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

Location: European Biological Control Laboratory

2016 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
Objective 1 We conducted foreign exploration in Europe, Africa and Asia and collected beneficial insects (parasitoids) that attack a variety of target pests in support of various biological control projects. Explorations for Bagrada bug were conducted by CABI cooperators in Pakistan during spring 2016. Two egg parasitoids were shipped to EBCL and identified by US taxonomists as GRYON nr GONIKOPALENSE and TRISSOLCUS HYALINIPENNIS (Platigastridae). Colonies of the two parasitoids are now established both at EBCL and Stoneville, MS inside containment facilities to be used for host specificity studies. An initial exploration trip to South Africa was conducted, resulting in collection of Bagrada bugs and establishment of plans for further exploration next year. Samples of olive psyllid were collected in Spain to rear out parasitioids (PSYLLAEPHAGUS EUPHYLLURAE) that were shipped to collaborators in California for use in host specificity evaluations. Populations of the tarnished plant bug parasitoid, PERISTENUS DIGONEUTIS, were collected by a CABI cooperator in Austria for genetic characterization to determine which strains contributed to successful biological control of lygus bugs in the US. Exploration for parasitoids of cattle fever tick in the Balkan region (southern Albania) and the region of Izmir in Turkey began this year and agreements with local farmers to collect ticks were made. A planned five-week exploration for parasitoids of the Asian longhorned beetle in South Korea was organized, but had to be cancelled at the last minute because of health problems. An egg parasitoid (APROSTOCETUS sp.) and 10 species of mites associated with egg masses were collected in France in a survey of natural enemies of the viburnum leaf beetle (PYRRHALTA VIBURNI), which is an invasive species in northeastern USA. Objective 2 Molecular genetic analysis was used to "bar code" various insect pest and parasitoids to help clarify their identification and to pinpoint the geographic origin of invasive populations. For example, populations of Bagrada bugs collected on cabbage or caper plants by EBCL scientists and collaborators in Africa (Morocco and South Africa) and in East Asia (Pakistan and Myanmar) were compared with those from California. We determined that the Bagrada bug invading California probably originated from western Asia because of genetic similarity to specimens from Pakistan. We pursued integrative taxonomy, non-destructive molecular genetic analysis combined with morphological analysis by ARS collaborators at the Smithsonian Institute, to distinguish twenty species of parasitoids in the genus TRISSOLCUS that attack brown marmorated stink bug (BMSB), some being new to science. Genetic fingerprinting (microsatellites) was used to identify an accidentally introduced parasitoid of BMSB (TRISSOLCUS JAPONICUS) that was collected in the western USA and we determined that it was an accidental introduction that did not originate from any of the colonies maintained in USDA quarantines. This population also was genetically different from a parasitoid population accidentally recovered in 2015 near BARC in Beltsville, MD, thus indicating two accidental introductions of the parasitoid in the USA, originating from two different regions: probably China and Japan. Objective 3 Colonies of two parasitoids (GRYON nr GONIKOPALENSE and TRISSOLCUS HYALINIPENNIS) of Bagrada bug collected by collaborators in Pakistan were established at EBCL. Experiments indicated that both species can complete development on eggs of Bagrada bug from colonies that were established from California, Pakistan and South Africa, indicating that they could be useful for biological control if they prove to be sufficiently host specific. Experiments to improve efficiency of mass-rearing of the olive fruit fly parasitoid, PSYTTALIA LOUNSBURYI, were conducted to measure the effect of crowding on adult survivorship. Other experiments showed that females did not lay more eggs per exposure period if there was a longer interval between successive periods, further helping to optimize production. We shipped 16,395 adult parasitoids to California to release in fall 2015 to help control the olive fruit fly. In research funded by an Italian agency, the seasonal abundance of an egg parasitoid (APROSTOCETUS FUKUTAI) of the citrus longhorned beetle (CLB) was monitored at several sites in Italy where the parasitoid appears to be impacting the pest population. The virulence of five entomopathogenic fungi (EPF) isolated from olive orchard soils, which were preserved in liquid nitrogen at EBCL, was evaluated on larvae and pupae of the olive fruit fly under laboratory conditions. Results showed that two strains caused more than 60% mortality of larvae at a dose of 10^8 spores/ml. We began searching for the presence of bacterial endosymbionts which might affect the reproductive success of an egg parasitoid of BMSB (TRISSOLCUS JAPONICUS) in colonies maintained in quarantine at the ARS laboratory in Newark, DE using classical PCR to help improve rearing these colonies. Objective 4 Mosquito surveys with CDC (Center for Disease Prevention & Control) light traps have been deployed in Central and East Macedonia to collect mosquitoes. Mosquitoes were identified to species level, and this year’s data along with data from previous years have been used in collaboration with other European partners and European authorities (the European Center for Disease Prevention & Control – ECDC) to create mathematical models for predicting mosquito population dynamics and disease outbreaks (particularly West Nile virus [WNV] outbreaks). The development of mosquito resistance to insecticides was studied and a new mutation associated with resistance to pyrethroids was detected for the first time in Greece. Mosquito-breeding habitats in the rice-field region of Central Macedonia were surveyed for the presence of aquatic insects which were categorized as follows: a) harmful insects b) non-target insects, and c) mosquito predators. Information on operational mosquito spray activities were retrieved from the Region of Central Macedonia and surveys to compare the insect biodiversity were made simultaneously in unsprayed areas and areas that were sprayed regularly. A sand fly monitoring network using CDC light traps was deployed in animal facilities of central Macedonia to survey sand fly populations. Novel protocols for sand fly identification and their blood-feeding preferences have been developed. The behavior (flight height preference, diel activity patterns) of the major Leishmania vectors were investigated under field conditions for designing efficient species-specific vector control treatments. Evaluation of ultra-low volume (ULV) space spray treatments against sand fly populations in the field were initiated. Residual spraying trials for mosquito and sand fly control were initiated in partnership with the USDA Center of Medical and Veterinary Entomology, Florida. Residual treatments are being conducted simultaneously in other countries with different climatological profiles in order to determine the impact of weather on the efficacy of the spraying treatments. 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 oversees.

