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Research Project: Biological Control and Integrated Management of Invasive Arthropod Pests from Europe, Asia, and Africa

Location: European Biological Control Laboratory

2022 Annual Report

Objective 1: Explore across Europe, Asia and Africa for natural enemies of invasive arthropod pests identified as high priority targets by the ARS Office of National Programs, that include but are not limited to stink bugs, fruit flies, beetles, planthoppers, ticks and mosquitoes. Objective 2: Characterize target pests and their natural enemies to enable the successful search of prospective biocontrol agents. • Sub-objective 2a. Investigate the phylogeography of target pests to trace the geographic origin of US invasive populations. • Sub-objective 2b. Characterize genetically the target pests and associated natural enemies in order to support taxonomic identification and facilitate selection of best candidates for biological control. • Sub-objective 2c. Characterize microbiomes associated with target pests and candidate biocontrol agents of interest, with the support of whole genome sequencing when needed. • Sub-objective 2d. Characterize and compare the full genome sequences for biocontrol agents of the olive fruit fly and for a hemipteran invasive pest, bagrada bug, to probe the genetic bases of invasiveness or potential for biological control. Objective 3: Prevent adverse environmental impacts of biological control by in-depth evaluation of efficacy and safety of prospective natural enemies. • Sub-objective 3a. Determine biological and ecological parameters that affect the efficacy and safety of parasitoids of target pests. • Sub-objective 3b. Investigate the chemical ecology of target pests, including the viburnum leaf beetle and the olive fruit fly, and their natural enemies. • Sub-objective 3c. Synergism between Sterile Insect Technique (SIT) and biological control for fostering management of the bagrada bug and research of its new natural enemies. Objective 4: Develop surveillance strategies for blood feeding arthropods and design novel insecticide application methods for better protection of human health, ecosystems and wildlife. • Sub-objective 4a. Evaluate novel and optimize existing vector surveillance tools for increasing treatment-precision and quality assessment of vector control applications including but not limited to Mosquitoes, sand flies and ticks). • Sub-objective 4b. Evaluate novel and optimize existing vector control strategies under field conditions including but not limited to Mosquitoes, sand flies and ticks).

Biological invasions by nonnative arthropod pests are on the rise in the U.S., causing adverse impacts on U.S. agriculture, natural ecosystems, and human and animal health. Without improved strategies based on innovative scientific advances and increased investments to counter biological invasions, harm is likely to accelerate. The USDA emphasizes environmentally friendly management of invasive arthropod pests for which classical biological control and vector control are two major components. Classical biological control by definition involves the intentional introduction of non-native, usually coevolved, natural enemies for permanent establishment and long-term pest control. Once established, natural enemies are self-perpetuating, conserving non-renewable resources and reducing management expenses. One of the main challenges of biocontrol is the long time required to discover appropriate agents and to determine that they will not create a problem when introduced. The European Biological Control Laboratory proposes to take advantage of its biologically strategic locations in Europe, and excellent facilities, including two quarantines, to develop efficient approaches in classical biological control and vector management. Research involves discovering natural enemies (insects or mites) that attack the target pest in its land of origin. Prospective agents will be characterized morphologically, genetically and biologically, and their degree of specificity toward the target pest will be assessed before shipment to U.S. cooperators. Research incorporates the most advanced tools in chemical ecology, microbiomics, and genomics that shall improve the predictability and safety of the agents. Priority targets currently include the bagrada bug, spotted lanternfly, olive psyllid, viburnum leaf beetle, allium leaf miner, roseau cane scale, cattle fever tick and Asian longhorned tick. Vector control involves chemical and non-chemical strategies to control target vectors in the most efficacious and environmentally safe way possible. Research improves methods for detecting and monitoring vector populations such as mosquitoes, sand flies and ticks and designs novel vector control technologies under the umbrella of Integrated Pest Management (IPM) for better protection of human health, ecosystems and wildlife.

