Diagnosis of Lymantria species using DNA barcodes

Brief summary of detection methods

DNA barcoding is a technique for characterizing species of organisms using a short DNA sequence from a standard and agreed-upon position in the genome. Specimens to be used for DNA barcoding are deposited in museums for future reference and a tissue sample is removed for DNA extraction, PCR and DNA sequencing. DNA sequences are deposited in a public database (GenBank or the Barcode of Life Data System). Once sufficient data has been collected for a species and its close relatives, and validated, it is a simple matter to identify an unknown specimen using routine molecular evolutionary analyses available at the click of a button on the website.

Storage of sample

Insect samples to be used for DNA barcoding can be stored dried/pinned, in 70-100% ethanol, or in propylene glycol.

Transport of sample

There are no special requirements for transportation, other than those dictated by airlines if transporting specimens in ethanol.

Detection methods

Lymantria molecular identification (DNA barcoding)

"DNA barcodes" are short DNA sequences, derived from the mitochondrial gene cytochrome c oxidase subunit 1 (COI) that have been shown to be useful for species identification in many taxa. COI sequences can be identical among closely related species therefore the diagnostic utility of DNA barcodes must be established on a case-by-case basis. Published data indicates that DNA barcoding will be a powerful approach to species diagnosis in lymantriid moths: Bogdanowicz et al. (2000) demonstrated high levels of COI variability among populations of Lymantria dispar while Ball & Armstrong (2006) used DNA barcode data to correctly identify 20 species of Lymantriidae, including L. dispar, with 100% success.

DNA extraction

DNA extraction is most efficiently performed using commercial nucleic acid extraction kits based on proteinase-K digestion of tissue followed by silica-based spin column technology, e.g. Machery Nagel NucleospinÒ Tissue Kit, Qiagen DNeasy Tissue Kit, Sigma GenEluteÔ Mammalian Genomic DNA Extraction Kit, etc. Up to 20mg of tissue (a single leg from adult moths, an equivalent amount of tissue for larvae, or even single ovum) is macerated in buffer and digested with proteinase-K for 1-3 hours before being applied to a spin column, washed and eluted.

PCR & DNA sequencing

The PCR primers used by Ball & Armstrong (2006) were LCO1490 (5'-GGTCAACAAATCATAAAGATATTGG-3') and HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3'). PCR amplification consisted of 2 min at 94°C followed by 40 cycles of (94°C for 15 s, 52°C for 30 s, 72°C for 1 min) and finally 72°C for 5 min. PCR products were visualised by agarose gel electrophoresis, and cycle-sequenced in one direction. The above protocol is adequate for research purposes but arguably not for diagnostics. To maintain a high standard of data quality it is desirable to purify PCR products before DNA sequencing using any of the commercially available kits, e.g. Sigma GenEluteÔ PCR Clean-Up Kit, and sequence the products in both directions.

Data Analysis

Forward and reverse direction DNA sequencing reactions should be assembled into contiguous arrays using an appropriate software package. The consensus sequence is exported in FASTA format. To identify an unknown specimen based on its COI sequence one must use the Barcode of Life Database (www.barcodinglife.com) which contains diagnostic COI sequences for Lymantria dispar and a number of other Lymantria species. Under the "Identification Engine" heading on the main page of this website one must select the link "Request identification," paste in the FASTA-formatted COI sequence and click on "Submit." A results window then displays a list of the 100 most similar sequences found in the database, their percentage similarity and their identity. In addition, by clicking on the "Tree Based Identification" button one can view a neighbour-joining tree of one's unknown sequence and the 100 closest sequences. This protocol has proved sufficient for species diagnosis in all Lymantria species examined to date. Importantly, L. dispar is easily differentiated from other Lymantria species using this method.

Though unlikely, it is possible that this conclusion will not hold true for all Lymantria species when data is collected for other species. Therefore to validate this protocol for use in Australia it is essential to collect COI sequence data from all Australian species of Lymantria. This is currently underway. However, L. antennata and L. pelospila are by far the most likely species to be caught in quarantine pheromone traps.