1. Use of molecular genetics to identify mosquitoes and sandflies. Mosquitoes and sandflies are important pests and vectors of disease of humans and domestic animals, but it is extremely difficult to identify species of adults and often impossible for larvae. Scientists at the USDA-ARS European Biological Control Laboratory in Thessaloniki Greece developed a new technique, called Mild-Vectolysis, which permits extracting DNA from mosquito or sandfly specimens without damaging their external morphological characters. DNA barcoding (COI) correctly identified all three mosquito and six sandfly species analyzed. This provides a relatively fast and cheap method to identify specimens that can be retained for morphological study or preserved as museum vouchers. This technique will be useful to researchers and pest managers who need to know what species they are targeting so that they can use appropriate techniques and strategies to manage them.

2. Determination of the geographic origin of wheat stem sawfly. Wheat stem sawfly (CEPHUS CINCTUS) is an important pest of wheat in the Northern Plains. It has long been suspected that this species was introduced from Asia, which is critical to know in order to direct where to search for prospective biological control agents. Molecular genetic techniques (sequencing of COI and 16S and microsatellites) were used to determine that North American wheat stem sawflies are distantly related to species found in Asia, and thus are not an invasive alien species. Furthermore, results suggest that recent outbreaks in Colorado are caused by local adaptation of the pest, and that there are three genetically distinct populations of the pest in the USA. Biological control efforts should focus on the conservation and possible augmentation of indigenous biological control agents rather than exploration for agents in Asia.

Review Publications
Bon, M., Hoelmer, K.A., Pickett, C.H., Kirk, A., He, Y., Mahmood, R., Daane, K.M. 2015. Populations of Bactrocera oleae (Diptera: Tephritidae) and Its Parasitoids in Himalayan Asia. Annals of the Entomological Society of America. 109:81-91. doi: 10.1093/aesa/sav114.
Brabbs, T., Collins, D., Herard, F., Maspero, M., Eyre, D. 2015. Prospects for the use of biological control agents against Anoplophora in Europe. Pest Management Science. 71:7-14.
Cameron, M., Acosta-Serrano, A., Bern, C., Boelaert, M., Den Boer, M., Chapman, L., Chaskopoulou, A., Coleman, M., Croft, S., Courtney, O. et al. 2016. Understanding the transmission dynamics of Leishmania donovani to provide robust evidence for interventions to eliminate visceral leishmaniasis in Bihar, India. Parasites & Vectors. 9:25 DOI: 10.1186/s13071-016-1309-8.
Chaskopoulou, A., Giantsis, I., Demir, S., Bon, M. 2016. Species composition, activity patterns and blood meal analysis of sand fly populations (Diptera: Psychodidae) in the metropolitan region of Thessaloniki, an endemic focus of canine leishmaniasis. ACTA TROPICA. 158: 170-176.
Deletre, E., Schatz, B., Bourguet, D., Chandre, F., Williams Iii, L.H., Ratnadass, A., Martin, T. 2016. Prospects for repellent in pest control: current developments and future challenges. Chemoecology. 26:1-16.
Giantsis, I., Chaskopoulou, A., Bon, M. 2016. Mild-Vectolysis: A nondestructive DNA extraction method for vouchering sand flies and mosquitoes. Journal of Medical Entomology. 53:3, 692-695.
Hongsheng, P., Yanhui, L., Chunli, X., Huihui, G., Xiaoming, C., Xiaoling, S., Yongjun, Z., Williams, L., Wyckhuys, K.A., Kongming, W. 2015. Volatile fragrances associated with flowers mediate the host plant alternation of a polyphagous mirid bug. Scientific Reports. 14805; doi: 10.1038/srep14805.
Jacquet, S., Garros, C., Lombaert, E., Walton, C., Restrepo, J., Allene, X., Baldet, T., Cetre-Sossah, C., Chaskopoulou, A., Delecolle, J. 2015. Colonization of the Mediterranean Basin by the vector biting midge species Culicoides imicola: an old story. Molecular Ecology. 24(22):5707-25.
Roy, L., Bon, M., Cesarini, C., Serin, J., Bonato, O. 2016. Pinpointing the level of isolation between two cryptic species sharing the same microhabitat: a case study with a scarabaeid species complex. Zoologica Scripta. 45(4):407-420.
Saadat, D., Seraj, A., Goldansav, S., Williams III, L.H. 2016. Factors affecting reproductive success and life history parameters of Bracon hebetor Say (Hymenoptera: Braconidae) from three host-associated populations. Biological Control. 96:86-92.
Auge, M., Bon, M., Hardion, L., Le Bourgeois, T., Sforza, R. 2016. Genetic characterization of a red color morph of Euphorbia esula subsp. esula (Euphorbiaceae) in the floodplains of Saône (Eastern France). Botany. 10.1139/cjb-2016-0067.