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

Location: European Biological Control Laboratory

2017 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 four 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 and its natural enemies was begun by Agricultural Research Council (ARC) cooperators in South Africa. Samples of the target pest were collected at seven new sites for genetic analysis. A collaboration was established with scientists at the Kenya Agricultural and Livestock Research Organisation (KALRO) to survey for bagrada bug and its natural enemies in Kenya. The nymphal parasitoid PERISTENUS DIGONEUTIS was reared out of 111 cocoons of the tarnished plant bug LYGUS that were collected in Austria in 2016 for DNA analysis to help determine which strains contributed to successful biological control of Lygus bugs in eastern USA. 576 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. Laboratory-reared sentinel cattle ticks (RHIPICEPHALUS ANNULATUS) were exposed to collect natural enemies at one location in northern Greece and one in Bulgaria for 21 to 25 days. Engorged ticks were collected in glass vials and held for the emergence of possible parasitoids and entomopathogens. In both countires tick larvae were monitored by digital cameras under natural conditions to detect possible predation. Engorged ticks were also collected in Albania (Korca) and Turkey (Izmir, Kutahya and Nesehir) and are being incubated for possible emergence of natural enemies. Under Objective 2, molecular genetic analysis was used to "barcode" various insect pests and parasitoids to help clarify their identification and to pinpoint the geographic origin of invasive populations. Genetic comparison of populations of bagrada bugs collected by EBCL collaborators in Hawaii, California and from several countries in the native range showed that bugs invading two Hawaii islands probably originated from the same region in western Asia (Pakistan) as those in California. We started an integrative taxonomy study using non-destructive molecular genetic analysis combined with quantitative morphological analysis by the curator of Hymenoptera of the Florida Department of Agriculture and Consumer Services (FDAC), to delineate species of parasitoids in the genus GRYON that attack two bugs targeted for biological control: bagrada bug (BAGRADA HILARIS) and brown marmorated stink bug (HALYOMORPHA HALYS). Results will be used to identify the species of GRYON that is currently being evaluated at EBCL and the ARS laboratory in Albany, CA for biological control of bagrada bug. We used genetic fingerprinting (microsatellites) to determine that specimens of the parasitoid of BMSB (TRISSOLCUS JAPONICUS) that were recovered in the eastern and western U.S. are genetically different from all parasitoid populations recovered so far, indicating that the parasitoid has been accidentally introduced multiple times. Genetic barcoding analysis of the parasitoid PSYTTALIA LOUNSBURYI that was released in California for biological control of the olive fruit fly (BACTROCERA OLEAE) showed that the South African strain is widely established in northern and central coastal California, and the Kenyan strain is established in southern coastal CA. This result has shifted emphasis towards releasing the S. African strain at additional sites in Northern California. Under Objective 3A, life history studies were conducted on two egg parasitoids (TRISSOLCUS HYALINIPENNIS and GRYON sp.) of bagrada bug (BAGRADA HILARIS) originating from Pakistan to facilitate maintenance of laboratory colonies and to inform the design of host specificity experiments. We also determined that the age of host eggs, from 0 to 4 days-old, did not affect their suitable for parasitism by either parasitoids. However, frozen eggs were much less suitable, which indicates that frozen eggs would not be suitable to use as sentinel eggs to monitor the presence of either of these parasitoids. The fecundity and longevity of adults of the Asian longhorned beetle (ANAPLOPHORA GLABRIPENNIS) and the citrus longhorned beetle (ANAPLOPHORA CHINENSIS) were measured in the laboratory to evaluate the fitness of various colonies. A field experiment in Italy to measure parasitism of the citrus longhorned beetle egg parasitoid (APROSTOCETUS FUKUTAI) was cancelled because Italian authorities failed to provide a permit for us to transport quarantine material. Under Objective 3B, laboratory experiments were conducted to evaluate the virulence of entomopathogenic fungi (EPF) from our collection on the olive fruit fly (BACTROCERA OLEAE). One strain of Metarhizium anisoplae and four of Beauveria bassiana previously isolated from soil in olive orchards were tested. Larvae were placed on sand inoculated with different concentrations of conidial