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Research Project: Biological Control of Invasive Arthropod Pests from the Eastern Hemisphere

Location: European Biological Control Laboratory

2018 Annual Report

Objective 1: Explore for natural enemies, including microorganisms, of invasive arthropod pests identified as high priority targets by the ARS Office of National Programs, performing collections, importations and exportations in compliance with local and international regulations. High priority pests include but are not limited to Asian longhorned beetle, Bagrada bug, Lygus plant bugs, Rhipicephalus (Boophilus) ticks. Objective 2: Perform taxonomic, population genetic and phylogeographic studies as necessary to support successful development of biological control agents. Objective 3: Determine biological and physical parameters that affect the efficacy and safety of potential biocontrol agents, including host specificity, chemical ecology, climate, geography, and microbiological associations of symbionts and pathogens. Objective 4: Evaluate effects of integrated vector management on natural and agro-ecosystems, including effects on population dynamics of the target pests and beneficial/non-target taxa associated with these environments.

Invasions by non native arthropod pests are increasing in number and the area affected, and the damage to ecosystems, economic activity, and human welfare is accumulating. Without improved strategies based on recent scientific advances and increased investments to counter invasions, harm is likely to accelerate. The USDA emphasizes biologically-based integrated pest management systems of arthropod pests, and classical biological control is a major component. 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 France and Greece, and excellent facilities, including one molecular genetic unit and two quarantines, to develop practical approaches to manage invasive pests. Research involves discovering natural enemies (insects, mites or pathogens) that attack the target pest in its land of origin. Prospective agents are characterized morphologically, genetically and biologically, and their degree of specificity toward the target pest is assessed before shipment to US cooperators. Priority targets currently include the Asian longhorned beetle, mirid plant bugs, the bagrada bug, the brown marmorated stink bug, olive fruit fly, and cattle fever ticks. A new research objective involves the implementation of vector management practices to effectively control populations of mosquitoes and sand flies.

