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

Location: European Biological Control Laboratory

2020 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
Obj 1: Exploration for bagrada bug (BAGRADA HILARIS) and its natural enemies was pursued in South Africa. Hymenopteran parasitoids (TRISSOLCUS SP., GRYON SP.) were collected and shipped to US cooperators for morphological and genetic analyses. Egg masses of the viburnum leaf beetle PYRRHALTA VIBURNI were collected in France and Switzerland, resulting in the collection of one egg parasitoid, APROSTOCETUS SP. These parasitoids were used for taxonomic and genetic studies. Collections of egg masses of the elm leaf beetle XANTHOGALERUCA LUTEOLA, were conducted in the south of France in order to compare the egg parasitoid assemblages of the two leaf beetles resulting in the collection of > 1,000 egg parasitoids of the elm leaf beetle. Pupae of the allium leaf miner PHYTOMYZA GYMNOSTOMA were collected in France in organic leek fields. These collections resulted in the collection of one pupal parasitoid, PTEROMALUS SP.Pupae of the olive fruit fly BACTROCERA OLEAE (OFF) were collected by CABI in Pakistan in 2019 and shipped to EBCL where parasitoid emergence was monitored. The braconid wasp PSYTTALIA PONEROPHAGA represented 99% of the parasitoids emerged, but two additional parasitoids, still unidentified, were also collected. Following expositions of laboratory-reared sentinel cattle ticks (RHIPICEPHALUS ANNULATUS) in Vietnam, we evidenced for the first time that IXODIPHAGUS HOOKERI, one nymphal parasitoid attacks either the unfed or fed nymphs, and that eggs of the parasitoid hatch and develop within the tick nymph. A new method for easily collecting engorged tick nymphs was set up. A method for collecting IXODIPHAGUS HOOKERI while the tick is still attached to the skin of the cattle and a trapping method for collecting this parasitoid were initiated and are in the process of development. Obj 2A: Preliminary genetic comparisons were made between the allium leaf miner (PHYTOMYZA GYMNOSTOMA) collected in France and in the US. Analysis evidenced a genetic match between the French population and some populations occurring in Pennsylvania, indicating the potential origin of some Pennsylvania invasive populations and suggesting where to look for promising natural enemies. The host specificity of a parasitoid of OFF, PSYTTALIA PONEROPHAGA, native to Pakistan, was tested with two non-target TEPHRITIDAE species present in North America: the black cherry fruit fly (RHAGOLETIS FAUSTA) and the Cape-ivy gall fly (PARAFREUTRETA REGALIS). To complement findings from parasitoid emergence, we monitored for presence of PSYTTALIA PONEROPHAGA DNA within pupae of the non targets that did not develop, further indicating that the parasitoid attempted to infest the pupa but failed to complete its development. Genetic analyses allowed us to estimate the frequency of failed parasitism attempts. The results indicated that the number of parasitism attempts were higher for both non targets than what could have been estimated based on parasitoid emergence alone. Obj 2B: Genetic fingerprinting of new adventive populations of two egg parasitoids (TRISSOLCUS JAPONICUS and TRISSOLCUS MITSUKURII) of the brown marmorated stink bug (HALYOMORPHA HALYS) was pursued on populations recovered using sentinel eggs in 2019 in the Trentino-Alto Adige region in Italy. Results showed that the populations of these two parasitoids recovered in 2019 are similar to those recovered in 2017-2018, suggesting a rapid spread of TRISSOLCUS JAPONICUS and TRISSOLCUS MITSUKURII across Northern Italy. A similar pattern of dispersion of TRISSOLCUS JAPONICUS across the US was observed in previous analyses. Such pattern of successful establishment and dispersion of these parasitoids in invaded ranges is encouraging for the future of the biological control program of HALYOMORPHA HALYS. Obj 3A: Life history studies were pursued on the most promising egg parasitoid (GRYON GONIKOPALENSE) of BAGRADA HILARIS. Supported by video recordings, we showed that GRYON GONIKOPALENSE exhibited a well-adapted foraging behaviour by tracing and successfully parasitizing 2mm-deep buried eggs of bagrada. Our data suggested that host egg detection could be based on chemical cues with parasitism success reduced up to 20% when the substrate in which eggs were buried was clean or disturbed. The host-specificity and parasitism efficiency of a parasitoid of OFF, PSYTTALIA PONEROPHAGA was evaluated in quarantine. A study was designed to test the hypothesis that its rearing on an alternative host, the medfly (CERATITIS CAPITATA), for several years has decreased the specificity of P. PONEROPHAGA towards OFF. The lab colony of the parasitoid was compared with a wild population from Pakistan in host specificity trials under choice and no-choice conditions with two non-target TEPHRITIDAE species present in North America: the black cherry fruit fly (RHAGOLETIS FAUSTA) and the Cape-ivy gall fly (PARAFREUTRETA REGALIS). Parasitoids