Location: Crop Bioprotection Research
Project Number: 5010-22410-023-009-I
Project Type: Interagency Reimbursable Agreement
Start Date: Apr 1, 2025
End Date: Mar 31, 2026
Objective:
Introduce and establish O. popilliae at cargo airports and other locations in the Midwest where Japanese beetle (JB) has become established and is a problem. Collect JB samples from at least 12 states to survey for presence of the JB pathogen. Identify best methods for infecting adult Japanese beetles and conduct a microbiome survey to identify potential novel Japanese beetle entomopathogens acting in synergy with O. popilliae infection.
Objectives:
1. Collect Japanese beetles at five cargo airports in Midwestern United States (AR, IA [2], MO, NE) for pathogen analysis (qPCR) to verify that an epizootic infection has taken hold following previous pathogen releases at these sites.
2. Collect Japanese beetles infected with O. popilliae at epizootic sites in the Midwest and prepare and send spore solutions to cooperators for introduction at their sites.
3. Establish cooperators from multiple locations and states using field staff and others (CT, DE, IL, KY, MA, NJ, OH, PA, TN, VA, WI, WV). Conduct yearly collections of Japanese beetles during peak adult emergence (June or July) from these sites for pathogen analysis to confirm the infection baseline and to monitor pathogen establishment (microbiome survey).
4. Test two inoculation methods (adult feeding on spores and soil drenches of grubs with spores) to identify the best approach to achieve infection by the pathogen.
5. Determine the microbiome associated with microsporidia infections in Japanese beetles to identify novel microbial entomopathogens that could be harnessed to enhance Japanese beetle control.
6. To sequence and provide a draft genome of Ovavesicula popilliae to enhance our understanding of its biology and interactions with Japanese beetles.
Approach:
1. Collect Japanese beetle at introduction sites at the five cargo airports in the Midwest (AR, IA [2], MO, NE) and collect JB for an O. popilliae survey from an additional 12 states to determine the status of pathogen establishment. APHIS Field Operations staff and state cooperators will set Japanese beetle traps at each study site starting after the first emergence of Japanese beetle adults. Frozen beetles will be sent to the Forest Pest Methods Laboratory (FPML) and to the Crop Bioprotection Research Unit (ARS) for pathogen analysis at the end of the trapping period. These samples will be processed for O. popilliae infection, and a portion of any infected samples will be forwarded to the ARS-NCAUR lab for further microbiome analysis.
2. Infected Japanese beetles will be collected and processed into spore solutions in Illinois and shipped to cooperators for introduction of the pathogen at approved release sites in their state. Field staff in central Illinois will collect infected JB in traps and use written protocols to prepare the spore solutions and will send them to cooperators within the state and to valid permit holders in other states.
3. Inoculation studies: Japanese beetles will be exposed to varying levels of pathogen spores on grape leaves; adult beetles, exposed as grubs to spore solutions, will be assessed for infectivity. Adult beetles will be exposed to grape leaves with varying concentrations of spores to determine if pathogen spores can result in infection of the adult stage. A replicated study using male and female beetles will be conducted. Both frass and beetles will be tested for the pathogen by qPCR and spore presence determined by dissections of the Malpighian tubules. Efficacy of spring soil drenches using pathogen spore solutions will be evaluated at twenty vineyards where Japanese beetle populations have been historically high. Three concentrations and a control will be used and adults emerging from the plots will be evaluated to obtain a percent beetle infection rate.
4. Microbial profiling of O. popilliae-infected Japanese beetles to identify novel microbial entomopathogens acting in synergy with O. popilliae. Japanese beetles collected from several locations (above) will be assessed for microsporidia infection via qPCR. We will conduct targeted 16s rRNA gene and shotgun metagenomic sequences to profile the microbiome of infected and uninfected Japanese beetles.
5. Provide a draft genome sequence of O. popilliae. Ovavesicula popilliae spores will be isolated from the Malpighian tubules of Japanese beetles collected from three established O. popilliae infections in Illinois. High-quality DNA will be extracted from purified O. popilliae spores and outsourced to a sequencing core facility for whole genome sequencing.