suspension. A dosage effect on mortality was observed, and two strains were more virulent than the others at the highest concentration (1.0 X 10^8 conidia/ml). Under Objective 3C we investigated the diversity of endosymbiotic bacteria of four populations of olive fruit fly parasitoids, PSYTTALIA spp., maintained in our quarantine laboratory using 16S rRNA gene sequencing. All the PSYTTALIA LOUNSBURYI individuals from the same geographic origin have similar internal bacterial communities. PSYTTALIA PONEROPHAGA harbors very few Wolbachia bacteria compared to PSYTTALIA LOUNSBURYI. Populations of the Asian longhorned beetle (ANAPLOPHORA GLABRIPENNIS) from Korea (Pocheon and Oknyo) do not develop well in the quarantine laboratory. A comparison of microbial endosymbionts of the different lineages of Anaplophora glabripennis is ongoing to determine if the bacterial communities (including Wolbachia) can explain differences in the success of rearing the different populations. Under Objective 4, mosquito surveys with CDC light traps were continued in Central and East Macedonia Thrace, Greece in agricultural areas where mosquito control interventions are applied. Surveys were also conducted in irrigated rice-fields for mosquito larvae. The survey results were used to monitor the efficacy of mosquito control applications (adulticiding and larviciding treatments) in those regions. A low application rate of diflubenzuron and a medium application rate of deltamethrin, following label instructions, provided significantly high mortality of both larval and adult stages of mosquitoes associated with rice production (AEDES CASPIUS, CULEX PIPIENS, ANOPHELES HYRCANUS). In parallel, studies on mosquito resistance to pyrethroid insecticides were continued and expanded to include insecticides other than pyrethroids (diflubenzuron – an insect growth regulator, and temephos – an organophosphate). This research has shown that resistant genotypes of Ae. albopictus individuals moved between Florida (USA) and Athens (Greece), highlighting the importance of passive transportation of disease vectors carrying resistance mechanisms. The rapid global dispersal of resistant populations of mosquitoes is likely to accelerate the decline in efficacy of existing insecticides. Mosquito-breeding habitats in the rice-field region of Central Macedonia were surveyed for a second year to collect and identify aquatic insects. Insects were categorized as: a) harmful insects b) non-target insects, and c) mosquito predators. Information on operational mosquito spray activities were obtained 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. One invasive rice-pest, the rice water weevil LISSORHOPTRUS ORYZOPHILUS (the most destructive rice pest globally), was recorded for the first time in the Balkan region. Evaluation of ultra-low volume (ULV) space spray treatments against sand fly (PHLEBOTOMUS spp.) populations in animal facilities (kennels) were completed. Overall, the ULV adulticiding application of deltamethrin at 2 different application rates provided significant sand fly mortality. The results of this study provide strong evidence that ground ULV applications of water-based pyrethroids can result in high sand fly control levels, even in a heavily infested sand fly environment such as the animal facilities used in this study. Residual spraying trials for mosquito and sand fly control were continued in partnership with the USDA Center of Medical and Veterinary Entomology (CMAVE), 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. Determination of the life history traits of egg paraitoids 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. Two species of egg parasitoids (TRISSOLCUS HYALINIPENNIS and GRYON SP.) were collected in Pakistan and are being evaluated for their potential as classical biological control agents 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 of different ages. The results provide a basis for maintaining laboratory colonies and for designing host specificity experiments. Furthermore, it was shown that these parasitoids usually cannot complete development on frozen host eggs, so the current practice of using frozen sentinel eggs to monitor parasitism in the field is not suitable for these two species. Successful development of classical biocontrol will benefit organic farmers who have no effective methods to control bagrada bug in cole crops and consumers of these products.

Review Publications
Lesieur, V., Martin, J., Weaver, D.K., Hoelmer, K.A., Shanower, T.G., Smith, D.R., Morill, W.L., Kadiri, N., Cockrell, D., Randolf, T.L., Waters, D.K., Bon, M. 2016. Origin and phylogeography of the wheat stem sawfly, Cephus cinctus Norton (Hymenoptera : Cephidae): implications for pest management. PLoS One. 11(12):e0168370. doi:10.1371/journal.pone.0168370.