Progress Report
3a. Under Objective 1, exploratory research was conducted on several targets. Sentinel eggs of Bagrada bug were exposed in South Africa by a local student at the University of Stellenbosch. No additional Hymenopteran egg parasitoids were collected. Additionally, under objective 1, a total of 2,040 of viburnum leaf beetle larvae were collected across the southern half of Sweden in May-June 2022 and are currently maintained inside EBCL quarantine to monitor the emergence of larval parasitoids. Collections of egg masses made in France, Belgium, Switzerland, and Luxemburg in February-March 2022 resulted in the total emergence of 4,538 larvae and 389 egg parasitoids. This is the first-time egg parasitoids were obtained from Luxemburg. The presence of viburnum leaf beetle was not detected in all sites examined in March 2022 in Greece and Bulgaria. Additionally, under objective 1, sentinel plants infested with allium leaf miner adults originating from EBCL were exposed in 12 sites near EBCL and close to organic leek fields. In total, 23 larval parasitoids emerged from these infested plants, among which 22 have been identified as Miscogaster sp. and one as an unidentified species in the genus Pseudopezomachus. This specimen is currently being examined for identification at the species level by a USDA collaborator. Exposure of larvae in leek plants to two parasitoids of leaf mining flies, Diglyphus isaea and Dacnusa sibirica obtained from KOPPERT company showed that D. sibirica can successfully complete its development using allium leaf miner as a host. This is a first record of parasitism of this pest for this parasitoid. Results with D. isaea were inconclusive. Additionally, under objective 1, engorged nymphs of cattle fever ticks were collected on cattle in Northern Greece and questing larval and nymphal ticks were sampled using carbon dioxide-baited tick traps in Crete. No parasitoids emerged. However, molecular identification of the ticks confirmed that they all are closely related to cattle fever ticks in Texas, hence broadening the current area where to explore for most coevolved natural enemies. Additionally, under objective 1, two regions in Northern Vietnam (Thai Nguyen and Ninh Binh) were surveyed in May 2022 for Asian longhorned tick and its natural enemies. All ticks were preserved in ethanol for species identification and detection of potential parasitoids parasitizing these hard ticks. In December 2021, our collaborators at NIVR in Vietnam shipped to EBCL ticks collected either directly from cattle, goats and dogs or using CO2 traps and preserved in ethanol. None were identified as Asian longhorned tick, but some were assigned to another EBCL target, cattle fever tick. Additionally, under objective 1, surveys for endoparasitoids of olive psyllid were accomplished in Crete and Northeastern Peloponnese in Greece in May and July 2022. Samples are currently maintained inside EBCL quarantine to monitor endoparasitoid emergence. 3b. Under Sub-Objective 2A, the Bagrada sampled in Malta in 2021 were found genetically similar to all Bagrada occurring on caper plants in Sicily and Morocco, distant from Bagrada in the U.S. but closely related to Bagrada in South Africa where they attack cole crops. The genetic match between Pakistan/Indian populations of Bagrada compared to populations occurring in California, Hawai and Chili was confirmed, indicating Pakistan/India as the likely origin of Bagrada invasive populations in the U.S. Under Sub-Objective 2B, taxonomic investigation of one promising biocontrol agent of Bagrada, revealed that an incorrect name was assigned to this agent. This agent is now Gryon aetherium and has been recently detected in California and Chili. Under Sub-Objective 2C, Wolbachia was detected in seven populations of P. lounsburyi recovered in 2015 in California after release. The two Wolbachia variants present in the historical EBCL colonies of P. lounsburyi are persisting after release in CA and not affected by the host switching (from alternative host for mass-rearing to natural host). Additional samples recovered from 28 localities are being analyzed. Additionally, under Sub-Objective 2C, sequencing of the Spliced Leader rRNA gene of the trypanosomatids associated with Bagrada EBCL colonies originating from U.S. and South Africa confirmed that these trypanosomatids belong to one single species of the Blastocrithidia genus. An in vitro culture method and a counting procedure using a hemocytometer were successfully developed, thus facilitating research on these parasites. The in vitro development of trypanosomatids from infected Bagrada host faeces strongly indicated that trypanosomatids are horizontally transmitted to the offspring via the ingestion of specialized resting stage (cyst-like amastigotes) from the faeces of other infected hosts. Under Sub-Objective 2D, Genome contig assemblies of Bagradaspecimens originating from U.S. and South Africa provided by ARS scientists in Beltsville and France (EBCL) have been generated by the Ag100Pest Initiative. All assemblies were generated using PacBio HiFi data generated from DNA from a single specimen. Hi-C libraries have been generated and are in queue for sequencing in order to scaffold the assemblies. 