The Japanese beetle (Popillia japonica) is an invasive species that has become established in the eastern and midwestern U.S. and causes harm to numerous commercial crop species. To try to prevent the pest’s westward expansion, the U.S. Department of Agriculture’s Animal and Plant Health Inspection Service (APHIS) began implementing biocontrol of Japanese beetles in 2017 with release of a microsporidian parasite, Ovavesicula popilliae, at cargo airports in the Midwest. A group of researchers led by David Smitley, Ph.D., professor emeritus in the Department of Entomology at Michigan State University, refined a quantitative polymerase chain reaction (qPCR) technique for quantifying infection of Japanese beetles by O. popilliae, and they improved both the accuracy and the efficiency of the qPCR measurements. (Photo by David Cappaert, Bugwood.org)

By John P. Roche, Ph.D.

The Japanese beetle (Popillia japonica) was discovered in the U.S. in New Jersey in 1916, and by 2018 it had spread throughout the eastern U.S. and west as far as Colorado. Japanese beetles damage leaves and fruit of more than 300 plant species and cause harm to numerous commercial crop species, including fruit trees, blueberries and raspberries, soybeans, and hops. Pest managers don’t want this invasive species to spread further west because it could cause severe economic damage in agricultural regions along the west coast.

To try to prevent the pest’s westward expansion, the U.S. Department of Agriculture’s Animal and Plant Health Inspection Service (APHIS) began implementing biocontrol of Japanese beetles in 2017, using a microsporidian parasite, Ovavesicula popilliae. They released the microsporidian at cargo airports in the Midwest. Assessing the effectiveness of this biocontrol program depends on accurate measurement of the amount of infection of the beetles at airports. Previous work on assessing infection with O. popilliae used a DNA-measuring technique called quantitative polymerase chain reaction (qPCR), but the initial qPCR measurements were subject to inaccuracies. A group of researchers led by David Smitley, Ph.D., professor emeritus in the Department of Entomology at Michigan State University, conducted a study aiming to improve the accuracy and efficiency of the qPCR technique, and to evaluate the distribution of O. popilliae in airports in the midwestern U.S. They report their findings in a study published in October in the Journal of Economic Entomology.

Ovavesicula popilliae is a fungal, microsporidian, spore-producing parasite that was first discovered in 1987 in Connecticut. Other microsporidian parasites have been used for biological control of other insect pests, with the impact mostly limited to weakening their host. Data from previous studies suggest that O. popilliae severely weakens Japanese beetle larvae by damaging their “kidneys” (Malpighian tubules) and reduces egg production in females by 50%, leading to population declines.

Japanese beetles (Popillia japonica) are widely distributed in the eastern United States but not yet present in the western U.S, as shown here in 2018 estimates by the U.S. Department of Agriculture. Circled numbers show  eight airports sampled in a recent study to refine techniques for quantifying infection of Japanese beetles by microsporidian parasite, Ovavesicula popilliae, deployed as a biological control agent. (Figure originally published in Smitley et al 2025, Journal of Economic Entomology)

The first step in assessing effectiveness of this microsporidian for Japanese beetle biocontrol is to measure how much O. popilliae is present at airports and other potential introduction sites. In the present study, Smitley and colleagues sampled eight airports in the Midwestern U.S. in June and July of 2019 and 2020. They found significantly more O. popilliae infection of Japanese beetles at airports east of the Mississippi River than in Japanese beetles west of the Mississippi.

In 2020, Daniel Hurlbert and colleagues developed a qPCR technique to assess O. popilliae infection of Japanese beetles. qPCR measures fluorescence coming from a dye that allows quantification of DNA amounts in real time. Using qPCR, they found that 12% of beetles were diagnosed incorrectly. They hypothesized that this was due to leaf material in the digestive system of the beetles that inhibited the signal from the qPCR measurements.

In the present study, Smitley and colleagues set out to refine the qPCR technique to reduce these errors. To try to increase the accuracy of sampling for O. popilliae, they diluted DNA to ratios of 1:3, 1:7, and 1:15. They then compared the amount of O. popilliae found via microscopic assessment of dissected beetles with that found by qPCR. With qPCR, the amount of O. popilliae DNA is indicated by the Ct value (cycle threshold) which is the number of PCR cycles at which a target nucleic acid sequence becomes visible with a fluorescent dye. The lower the Ct value, the higher the amount of targeted DNA that is present. When they compared the Ct values from undiluted samples with Ct values from diluted samples, they found evidence for reduced inhibition of detection in the diluted samples. When they compared O. popilliae detection in samples diluted by 1:7 with undiluted samples, the proportion of correct diagnoses improved from 28 out of 31 samples to 31 of 31 samples.

Exclusion netting keeps Japanese beetles (Popillia japonica) out of cargo planes during loading operations. The netting is used on both cargo and crew doors. (Photo courtesy of Phillip Lewis, USDA-APHIS)

Technicians with the U.S. Department of Agriculture’s Animal and Plant Health Inspection Service and the U.S. Air Force release Japanese beetles (Popillia japonica) infected with a microsporidian parasite, Ovavesicula popilliae, at Dover Air Force Base in August 2018. (Photo courtesy of Phillip Lewis, USDA-APHIS)

“It was Yoshiko Nomura in our lab that tested for PCR inhibition by diluting each purified DNA sample by 1:3, 1:7, and 1:15 before qPCR analysis,” Smitley says. “She was able to demonstrate that most of the PCR inhibition, most likely caused by tannins in the leaves, was removed by dilution. Because PCR accurately determines such small amounts of DNA, the diluted samples were still extremely accurate for determining the concentration of parasite DNA, giving us 100% accuracy in diagnosis of 31 individuals.”

Because of the need for widescale, efficient screening for O. popilliae at airports, Smitley and colleagues also conducted experiments to try to improve the efficiency of sampling. In this part of their study, they took DNA from 312 individuals and analyzed them individually for O. popilliae. Then they combined purified DNA from eight Japanese beetles into a single pooled sample. The team found that the Ct values from individual beetles and the Ct values from pooled samples were significantly correlated with each other. This means that the same degree of accuracy could be achieved more efficiently by pooling samples together into groups.

“We can now accurately determine how heavily infected a Japanese beetle is by the amount of O. popilliae DNA present in the beetle,” Smitley says. “This has allowed us to use pooled samples of eight Japanese beetles together to accurately find a single infected Japanese beetle among 12 samples of eight Japanese beetles (96 individuals) in less time and more accurately than the traditional method of dissection and tissue mounting on a slide for microscopic diagnosis.”

One of the huge benefits of this qPCR technique is its high degree of sensitivity. “This paper provides a sound foundation for future work where trace amounts of parasite DNA will allow us to detect invasion by an insect parasites three or four years earlier than we could when using dissection for diagnosis,” Smitley says. “We are now able to detect the pathogen in such small amounts that we know if it is active in a large area the size of an 18-hole golf course several years before a single infected beetle is found among a sample of 96.”

The investigators concluded that their study of detecting O. popilliae infection in Japanese beetles provided valuable tools for current introduction efforts by APHIS and university researchers in several states launching long-term biological control programs using O. popilliae.

John P. Roche, Ph.D., is an author, biologist, and science writer with a Ph.D. and postdoctoral fellowship in the biological sciences and a dedication to creating compelling narratives for readers. He authors books and writes materials for universities, scientific societies, and publishers. Professional experience includes serving as a scientist and scientific writer at Indiana University, Boston College, and the UMass Chan Medical School; as a visiting professor at four tier-one schools; and as editor of science periodicals at Indiana University and Boston College.