from the rearing and wild populations were able to successfully parasitize both non targets. The wild population was more effective at parasitizing OFF larvae. In cooperation with Nicholas Manoukis, a visiting ARS scientist, tests to adapt the augmentorium (tent-like structure where infested fruits can be deposited) to OFF management were conducted at EBCL aimed at finding the optimal mesh size and shape of the net covering the augmentorium. Promising results were obtained with one type of mesh retaining 90% of OFF adults and allowing 72% of PSYTTALIA LOUNSBURYI parasitoids to escape. The preferences of the two PSYTTALIA species for different types of visual cues were evaluated. Results revealed that parasitoids preferred to land on certain colors compared to others, with black being the color the most frequently landed on. However, the presence of visual cues (black circles the size of olives) did not increase parasitism rates on substrates containing OFF larvae. Obj 3B: Laboratory experiments to evaluate the virulence of entomopathogenic fungi (EPF) on the olive fruit fly BACTROCERA OLEAE were completed. Efficacy of five strains isolated from soils in olive orchards and the commercial GHA strain were evaluated and three modes of inoculation (immersion, ectopic inoculation, and soil inoculation) were tested. With the two first inoculation methods, results show a significant increase in mortality rate of pupae as concentrations increased but no difference in strain efficacy was observed. With soil inoculation method, results showed a significant dose effect on mortality rate of pupae and mortality was consistently higher with the METARHIZIUM ANISOPLIAE (strain 02068) at the highest concentration (108 conidia/g of soil). Results with the soil inoculation method validate the prospective use of the METARHIZIUM ANISOPLIAE strain 02068 as a microbial biocontrol agent of OFF. A study was designed to exploit the volatile organic compounds produced by microbes associated with both the midgut of OFF larvae and the leaf surfaces of the olive trees for their attractant or repellant properties. Olives infested by OFF larvae and leaves of olive trees of different varieties with contrasted susceptibility to OFF were collected in the field in order to isolate bacteria from the midgut of larvae and bacteria at the leaf surface. The attractiveness of the bacterial cultures to OFF is currently being tested using an olfactometer. Obj 3C: presence of bacterial endosymbionts such as WOLBACHIA was investigated in two promising egg parasitoids of BAGRADA HILARIS, GRYON GONIKOPALENSE and TRISSOLCUS HYALINIPENNIS. No WOLBACHIA or other endosymbiotic bacteria were detected in the tested parasitoids on contrary to their host. The two EBCL colonies of BAGRADA HILARIS originating from South Africa and the US were shown to be mono-infected with a same strain of WOLBACHIA although the prevalence of WOLBACHIA was higher in the South Africa colony. Dissection of the alimentary tract of BAGRADA HILARIS revealed the lack of crypt-associated symbiotic bacteria in the posterior midgut which makes BAGRADA HILARIS an exception among known phytophagous stinkbugs. Obj 4: EBCL scientists in Greece compared bio-efficacy of a conventional larvicide (insect growth regulator = IGR) and a biological larvicide (BACILLUS THURIGIENSIS ISRAELENSIS = BTI) against mosquitoes breeding in rice fields. The impact of both products was investigated on AEDES CASPIUS (major nuisance mosquito species in the Mediterranean) and CULEX PIPIENS (primary West Nile virus vector). Larval population densities and emergence inhibition were compared between the two treatments and the un-treated (control) fields. Both BTI and IGR resulted in significant larval mortalities, with the IGR product exhibiting a higher residual effect. Integrated WNV surveillance systems were established in North and South Greece to monitor viral circulation using sentinel chickens and mosquitoes. The system operated biweekly using Centers for Disease Control (CDC) mosquito light-traps targeting areas susceptible to WNV transmission. WNV infected mosquito pools belonging to the species CULEX PIPIENS were detected two weeks before the onset of human cases. Aerial ultra low volume (ULV) space spray treatments using a pyrethroid insecticide were evaluated against wild CULEX PIPIENS populations. Mosquito traps (CDC) were used to assess the impact of aerial interventions by comparing CULEX population abundance and WNV minimum infection rates (MIR = number of positive pools/total specimens tested x 1000) before and after treatments. Significant CULEX population reduction was observed in treated areas in parallel to a reduction in mosquito infection rates. No population/MIR reductions were observed in untreated areas where, on the contrary, an increase in WNV circulation levels was recorded. In collaboration with US cooperators, a colony of PHLEBOTOMUS PAPATASI, a major vector of leishmaniasis and a highly anthropophilic species, was established for the first time at EBCL in Greece.