Progress Report
Progress was made on all four objectives and their subobjectives, all of which fall under National Program 304, Crop Protection and Quarantine, Component 3: develop innovative control methods and integrated pest management (IPM) strategies for insect and mite pests. Objective 4 also relates to National Program 104, Veterinary, Medical, and Urban Entomology, Component 1 Medical Entomology for the Public and the Military. Under Objective 1, exploration for bagrada bug (BAGRADA HILARIS) and its natural enemies was pursued by European Biological Control Laboratory (EBCL) cooperators in South Africa, at the Agricultural Research Council (ARC) and in Kenya at the Agricultural and Livestock Research Organization (KALRO). In 2018, parasitized mummies of olive psyllid (EUPHYLLURA OLIVINA) were collected in Spain and shipped to cooperators at the California Dept. of Food and Agriculture (CDFA) to facilitate host specificity testing of the parasitoid PSYLLAEPHAGUS EUPHYLLURAE, which is a prospective biological control agent of the psyllid. Over 53,000 adults of the South African strain of the parasitoid PSYTTALIA LOUNSBURYI were mass-reared and shipped to California for release to control the olive fruit fly (BACTROCERA OLEAE). Over 500 adults of the parasitoid, PSYTTALIA PONEROPHAGA, which is a prospective agent for olive fruit fly, was sent to cooperators in California for host specificity studies. Twelve shipments of infested native olives were received from CABI Pakistan, containing over 5000 immature olive fruit flies and from which 693 adult PSYTTALIA PONEROPHAGA were reared out to augment the EBCL laboratory colony and use for host specificity studies. Laboratory-reared sentinel cattle ticks (RHIPICEPHALUS ANNULATUS) were exposed to collect natural enemies in Crete, Greece and in Bulgaria. Engorged ticks were held for the emergence of possible parasitoids and entomopathogens. Under Objective 2, genetic comparison of populations of Bagrada bug (BAGRADA HILARIS) in Chile, California and several countries in the native range showed that bugs invading Chile probably originated from the same region in western Asia (Pakistan) as those in California. Genetic barcoding of parasitoids accidentally recovered from sentinel eggs of Bagrada bug (BAGRADA HILARIS) in California, and of parasitoids trapped as part of the BioSCAN project in California, revealed that all these parasitoids were belonging to TRISSOLCUS HYALINIPENNIS, a potential biological control agent. Integrative taxonomy approaches in collaboration with the curator of Hymenoptera of the Florida Department of Agriculture and Consumer Services (FDAC), were pursued to delineate species in the genus GRYON that attack two bugs targeted for biological control: bagrada bug (BAGRADA HILARIS) and brown marmorated stink bug (HALYOMORPHA HALYS. Genetic fingerprinting of specimens of the parasitoid of BMSB (TRISSOLCUS JAPONICUS) recovered in 2017 in eastern U.S. (in New York) and western U.S (in Oregon) showed that they are genetically different from all parasitoid populations recovered so far, indicating that the parasitoid has been accidentally introduced multiple times. Under Objective 3A, ARS scientists at EBCL, Montpellier, France, conducted life history studies on the Pakistani egg parasitoid GRYON near GONIKOPALENSE of bagrada bug (BAGRADA HILARIS) to facilitate maintenance of laboratory colonies and to help designing host specificity experiments. GRYON near GONIKOPALENSE fecundity and foraging behaviour were further studied. Behavioral experiments were conducted with the olive fruit fly parasitoid PSYTTALIA PONEROPHAGA to examine its preferences for various fruits and fruit odors under laboratory conditions. Larvae of EBCL colonies of the Asian longhorned beetle (ANOPLOPHORA GLABRIPENNIS) and of the citrus longhorned beetle (ANOPLOPHORA CHINENSIS) were sent to cooperators in the USA and Canada, and specimens were sent to China, Austria and Poland for taxonomic work. Tree stumps infested with citrus longhorned beetle (ANAPLOPHORA CHINENSIS) were collected in Italy and the egg parasitoid (APROSTOCETUS FUKUTAI) was reared out to establish a laboratory colony. Under Objective 3B, ARS scientists at EBCL, Montpellier, France, pursued laboratory experiments to evaluate the virulence of entomopathogenic fungi (EPF) from our collection on the olive fruit fly (BACTROCERA OLEAE). One strain of METARHIZIUM ANISOPLIAE and four strains of BEAUVERIA BASSIANA previously isolated from soil in olive orchards were tested together with the commercial BEAUVERIA BASSIANA GHA strain. Preliminary results seem to confirm a dose effect on fly mortality as already observed in last year’s experiments, with the METARHIZIUM strain 02068 being more virulent than the others at the highest concentration (1.0 X 10^8 conidiae/ml). The METARHIZIUM strain 02068 showed the highest spores viability compared to the other strains tested. Under Objective 3C, ARS scientists at EBCL, Montpellier, France, investigated using 16S sequencing the diversity of endosymbiotic bacteria in quarantine colonies of olive fruit fly parasitoids, PSYTTALIA spp. All the PSYTTALIA LOUNSBURYI individuals from the same geographic origin shared similar endosymbiotic bacterial communities. PSYTTALIA PONEROPHAGA harbored very few WOLBACHIA compared to PSYTTALIA LOUNSBURYI although its overall microbial diversity was much higher. The opportunistic pathogenic bacteria SERRATIA MARCESCENS was detected in larvae of our PSYTTALIA colonies. These SERRATIA strains are being isolated from larvae and cultured. Their identification will be confirmed by sequencing. The differences in the microbial diversity observed between the PSYTTALIA colonies and the presence of the pathogenic SERRATIA might explain the differences in mass rearing success observed between the four colonies. Microbial endosymbionts of a colony of the pest ANOPLOPHORA GLABRIPENNIS originating from Korea, which is difficult to maintain in quarantine, were compared with those present in all the other EBCL colonies of ANOPLOPHORA GLABRIPENNIS to determine if differences observed in the mass rearing success could be associated with any difference in their microbial spectrum. Under Objective 4, ARS scientists at EBCL, Thessaloniki, Greece, pursued sand fly control because of their potential to vector disease-causing parasites to humans and domestic animals, including LEISHMANIA parasites. Evaluations of attractive toxic sugar baits (ATSB) applied singly and in combination with an ultra-low volume (ULV) application of a deltamethrin-based product for the control of wild sand fly populations were completed. The integration of ATSB and ULV resulted in significant vector control reduction for up to 2 weeks post treatment (versus 1 week with ATSB alone). Pyrethroid resistance mutations were investigated in wild sand fly populations in Greece and Turkey. The mutation 1014F (Phe), associated with pyrethroid-resistant phenotypes, was detected in PHLEBOTOMUS PAPATASI sand flies from Turkey at an allele frequency of 48%. This study represents the first investigation of resistance mutations in wild sand fly populations in Europe and Turkey and the first global detection of resistance mutations in the major leishmaniasis vector PHLEBOTOMUS PAPATASI. LEISHMANIA species DONOVANI and TROPICA were detected and identified in PHLEBOTOMUS PERFILIEWI, PHLEBOTOMUS TOBBI and PHLEBOTOMUS SIMICI of North Greece indicating late-stage, transmissible infection. This is the first time that these 2 parasites have been reported in Greece in naturally occurring sand flies. The efficacy of a new method to detect larvae of two species of sandflies in soil (as few as one larva in a 40-ml sample of soil) using a quantitative PCR was developed. This easy method will be very useful for conducting field studies on the biology and ecology of sand flies. Residual spraying trials integrated with ULV space spray treatments and spatial repellents for mosquito and sand fly control were continued in partnership with the USDA Center of Medical and Veterinary Entomology (CMAVE), Florida. The efficacy of a variety of different products against mosquito larvae of the species AEDES ALBOPICTUS (competent vector of dengue, ZIKA virus) as well as other mosquito vectors are currently being evaluated. This project is partly funded by the US Department of Defense and results will be of high importance for the protection of US troops deployed overseas.

