Abstract
DNA barcoding surveys of small insects usually extract DNA from either a complete insect or a leg. Little is known about how to optimize DNA quantity and quality from different insect parts while preserving a morphological voucher. Here, we quantify DNA yield from different body parts (antenna, hind leg, forewing, hind wing and abdomen) of the micro-moth Cameraria ohridella (Lepidoptera: Gracillariidae) using fluorescent nucleic acid stain (PicoGreen). Samples were preserved in 100% ethanol or dried for three weeks. Our experiment was designed to encompass practical sampling options during fieldwork. DNA quality was assessed by PCR amplification of the mitochondrial COI barcode fragment. In addition, we compared PCR amplification using Platinum® Taq and Qiagen DNA Polymerase and quantified sequence success of amplified DNA. We show that overall, dry parts showed higher eluted DNA yields. PCR and sequencing success rate were slightly higher for dry tissue than ethanol-preserved parts. We also show that Platinum® Taq yielded the highest PCR success rate and that all dry tissues are sequenceable. The optimal strategy for DNA barcoding surveys is therefore to mount micro-Lepidoptera specimens in the field for morphological analysis and sample tissues (hind legs are favoured) from dried samples at a later time (several weeks) in the lab for DNA barcoding using preferentially Platinum® Taq. If larger amounts of DNA are required (i.e. for nuclear gene sequencing), several legs from one side of the specimen or the abdomen should be preserved in pure ethanol.
Highlights
Lepidoptera play a prominent role in biodiversity surveys as indicators of habitat disturbance and hotspots of biodiversity (Küper et al, 2004; Kristensen et al, 2007)
Thirty freshly-emerged adult moths collected in Mézières-lezCléry, France, were placed in a collection tube that was placed in boiling water for several seconds, a practical and convenient technique that does not require dangerous killing reagents (Dall’Asta et al, 2002)
The five samples from the right side were fixed in 100% ethanol and coded as follows: right antenna (RAN), right forewing (RFW), right hindwing (RHW), right hindleg (RHL), abdominal segments I–III (FAB)
Summary
Lepidoptera (butterflies and moths) play a prominent role in biodiversity surveys as indicators of habitat disturbance and hotspots of biodiversity (Küper et al, 2004; Kristensen et al, 2007) They constitute a mega-diverse order of insects with at the latest count 157,424 recognized species (Nieukerken et al, in press) and an additional estimated 230,000 species (www.lepsys.eu) still to be discovered, mostly in tropical areas. For freshly collected material, it is critical that voucher specimens used in genetic analyses are available for subsequent morphological study to corroborate identifications, and in some cases for those vouchers to become future types This is relevant to surveys of microlepidoptera fauna in the tropics where exceptionally high levels of singletons (species represented by single individuals) of undescribed species are found (Davis & Stonis, 2007)
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