3c. Under Sub-Objective 3A, the role of temperature treatments (-20°C, 5°C, 13°C) and six durations (one week to three months) was investigated on Bagradaeggs. Bagrada eggs stored at 5°C up to one month were optimal for successful parasitism by Gryon gonikopalense without negative effect on progeny. In addition, we assessed the effect of host deprivation on parasitoid fecundity and progeny development. The parasitoid did parasitize Bagrada eggs after up to 60 days of host deprivation, but the fecundity was reduced. One publication has been accepted. Additionally, under Sub-Objective 3A, the egg parasitoid Aprostocetus sp. was shown to parasitize viburnum leaf beetle as well as the elm leaf beetle, and adults emerging from the two species can parasitize either the host they developed in or the other host. In conclusion, this parasitoid species can attack other Chrysomelid species (5 non-host species with successful development of the parasitoid in total). Due to this lack of specificity, no petition to introduce this parasitoid to the U.S. will be submitted. Under Sub-Objective 3B, volatile compounds (VOCs) emitted by Viburnum plants were collected under different modalities during Fall 2021 and Spring 2022. Some samples collected in spring 2021 have been sent to a USDA collaborator for identification and others were analyzed locally towards the optimization for sampling and analysis of VOC’s samples with a volatile collection apparatus at EBCL. First results indicate that Viburnum plants consistently produce a variety of green leaf volatiles and terpenes when attacked by viburnum leaf beetle and that these compounds could be used to develop attractive lures. Additionally, under Sub-Objective 3B, ability to conduct experiments investigating the effects of chemical cues on host acceptance in Psyttalia spp., as part of a EBCL sabbatical program involving was impacted due to travel restrictions. However, the parasitoids needed for the planned experiments have been continuously reared at EBCL with successThe sabbatical research is expected is expected to resume in the summer or fall 2022. Under Sub-Objective 3C, a protocol for irradiating Bagrada adults was optimized with our cooperators in Rome where several irradiation doses were tested on eggs and adults of a Bagrada colony originating from Pantelleria island, IT. With the best dose set at 40 Grays, eggs remain suitable for parasitism up to 10 days. Irradiated sentinel eggs were exposed for the first time in outdoor conditions in South Africa. After ten days, none of the irradiated eggs exposed eclosed. 3d. Under Sub-Objective 4A, sand fly larvae were detected molecularly in 48 out of the 90 soil samples collected last year from a multi-species animal farm in Greece and identified. Phlebotomus simici was the predominant one, followed by P. perfiliewi and P. tobbi. In most of the examined samples more than one sand fly species co-existed. A seasonal larval population pattern was detected, in line with seasonal patterns of adult sand flies. Abiotic parameters analyses through soil particle size and pH measurements indicated higher densities of sand fly larvae in clay and silt soil types and neutral soils. NGS data about microbial diversity associated to different putative breeding sites of sandflies have been obtained lately due to COVID, but data analysis is ongoing. West Nile Virus infected mosquito pools and sentinel chickens were examined bi-weekly, and data was shared with public health authorities for designing timely and targeted vector control treatments. Under Sub-Objective 4B, after extensive literature review and consultation with drone pilots and insecticide application professionals two drone technologies (a cold-fogger and a thermal-fogger) were chosen and purchased. Studies are ongoing for the characterization of these two technologies (droplet size range, effective swath width).

