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

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.

Keywords

• gracillariidae
• cameraria ohridella
• dna barcoding
• dna preservation
• dna quantification
• picogreen