Spongy moth specimens collected from the trapping survey undergo molecular diagnostics to determine European or Asian ancestry.
Lymantria dispar asiatica/japonica (Asian spongy moth) and
Lymantria umbrosa (Hokkaido spongy moth) cannot reliably be distinguished morphologically from
Lymantria dispar dispar (European spongy moth). The Standard Spongy Moth Diagnostic Assay is used to distinguish Asian spongy moth from European spongy moth on a genetic level. DNA is extracted from each specimen and amplified using PCR (polymerase chain reaction). Two genetic markers are used in the assay - the nuclear FS1 and a mitochondrial marker - to determine the type of moth.
FS1 Marker – Inherited from both maternal and paternal lineage. The FS1 marker is present in two copies of DNA within the spongy moth genome and can occur in two variations: North American (NA) or Asian (A).
The two FS1 copies can be identical (homozygous) or different (heterozygous) in a specimen. Three genotypes are possible: a moth can be homozygous North American (possessing two North American copies), homozygous Asian, or heterozygous (containing one copy each of the North American and Asian FS1 markers).
| FS1 Marker Outcomes |
| FS1 Copy 1 + |
FS1 Copy 2 = |
Genotype |
| North American |
North American |
North American |
| North American |
Asian |
Heterozygous |
| Asian |
Asian |
Asian |
Mitochondrial Marker – inherited from the maternal lineage. The mitochondrial marker is composed of a single section of DNA. The amplified DNA is exposed to two restriction enzymes (Nla III and Bam HI-HF) that have the ability to cut DNA at specific sites.
| Mitochondrial Marker Outcomes |
| Nla III + |
Bam HI-HF = |
Haplotype |
| Nla- |
Bam- |
North American |
| Nla+ |
Bam- |
A1 |
| Nla+ |
Bam+ |
A2 |
| Nla- |
Bam+ |
A3 |
Final Diagnostic Outcome - The final determination for each specimen is made by interpreting the combined outcomes of the Standard Spongy Moth Diagnostic Assay for each marker. The table below details all possible outcomes.
| Final Molecular DIagnostic Outcomes |
| FS1 Genotype + |
Mitochondrial Marker = |
Final Diagnostic Outcome |
| North American |
North American |
Lymantria dispar dispar-European spongy moth |
| North American |
A1 |
Lymantria dispar dispar-European spongy moth |
| North American |
A2 |
Lymantria dispar dispar-European spongy moth |
| North American |
A3 |
Lymantria dispar dispar-European spongy moth |
| |
|
|
| Heterozygous |
North American |
Lymantria dispar dispar-European spongy moth |
| Heterozygous |
A1 |
Lymantria dispar dispar-European spongy moth |
| Heterozygous |
A2 |
Additional DNA analysis required** |
| Heterozygous |
A3 |
Lymantria dispar dispar-European spongy moth |
| |
|
|
| Asian |
North American |
Lymantria dispar dispar-European spongy moth |
| Asian |
A1 |
Additional DNA analysis required** |
| Asian |
A2 |
Lymantria dispar asiatica/japonica-Asian spongy moth |
| Asian |
A3 |
Lymantria dispar asiatica/japonica-Asian spongy moth |
**Diagnostics that result in Heterozygous, A2 or Asian, A1 requires DNA barcoding to make a final determination. DNA barcoding is a molecular tool that uses PCR and DNA sequencing to identify a specimen. The tool is based on the COI marker, a section of mitochondrial DNA that is effective in distinguishing animals of different species. The technique relies on obtaining the unknown specimen’s COI DNA sequence and comparing it to sequences of known species that are available in public databases, GenBank and BOLD Systems.