1. Simplified DNA Barcoding for ticks to facilitate survey tick borne disease. Developing an easy-to-use barcoding protocol for ticks to improve the efficacy and sensitiveness of detection will be highly beneficial to ARS laboratories, and inspection services such as USDA-APHIS. As a result, EBCL modified the classical conditions of the Polymerase Chain Reaction for the genetic analysis of one single leg of individual ticks at the junenile stage. This protocol proved successful to amplify the barcode region in primary vectors of important tick-borne diseases in North America belonging to the genera Amblyomma, Dermacentor, Ixodes and Rhipicephalus. This protocol greatly reduces the chance to amplify mixed heterogenous DNAs including tick microbiome that is happening when extracting the entire tick larva or when pooling tick larvae because they do not provide enough genetic material for downstream reactions.

2. Potential utilization of mosquitoes as an alternative source of nutrients in animal diets. Increasing pressure on the world’s livestock production sector has stirred interest towards the exploration of insects as an alternative, nutritional feed source. However, decisions concerning the safe introduction of insects in animal diets have to be based on sound research. A nutritional balanced diet is essential for the growth, development, and performance of animals. For these goals to be met, feed components should be characterized by high protein content, a balanced amino profile, high digestibility, and palatability. EBCL scientists and collaborators produced the first study on the nutritional value of mosquitoes harvested from intensive agricultural settings (rice-fields) to be utilized as poultry feed. Mosquitoes exhibited an overall highly nutritious profile in terms of proteins, lipids, minerals, and antioxidant activities, comparable and even superior to common feedstuff (soybean meal, fishmeal) and widely consumed insects. Food pathogens were present in mosquitoes at similar levels as in fresh food harvested from soil. Mosquitoes harvested from the wild can complement crop-derived feed and support animal livestock production while contributing to the reduction of GHG emissions. This research opens the door to the recycling of wild-caught mosquitoes into the agricultural industry as an alternative, nutritional source for animal feed.

Review Publications
Hanache, P., Thomann, T., Caron, V., Desurmont, G. 2021. Can estivation preferences be used to develop novel management tools against invasive mediterranean snails?. Insects. 12:1118.
Talamas, E., Bremer, J.S., Moore, M.R., Bon, M., Lahey, Z., Roberts, C.G., Combee, L.A., McGathey, N., van Noort, S., Timokhov, A.V., Hougardy, E.H., Hogg, B.N. 2021. A maximalist approach to the systematics of a biological control agent: Gryon aetherium Talamas, sp. nov. (Hymenoptera, Scelionidae). Journal of Hymenoptera Research. 87:323–480.
Martel, G., Sforza, R. 2021. Evaluation of three cold storage methods of Bagrada hilaris (Hemiptera: Pentatomidae) and the effects of host deprivation for an optimized rearing of the biocontrol candidate Gryon gonikopalense (Hymenoptera: Scelionidae). Biological Control. 163: 104759.
Desurmont, G.A., Tannieres, M., Roche, M., Blanchet, A., Manoukis, N. 2022. Identifying an optimal screen mesh to enable augmentorium-based enhanced biological control of the olive fruit fly Bactrocera oleae (Diptera: Tephritidae) and the Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritida. Journal of Insect Science. 22(3):1-7. Article 11.
Mann, L., Laplanche, D., Degen, T., Turlings, T.C., Desurmont, G.A. 2021. Plant volatile emissions induced by insect and gastropod herbivory: a comparative study. Scientific Reports. 11, 23698.
Wang, X., Ramualde, N., Desurmont, G., Smith, L., Gundersen, D.E., Grodowitz, M.J. 2021. Reproductive traits of the egg parasitoid Aprostocetus fukutai a promising biological control agent for invasive citrus longhorned beetle Anoplophora chinensis. Biocontrol.
Grodowitz, M.J., Gundersen, D.E., Elliot, B., Evans, R.M., Sparks, M., Reed, D.A., Miles, G.P., Allen, M.L., Perring, T.M. 2022. Trypanosomatids associated in the alimentary canal of Bagrada hilaris (Hemiptera: Pentatomidae). Journal of Insect Science. 22(1):1-6.
Fotakis, E., Orfanos, M., Couleris, T., Stamatelopoulos, P., Tsiropoulos, Z., Kampouraki, A., Kioulos, I., Mavridis, K., Chaskopoulou, A., Koliopoulos, G., Vontas, J. 2021. VectorMap-GR: A local scale operational management tool for entomological monitoring, to support vector control activities in Greece and the Mediterranean Basin. Current Research in Parasitology and Vector Borne Diseases. 1, 100053.
Rivers, A.R., Grodowitz, M.J., Miles, G.P., Allen, M.L., Elliott, B., Weaver, M.A., Bon, M., Rojas, M.G., Morales Ramos, J.A. 2022. Gross morphology of diseased tissues in Nezara viridula (Hemiptera: Pentatomidae) and molecular characterization of an associated microsporidian. Journal of Insect Science. 22(2):4.