1. Egg foraging behavior of a parasitoid in a complex environment revealed. Bagrada bug (BAGRADA HILARIS) is a major pest of cole crops that first appeared in California in 2008 and is spreading eastward and southward. One dominant species of egg parasitoid (GRYON GONIKOPALENSE) collected in Pakistan was evaluated for its potential as a classical biological control agent by ARS researchers at the European Biological Control Laboratory in Montpellier, France. After determining its life history traits, the interactions among three trophic levels (plant, pest, and parasitoid) were investigated under controlled conditions and the parasitism behavior of parasitoids was measured at plant scale. No significant differences in preference were observed among broccoli, false arugula, or mustard host plants with on average 91% of BAGRADA eggs laid below ground, meaning they were all equally attractive for bagrada. However, GRYON has the ability to attack BAGRADA eggs laid either below ground or on the plant tissue surface at ~35cm above ground. Therefore, GRYON showed no preference in buried egg parasitism among host plants. This is a major accomplishment for the future of the biocontrol program of bagrada as GRYON GONIKOPALENSE exhibits a foraging behavior that indicated a close coevolution with its target.

2. Species identification of egg parasitoids of targeted pests including PYRRHALTA VIBURNI. The viburnum leaf beetle (PYRRHALTA VIBURNI) is native to Eurasia and invasive in the northeastern U.S. where it defoliates native and ornamental viburnum plants in natural areas and managed landscapes. There are about twenty VIBURNUM species native to North America and most of them are susceptible to the beetle. One dominant species of egg parasitoid (APROSTOCETUS SP.) in Europe is being evaluated by ARS scientists in Montpellier, France, for its potential as classical biological control agent. Previous genetic analyses of 170 APROSTOCETUS SP. specimens collected from populations of PYRRHALTA VIBURNI throughout Europe revealed that they all belonged to a unique species. Recently genetic analysis combined with MALDI-TOF approach were conducted on another species of APROSTOCETUS that parasitizes eggs of elm leaf beetle (XANTHOGALERUCA LUTEOLA). The outcomes of this analysis was that the two APROSTOCETUS species belonged to the same species and that this egg parasitoid was not specific to PYRRHALTA VIBURNI and naturally attacked eggs of other beetles in the field. These results confirmed prior results of host specificity trials conducted in 2019 at EBCL and further confirmed that this egg parasitoid lacks sufficient specificity and thus should not be considered an adequate classical biological control agent.

3. Development of a laboratory-rearing method for allium leafminer target pest. The allium leafminer, PHYTOMYZA GYMNOSTOMA, is an emerging invasive pest in Eastern U.S and native to Europe. It was first detected in 2015 in Pennsylvania and has since been recorded in New York State, Maryland, and New Jersey. The allium leaf miner attacks a wide range of species belonging to the ALLIUM genus including cultivated crops such as onion, garlic, leek, scallions, shallots, and chives. ARS scientists in Montpellier, France, developed a protocol to continuously rear the allium leaf miner on different ALLIUM species under controlled laboratory conditions. The protocol consisted of keeping the insect at 15°C temperature and a 12:12 photoperiod to prevent the pupae from going into diapause. Using this method, three to four generations of this insect could be obtained per year instead of the two generations per year observed in the field. This protocol will greatly facilitate future studies on the allium leaf miner pest.

4. Integrated West Nile virus (WNV) early warning surveillance system developed. West Nile fever/encephalitis is the most important mosquito-borne disease in the continental United States. The disease is caused by a flavivirus that is separated into distinct lineages, with lineage 1 (L1) and lineage 2 (L2) encompassing all WNV known isolates associated with human and veterinary disease. Currently, all United States (US) WNV known isolates belong to lineage 1. Lineage 2 isolates recently spread out of sub-Saharan Africa and were found in Europe causing large human and equine WNV outbreaks. The invasive threat and risk of WNV L2 invading the US is significant as recent evidence demonstrated that North American mosquito species are competent vectors of WNV L2 isolates from Africa and Europe. ARS scientists in Greece designed an integrated West Nile virus (WNV) early warning surveillance system specifically targeting the threatening WNV lineage 2 strains. The system relies on detecting viral-RNA in field-collected mosquitoes and screening of sentinel-chickens for WNV specific antibodies. This surveillance system was successfully implemented and provided information on WNV mosquito circulation and enzootic transmission one month prior to human cases allowing for targeted and proactive vector control interventions. Knowledge of WNV L2 ecology in Europe combined with optimized field-based surveillance systems and laboratory diagnostic tools can be applied to enhance capacities in the United States for early detection and early warning to control and reduce this emerging threat.

