of nuclear and mitochondrial DNA, thus the major problem is fragmentation of DNA and not inhibition of PCR primers. Effects are also larger for the nuclear gene than for the mitochondrial gene, which is not unlikely since the mitochondrial gene is present as multiple copies in every cell, whereas nuclear DNA only in two copies. Mitochondria are also structurally strong which might lead to better preservation of mitochondrial DNA than its nuclear counterpart . The concentration of insecticide used is also important with higher concentration resulting in increased damage of DNA. The dosages of dichlorvos used in this study might seem extremely high, but they (even the high dose) are probably closer to reality than the recommended dose. The pesticide is very potent even in small doses, and it is almost impossible not to use more than necessary. It is also possible that we will see similar results of DNA fragmentation for paradichlorobenzene and naphthalene when used in higher doses. Dichlorvos is a potent acetylcholinesterase inhibitor and can cause DNA damage in human cells at low concentrations, even after short exposure [40, 41], and it is putatively carcinogenic in humans . It has also been shown to cause severe damage on museum material, such as bleaching of colour, and even corrosion of metal [32, 33]. Because of its deleterious effects to both human and insect DNA the use of dichlorvos for pest prevention in natural history collections should be strongly avoided. Even naphthalene and paradichlorbenzene, are suspected carcinogens [43, 44]. They also effect colours and soften resins , and are documented less effective in killing the pests than dichlorvos . Therefore they are not recommended for use in museums. Non-toxic methods such as freezing [21, 22], or anoxic treatment  should be recommended if infestation has occurred since they are effective against pests and at the same time little hazardous to humans and items. On the other hand we wholeheartedly agree with Blyth & Smith , that prevention is better than the cure.
The use of dichlorvos for pest eradication in natural history collections should be strongly avoided due to deleterious effects on DNA. Chemical eradication methods in general should be avoided since they can cause damage to specimens and are associated with putative health issues.
We are grateful to Keyvan Mirbakhsh, Mattias Myrenås, Pia Eldenäs and Bodil Cronholm at the Molecular Systematics laboratory (Swedish Museum of Natural History) for discussions about molecular lab procedures. Tobias Malm kindly helped with DNA extractions. We also thank Anticimex for providing the dichlorvos. The study was funded by the Swedish Museum of Natural History.
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