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- Shatters, Robert - Bob
- 2023
- 2022
- 2021
- Publications
- Crop Protection & Quarantine (304)

Research Project: Biological Control and Integrated Management of Invasive Arthropod Pests from Europe, Asia, and Africa **Location:
                            European Biological Control Laboratory** 2023 Annual Report **Objectives**
                                

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). **Approach**
                                

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**
                                

Objective 1. Exploration for natural enemies of bagrada bug was pursued in South Africa, however, no additional Hymenopteran egg parasitoids were collected.

Objective 1. A collecting trip was conducted, Sept. 2022, to search for natural enemies of Allium leaf miner (ALM) in conventional and organic farms/gardens in northern France. The ALM was found in two of 8 sites, but no natural enemies emerged from collected samples. Also contacts were made with FREDON (France), CABI (Switzerland), and KOPPERT (The Netherlands) to locate new collection sites in Western Europe.  

Objective 1.  A CDFA funded project was established to find new biological control agents for the Diamondback Moth (DBM) because of issues of development of insecticide resistance. Foreign explorations in 3 Balkan countries were undertaken (Albania, Bulgaria and Greece) resulting in collection of various parasitoids including two new records of a parasitoid for the DBM.  Parasitoids included:
1.	Diadegma semiclausum
2.	Diadromus collaris
3.	Oomyzus sokolowskii (New)
4.	Dolichogenidea appalator (New)
Our collaborators at Plant Protection Research Institute in Ha Noi, Vietnam found DBM in Cole crops in Vietnam and that there were exotic parasitoids not present in Europe. A collection trip has been planned. 

Objective 1.  Collection of Olive Psyllid parasitoids was conducted in Greece and Albania.  Species of the Olive Psyllid in California, E. olivine, were not present in the Balkans but a related species, E. phillyreae was observed.  This species is associated with the same parasitoids that attack E. olivine.  If there is interest from CDFA for further work on this Psyllid/parasitoids, further collections will be done.
Objective 2A. The olive psyllid populations (Euphyllura olivine) sampled in Valencia and Murcia Spain, 2022, by EBCL and CA collaborators were characterized genetically to assess the native range diversity. The populations are genetically distinct between these two regions, and the olive psyllid occurring in Valencia was too genetically distant from olive psyllid in CA to be their likely origin. A better genetic match, though not complete, was between Murcia populations and the CA populations.  The results indicate that more southern regions in Eastern Spain should be explored for coevolved natural enemies. Genetic analysis of the olive psyllids collected in April 2022 in Greece and Crete revealed them all to be Euphylllura phillyreae, a different species than the target, olivine species.

Objective 2B. Genetic barcode data of olive psyllid parasitoids collected in the Valencia and Murcia regions in Spain in 2022 confirmed that all were the endoparasitoid, Psyllaephagus euphyllureae (Hymenoptera: Encyrtidae) for which the CA field release was officially permitted in 2022. 

Objective 2B.  A survey of pupal parasitoids associated with the allium leaf miner was conducted in 2022 at two sites in Southern France using a sentinel plant approach. DNA barcoding confirmation the presence of a Miscogaster species, family Pteromalidae. The Miscogaster populations were genetically similar to the populations previously sampled in three sites in Switzerland by a local cooperator and at a site in Lyon in France in 2019.  A parasitoid belonging to the Braconidae family was also observed for the first time in one site in France, increasing our knowledge on the diversity of allium leaf miner natural enemies.

Objective 2D.  Genome sequence data of Bagrada specimens from U.S. and South Africa, provided by ARS scientists in Beltsville and France (EBCL), was generated by the Ag100Pest Initiative using PacBio HiFi data sequencing of a single specimen.  Hi-C libraries were generated last year and are in queue for further sequencing/assembly. 

Objective 2D.  Frozen materials of F1 isofemale line progenies of two olive fruit fly parasitoid origins (South Africa and Kenya) were shipped to Hilo ARS facility where the whole genome assemblies will be generated. 

Objective 3A.  Investigated the role of temperature treatments (-20°C, 5°C, 13°C) and six durations (one week to three months) on Bagrada bug eggs storage. Eggs stored at 5°C up to one month were optimal for successful parasitism by the egg parasitoid Gryon aetherium. 