Review Publications
Desurmont, G.A., Morelon, S.P., Benrey, B. 2020. First insights into the chemical ecology of an invasive pest: Olfactory preferences of the viburnum leaf beetle Pyrrhalta viburni. Environmental Entomology. 49(2):364-369.
Desurmont, G.A., Von Arx, M., Turlings, T.C., Shiestl, F.P. 2020. Floral odors can interfere with the foraging behavior of parasitoids searching for hosts. Frontiers in Ecology and Evolution. 8:148.
Fotakis, E., Giantsis, I., Castells Sierra, J., Tanti, F., Balaska, S., Mavridis, K., Kourtidis, S., Vontas, J., Chaskopoulou, A. 2020. Fighting vector borne diseases in refugee camps, Greece: mosquito and sand fly population structure, pathogen circulation and insecticide resistance. Journal of Infectious Diseases. 9:30.
Fotakis, E., Mastantonio, V., Grigorika, L., Porretta, D., Puggioli, A., Chaskopoulou, A., Osorio, H., Weill, M., Bellini, R., Urbanelli, S. 2020. Identification and detection of a novel point mutation in the Chitin Synthase gene of Culex pipiens associated with diflubenzuron resistance. PLOS Neglected Tropical Diseases. 14(5): e0008284.
Groen, T.A., L'Ambert, G., Bellini, R., Chaskopoulou, A., Petric, D., Zgomba, M., Marrama, L., Bicout, D.J. 2017. Ecology of West Nile virus across four European countries II: Empirical modelling of the dynamics of Culex pipens abundances in different regions. Parasites & Vectors. 10(1):524.
Sabbatini Peverieri, G., Mitroiu, M., Bon, M., Balusu, R., Benvenuto, L., Bernardinelli, I., Fadamiro, H., Falagiarda, M., Fusu, L., Grove, E., Haye, T., Hoelmer, K.A., Lemke, E., Malossini, G., Marianelli, L., Moore, M.R., Pozzebon, A., Roversi, P., Scaccini, D., Shrewsbury, P., Tillman, P.G., Tirello, P., Waterworth, R., Talamas, E. 2019. Surveys of stink bug egg parasitism in Asia, Europe and North America, morphological taxonomy, and molecular analysis reveal the Holarctic distribution of Acroclisoides sinicus (Huang & Liao) (Hymenoptera: Pteromalidae). Journal of Hymenoptera Research. 74: 123-151.
Bon, M., Talamas, E., Hoelmer, K.A., Buffington, M.L. 2019. Molecular phylogeny of Trissolcus wasps (Hymenoptera: Scelionidae), natural enemies of stink bugs. Journal of Hymenoptera Research. 73:201-217.
Trunz, V., Lucchetti, M., Benon, D., Dorchin, A., Desurmont, G., Kast, C., Rasmann, S., Glauser, G., Praz, C. 2020. To bee or not to bee – the “raison d’être” of toxic secondary compounds in the pollen of Boraginaceae. Functional Ecology. 34(7):1345-1357.
Balaska, S., Fotakis, E., Kioulos, I., Grigoraki, L., Mpellou, S., Chaskoupoulou, A., Vontas, J. 2020. Bioassay and molecular monitoring of insecticide resistance status in Aedes albopictus populations from Greece, to support evidence-based vector control. Parasites & Vectors. 13:328.
Desurmont, G., Bon, M., Kerdellant, E., Guermache, F., Pfingstl, T., Tixier, M. 2020. An integrative approach combining molecular analyses and experiments to investigate predation of insect eggs by a mite. Ecosphere. 11(3).
Hlavackova, K., Dvorak, V., Chaskopoulou, A., Volf, P., Halada, P. 2019. A novel MALDI-TOF MS-based method for blood meal identification in insect vectors: a proof of concept study on phlebotomine sand flies. PLOS Neglected Tropical Diseases. 13(9): e0007669.
Jourdan, M., Thomann, T., Richet, R., Fendane, Y., Bon, M., Sheppard, A., Baker, G. 2019. Genetic variability in the parasitic fly, Sarcophaga villeneuveana, in south-western Europe and Morocco. Biocontrol. 65:59-70.