Xu, H., Desurmont, G., Degen, T., Laplanche, D., Henryk, L., Turlings, T.C. 2016. Combined use of herbivore-induced plant volatiles and sex pheromones for mate location in braconid parasitoids. Plant Cell and Environment. Vol 39, Pages 1920-1927. DOI: 10.1111/pce.12818.
Giantsis, I., Chaskopoulou, A., Bon, M. 2017. Direct multiplex PCR (dmPCR) for the identification of six Phlebotomine sand fly species (Diptera: Psychodidae), including major Leishmania vectors of the Mediterranean. Journal of Economic Entomology. tow269. DOI:
Desurmont, G., Zemanova, M., Turlings, T.C. 2016. The gastropod menace: slugs on Brassica plants affect caterpillar survival through consumption and interference with parasitoid attraction. Journal of Chemical Ecology. Vol 42, Issue 3, Pages 183-192.
Desurmont, G., Xu, H., Turlings, T.C. 2016. Powdery mildew suppresses herbivore-induced plant volatiles and interferes with parasitoid attraction in Brassica rapa. Plant Cell and Environment. Vol 39, Pages 1920-1927. DOI: 10.1111/pce.12752.
Danner, H., Desurmont, G.A., Cristescu, S.M., Van Dam, N.M. 2017. Herbivore-induced plant volatiles accurately predict history of coexistence, diet breadth, and feeding mode of herbivores. New Phytologist. 220(3):726-738. 10.1111/nph.14428.
Fotakis, M., Chaskopoulou, A., Grigoraki, L., Tsiamantas, A., Kounadi, S., Georgiou, L., Vontas, J. 2017. Analysis of population structure and insecticide resistance in mosquitoes of the genus Culex, Anopheles and Aedes from different environments of Greece with a history of mosquito borne disease transmission. ACTA TROPICA. 174:29-37. doi: 10.1016/j.actatropica.2017.06.005.
Grigoraki, L., Pipini, D., Lappe, P., Weill, M., Chaskopoulou, A., Vontas, J. 2017. Carboxylesterase gene amplifications associated with insecticide resistance in Aedes albopictus: geographical distribution and evolutionary origin. PLOS Neglected Tropical Diseases. 11(4), p.e0005533.
Kim, M., Lee, H., Ku, D., Herard, F., Gould, J.R., Williams, D.W., Kim, I., Hong, K. 2016. Discovery of Spathius ibarakius Belokobylskij et Maeto (Hymenoptera: Braconidae) as a larval ectoparasitoid of citrus longhorned beetle in Korea. Korean J. Appl. Entomol. 55(3): 285-291 DOI:
Giantsis, I., Sierra, J.C., Chaskopoulou, A. 2017. The distribution of the invasive pest, rice water weevil Lissorhoptrus oryzophilus (Coleoptera: Curculionidae), is expanding in Europe: First record in the Balkans, confirmed by CO1 DNA barcoding. Phytoparasitica. 45: 147.
Desurmont, G., Kholer, A., Maag, D., Laplanche, D., Xu, H., Baumann, J., Demaire, C., Devenoges, D., Glavan, M., Mann, L., Turlings, T.C. 2017. The spitting image of plant defenses: effects of plant secondary chemistry on the efficiency of caterpillar regurgitant as an anti-predator defense. Journal of Chemical Ecology. DOI 10.1002/ece3.3174.
Hily, J., Demaneche, S., Poulicard, N., Tannieres, M., Djennane, S., Beuve, M., Vigne, E., Demangeat, G., Komar, V., Gertz, C. 2017. Metagenomic-based impact study of transgenic grapevine rootstock on its associated virome and soil microbiome. Plant Biotechnology Journal. doi:10.1111/pbi.12761.
Carrasco, D., Desurmont, G., Laplanche, D., Proffit, M., Gols, R., Becher, P.G., Larsson, M.C., Turlings, T.C., Anderson, P. 2017. The multitrophic consequences of concurrent insect invasions: a range-expanding herbivore and its associated parasitoid affect native tritrophic interactions. Global Change Biology. DOI: 10.1111/gcb.13836.
Desurmont, G., Guiguet, A., Turlings, T.C. 2017. Invasive insect herbivores as disrupters of chemically-mediated tritrophic interactions: effects of herbivore density and parasitoid learning. Biological Invasions. DOI:10.1007/s10530-017-1526-x.