A Fast, Frassy Way to Detect the Asian Longhorned Beetle
By Andrew Porterfield
The Asian longhorned beetle (Anoplophora glabripennis) is known for causing damage to a variety of tree species. From its east Asian roots, the beetle is now common in nearly all of China and Korea and has spread to Japan, nine European countries, Canada, and some states in the U.S.
Anoplophora glabripennis is known to damage 34 species of trees, including poplars, several species of maple, paper birch, willows, and elms. It is considered a “quarantine” pest in the U.S. and Europe and has caused significant economic losses there and in Asia. Females can lay between 30 and 178 eggs under the host plant’s bark, and the larvae feed on the phloem and sap-containing wood of the host tree, causing damage to the central and upper trunk and main branches.
Eradication of the beetle has hinged on early diagnosis of infestation, in addition to monitoring for signs of beetle damage (particularly frass, a combination of wood debris and feces left behind by the beetle larvae). Detection methods based on pheromones and other airborne chemicals emitted by the beetles and infested plants only detect adult beetles post-infestation. DNA-based techniques, however, are sensitive and precise enough to detect beetles in the early stages of an infestation. Low concentrations of DNA can still be detected using these techniques.
Loop-mediated isothermal amplification (LAMP) has been attracting the attention of biologists because it can easily detect DNA to identify a wide range of species, and it can detect insect pests and fungi on plants. It also is easier to use and less costly than its more familiar DNA-detection cousin, the polymerase chain reaction (PCR).
To test whether LAMP can provide easy, early detection of A. glabripennis larvae, a team of Italian scientists developed a protocol using visual and real-time LAMP. Their rapid diagnostic protocols proved to be a reliable, accurate, and promising tool to help biologists detect the presence of Anoplophora. Their results were published in September in the Journal of Economic Entomology.
Elisabetta Rossi, Ph.D., associate professor of agriculture, food, and environment at the University of Pisa, and her colleagues developed the two LAMP techniques on frass left behind by beetle larvae. After collecting target species in Lombardy, Italy, from 2014 to 2018, they also collected beetle frass as well as non-target samples for comparison.
Six LAMP primers (used to begin identifying the target DNA strand in the samples) were designed for the beetle tests. Real-time LAMP was used to amplify DNA thermocycling and analyzed. At the same time, a visual LAMP assay was developed to determine rates of color change (from purple to blue, respectively indicating negative to positive reactions). Sensitivity, specificity, and accuracy were calculated for both assays.
For specificity, none of the tests showed non-specific signals—only the beetle DNA was detected. In visual LAMP tests, the color change from purple to blue was easily and definitively detected. Sensitivity and accuracy for the LAMP methods were 100 percent, the researchers found, with no cross-reaction with a similar species of beetle (A. chinensis). A blind panel test carried out in two laboratories confirmed the sensitivity, specificity, and accuracy results. The LAMP methods were faster than traditional PCR: 80 minutes for real-time LAMP, and 30–60 minutes for visual LAMP.
“These results and the rapid execution of the tests suggest that this protocol could be a useful diagnostic tool for monitoring of plants in the nursery during phytosanitary inspections,” the researchers say, “and for the identification of management of outbreaks through a timely indirect diagnosis including the entry points of a region/nation.”
Journal of Economic Entomology
Andrew Porterfield is a writer, editor, and communications consultant for academic institutions, companies, and nonprofits in the life sciences. He writes frequently about agriculture issues for the Genetic Literacy Project. He is based in Camarillo, California. Follow him on Twitter at @AMPorterfield or visit his Facebook page.