Objective 3A.  In March 2023, 80 chive plants were infested with allium leaf miner (ALM) flies (from an EBCL ALM colony started in 2020). 60 of these plants were then exposed for 24 hours to the parasitoids Diglyphus isaea and Dacnusa sibirica, and 20 were not exposed and used as controls. Exposure to parasitoids was by either exposure to young ALM larvae (7 days after infestation) or exposure to older ALM larvae (14 days after exposure). The plants were then transferred to a growth chamber at 16°C to prevent ALM larvae from entering diapause to slow down their development and pupation time. Evaluation of emergences of flies and parasitoids from infested plants is still ongoing and should be finished by the end of summer 2023.  

Objective 3C. We investigated an optimized irradiation protocol for irradiating bagrada bug adults with our cooperators in Rome (BBCA). This resulted in a publication.

Objective 4.  Cattle Fever Tick (Rhipicephalus annulatus and R. microplus) collections were made in Greece and Vietnam for detection of the parasitic wasp Ixodiphagus sp. Unfortunately, no wasps were detected.  Even with manipulated exposure of a large number of tick larvae in nature to allow for parasitism, followed by placing ticks on cattle until the they become engorged nymphs, also did not result in detection of any parasitoids. This indicates that we should broaden our search of natural enemies to include egg parasites and nematodes or entomopathogenic fungi attacking tick larvae. 
Objective 4. DNA barcoding was applied to engorged nymphs of cattle fever ticks collected on cattle and dog in Vietnam and questing larval and nymphal ticks captured using carbon dioxide-baited tick traps to describe the full range of genetic diversity of cattle fever ticks occurring in three sites in Vietnam in May, June and July 2022. The cattle fever tick and another tick species, Rhipicephalus sanguineus were co-occuring in the three sites, and the cattle fever ticks were closely related to cattle fever ticks in Texas. The occurrence of parasitoid wasps parasitizing these tick populations was determined using quantitative PCR. Only 1.8 % of the ticks were parasitized by IXODIPHAGUS HOOKERI (Hymenoptera: Encyrtidae), a known parasitoid of ticks. 

Objective 4.  After extensive sampling from cattle, dogs, cats and goats in various regions of Vietnam, the Asian Longhorned Tick (Haemaphysalis longicornis) was detected in one region near Ha Noi and Haemaphysalis cornigera a close relative of Asian Longhorned Tick in 4 other regions. This will help us to concentrate on these regions for collection of tick natural enemies in Vietnam.
Objective 4A.  The EBCL-developed method (Giantsis and Chaskopoulou 2019) for detecting environmental insect DNA in soil samples, was modified for detecting, identifying, and quantifying ticks from soil samples. The protocol was optimized with the appropriate primer pairs to exclusively amplify Rhipicephalus, Ixodes and Amblyomma tick spp. DNA. The specificity and sensitivity of the protocol were tested in artificially prepared soil samples with different numbers and stages of the targeted tick species (alone and in combination). The method successfully identified all targeted species and at as low as 10 tick-eggs in 40 ml of soil. The method is being further optimized, while, in parallel, soil samples are being collected from a variety of field sites with known presence of ticks for further validation under field conditions. 
Objective 4A.  Microbiome analysis, of field-collected soil samples with high and low sand fly larval abundances, was completed. Significant differences in the abundance of certain bacterial taxa in soil with high larval concentrations versus soil with no larvae was observed. We identified twelve bacterial taxa significantly more abundant and nine less abundant in the high larval conditions versus soil with no larvae. Interestingly, a higher species richness and diversity was revealed in the no larvae samples, indicating that sand fly larvae might be influencing bacterial diversity. A publication is scripted combining all new knowledge on the field ecology and behavior of sand flies. 

Objective 4A.  Sand fly larvae bioassays were developed to ID novel attractants for new surveillance and control strategies (e.g., attract and kill systems). Sandfly larvae demonstrated a preference towards composted organic matter over non-composted matter. Two different bioassays developed showed a strong larvae olfactory response to a variety of substrates. Volatile compounds were collected using headspace (HS) and HS solid phase microextraction (SPME) and analyzed using GC-MS. A comparison of the molecular composition of the extracted products is being performed.
Objective 4A.  West Nile Virus (WNV) surveillance research continued for another year. WNV infected mosquito pools and sentinel chickens are being examined bi-weekly, and data are shared with public health authorities for designing timely and targeted vector control treatments.
Under Objective 4B.  An EBCL agricultural spray drone technology is being modified for public health applications (ultra-low volume sprays, known as space sprays) for the control of sand fly.  Trials are being conducted to evaluate the efficacy of the system in reducing sand fly populations (as well mosquitoes) under field conditions. **Accomplishments**
                                