1. Determinination of the life history traits of egg paraitoid biological control agents of bagrada bug. Bagrada bug (BAGRADA HILARIS) is a major pest of cole crops that first appeared in California in 2008 and is spreading eastward. The egg parasitoid, GRYON near GONIKOPALENSE was collected in Pakistan and evaluated for its potential as a classical biological control agent by ARS researchers at the European Biological Control Laboratory in Montpellier, France. Experiments were conducted to measure the longevity, fecundity and ability to parasitize host eggs buried in the soil by BAGRADA females, providing a basis for maintaining laboratory colonies and for designing host specificity experiments. It was further shown that this parasitoid can track BAGRADA eggs in the soil indicating a potential role of chemical attractants. Successful development of classical parasitoid-based biocontrol strategies will benefit organic farmers who have no effective methods to control bagrada bug in cole crops and consumers of these products.

Review Publications
Chaskopoulou, A., L'Ambert, G., Petric, D., Bellini, R., Zgomba, M., Groen, T.A., Marrama, L., Bicout, D.J. 2016. Ecology of West Nile Fever across four European countries: Review of weather profiles, vector population dynamics and vector control response. Parasites & Vectors. 9(1): 482. DOI: 10.1186/s13071-016-1736-6.
Daane, K., Middleton, M., Sforza, R., Kamp-Hughes, N., Almeida, R., Correa, M., Downie, D., Walton, V. 2018. Determining the geographic origin of invasive populations of the mealybug Planococcus ficus based on molecular genetic analysis. PLoS One. 13(3): e0193852. journal.pone.0193852.
Herard, F., Maspero, M. 2018. History of discoveries and management of the Citrus longhorned beetle, Anoplophora chinensis, in Europe. Journal of Pest Science. 92:117-130.
La-Spina, M., Pickett, C.H., Daane, K.M., Hoelmer, K.A., Blanchet, A., Williams III, L.H. 2017. Effect of exposure time on mass-rearing production of the olive fruit fly parasitoid, Psyttalia lounsburyi (Hymenoptera: Braconidae). Journal of Applied Entomology. 142(3):319-326.
Madeline, M., Goolsby, J., Vacek, A.T., Kirk, A., Moran, P.J., Cortes, E., Cristofaro, M., Bownes, A., Mastoras, A., Kashefi, J. 2018. Densities of the Arundo Wasp, Tetramesa Romana (Hymenoptera: Eurytomidae) across its native range in Mediterranean Europe and introduced ranges in North America and Africa. Biocontrol Science and Technology. 28(8):772-785.