1. Discovery and characterization of sand fly larval habitats. Sand flies are small biting insects, and can transmit a wide variety of infectious pathogens (such as Leishmania parasites) that may result in human and animal diseases. According to the World Health Organization (WHO), 350 million people worldwide are at risk of acquiring leishmaniasis (a disease caused by the Leishmania parasites) with 700,000- 1 million new cases annually. Due to the miniscule size of sand fly larvae - immature stages of sand flies occurring in soil - it has been almost impossible to detect them in nature, leading to a significant knowledge gap on the ecology of this species and preventing the development of effective control methods. ARS researchers in Thessaloniki, Greece developed a novel, labor-saving, and cost-effective molecular method to detect and quantify sand fly larvae in soil. The method was successfully applied for the first time under natural conditions and generated unprecedent knowledge on the precise abiotic (e.g., PH, humidity, soil texture) and biotic (microbial community) characteristics of the preferred sand fly larval habitats. This new knowledge will guide the design of novel, targeted control methods against sand flies to prevent the transmission of diseases affecting people, wildlife, and livestock. The method can be adapted to other soil-inhabiting insects (such as ticks) that impose a threat to humans and animals. **Review Publications**
                            *Christaki, A., Zinoviadou, K., Papoti, V., Miaoulis, M., Chaskopoulou, A. 2022. The nutrient composition of three mosquito species, Aedes caspius, Anopheles hyrcanus and Culex pipiens, harvested from nature. Sustainability. 14,13852. https://doi.org/10.3390/su142113852.*
                                *Desurmont, G., Blanchet, A. 2023. Florivory is an alternative but suboptimal diet for an invasive leaf-feeding beetle. Ecological Entomology. 1-4.  https://doi.org/10.1111/een.13237.*
                                *Tsikolia, M., Tabanca, N., Kline, D.L., Demirci, B., Yang, L., Linthicum, K., Bloomquist, J.R., Bernier, U.R. 2022. Studies on the volatiles composition of stored sheep wool, and attractancy toward Aedes aegypti mosquitoes. Insects. 13(2):1-9. https://doi.org/10.3390/insects13020208.*
                                *Ibáñez-Justicia, A., Van De Vossenberg, B., Warbroek, T., Teekema, S., Jacobs, F., Zhao, T., Bueno-Mari, R., Aranda, C., Flacio, E., Chaskopoulou, A., Albieri, A., Stroo, A. 2022. Tracking Asian tiger mosquito introductions in the Netherlands using Nextstrain. Journal of the Mosquito Control Association. 40 (1):11-21. https://doi.org/10.52004/JEMCA2021.0006.*
                                *Sofia, M., Giannakopoulos, A., Giantsis, I., Touloudi, A., Birtsas, P., Papageorgiou, K., Athanasakopoulou, Z., Chatzopoulos, D., Vrioni, G., Galamatis, D., Diamantopoulos, V., Mpellou, S., Petridou, E., Kritas, S., Palli, M., Georgakopoulos, G., Vassiliki, S., Athanassios, T., Chaskopoulou, A., Charalambos, B. 2022. West Nile Virus occurrence and ecological niche modeling in wild bird species and mosquito vectors; an active surveillance program in the Peloponnese Region of Greece. Microorganisms. 10(7),1328. https://doi.org/10.3390/microorganisms10071328.*
                                *Hoelmer, K.A., Sforza, R., Cristofaro, M. 2023. Accessing biological control genetic resources: the United States perspective. BioControl. 68: 269–280. https://doi.org/10.1007/s10526-023-10179-5.*
                                *Martel, G., Sforza, R.F. 2023. Development, survivorship and reproduction of Gryon aetherium Talamas (Hymenoptera: Scelionidae), an egg parasitoid of Bagrada hilaris (Burmeister) (Hemiptera: Pentatomidae), under eight constant temperatures. Biological Control. 180. https://doi.org/10.1016/j.biocontrol.2023.105185.*
                                *Mainardi, C., Peccerillo, C., Paolini, A., Cemmi, A., Sforza, R.F., Musmeci, S., Cristofaro, M. 2023. Using the irradiation technique to predict the sperm competition mechanism in Bagrada hilaris (Burmeister) (Hemiptera: Pentatomidae): Insights for a future management strategy. Insects. 14,681.  https://doi.org/10.3390/insects14080681.*


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