Location: European Biological Control Laboratory
2021 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
Under Objective 1A (Explore across Europe, Asia and Africa for natural enemies of invasive arthropod pests) exploratory research was conducted on several targets:
1. Several wasp parasitoids (Trissolcus sp. and Gryon sp.) of Bagrada eggs were collected in South Africa and their identification using both morphological and molecular tools is continuing.
2. Surveys for the natural enemies of the viburnum leaf beetle (Pyrrhalta viburni) were intensified in France. This is being accomplished by collecting large numbers of field collected egg masses and larvae and observing these for parasitoid emergence. 171 egg parasitoids have emerged, all belonging to the species Aprostocetus celtidis, but no larval parasitoids. A database of the presence and damage of Pyrrhalta viburni in the south of France has been assembled. This database based on observations made in the south of France will be used to target more precisely areas to survey for natural enemies in the native range as well as predicting the spread of the pest in its invasive range.
3. Eight species of pupal parasitoids associated with the allium leaf miner (Phytomyza gymnostoma) were identified through the collection and monitoring of large numbers of pupae obtained from both cultivated and natural field sites. Species identification of these parasitoids is currently ongoing through a classical taxonomic approach and DNA characterization (barcoding). Discovery of pupal parasitoids is encouraging as it shows that this pest has a diverse natural enemy assemblage in its native range, which increases the likelihood of finding suitable biocontrol agents.
4. A CO2 baited trap based on dry ice, initially developed for mosquitoes, was tested and found promising to identify natural enemies of ticks. In a preliminary trial conducted in France, this method proved more successful than live-baited traps as a large number of questing tick larvae and nymphs were captured. This method will greatly maximize the opportunity to sample parasitized ticks including the Asian longhorned tick (Haemaphysalis longicornis), and identify associated natural enemies.
5. Under Objective 2A (Investigate the phylogeography of target pests to trace the geographic origin of U.S. invasive populations) a genetic match was observed between European populations of Phytomyza gymnostoma compared to populations occurring in Pennsylvania. This indicates the potential origin of Pennsylvania invasive populations and identifying native ranges where to explore for promising natural enemies. Microsatellite-enriched libraries were purposely prepared from a pool of DNA extracted from Pyrrhalta viburni sampled in 4 sites in France and 2 sites in Pennsylvania from which a total of 265 microsatellite loci was obtained. Testing of the polymorphism of these microsatellite loci is ongoing as a prerequisite of their validation as population genetic markers for Pyrrhalta viburni.
6. Under 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) genetic characterization using barcode data of pupal parasitoids of Phytomyza gymnostoma collected in Switzerland indicated that all pupal parasitoids were most closely related to the genus Miscogaster in the family Pteromalidae.
7. The full genome of bacteria isolated from two biocontrol agents of the olive fruit fly, Psyttalia lounsburyi and Psyttalia ponerophaga was sequenced and characterized. (Objective 2C, Characterize microbiomes associated with target pests and candidate biocontrol agents of interest, with the support of whole genome sequencing when needed). These intracellular bacteria appeared to belong to a novel Serratia species, and most closely related to Serratia marcescens. Functional characterization of this species' gene content is ongoing.
8. Isofemale lines were created in quarantine for bagrada bug colony derived from South Africa, Psyttalia lounsburyi derived from Kenya and South Africa and Psyttalia Ponerophaga derived from Pakistan (Objective 2D, Characterize and compare the full genome sequences for biocontrol agents of the olive fruit fly and for a hemipteran invasive pest, Bagrada). At least five isofemale lines were founded for all listed targets and frozen materials of F1 progenies were shipped to Hilo where the whole genome assemblies will be generated.
9. Bagrada bug eggs stored at 5°C up to one month were optimal for successful parasitism by Gryon gonikopalense without negative effect on offspring (Objective 3A, Determine biological and ecological parameters that affect the efficacy and safety of parasitoids of target pests). Such information makes a real progress for rearing of Gryon gonikopalense under laboratory and potentially mass-rearing conditions. In addition, published results have showed that, under laboratory conditions, Gryon gonikopalense preferred parasitizing buried Bagrada bug eggs (representing 87 % of all parasitized eggs versus eggs laid on the foliage). Under objective 3A, conservation of Pyrrhalta viburni eggs in presence of Potassium nitrate in the cage maintains a stable 93% humidity at 8°C for Pyrrhalta viburni egg masses. This conservation method represents a significant improvement of past methodologies, allowing parasitized eggs to remain viable for at least 3 months past their natural time of emergence in the field. A rearing method of Phytomyza gymnostoma has also been optimized to increase the number of flies produced per plant at each generation. A total of over 700 flies were produced over the period January-June 2021; this is equivalent to those reared for the whole of 2020. This increased efficacy in rearing will facilitate future studies on the allium leaf miner and its natural enemies.
10. Under Objective 3B (Investigate the chemical ecology of target pests, including the viburnum leaf beetle and the olive fruit fly, and their natural enemies), volatile compounds emitted by leaves of Viburnum tinus plants infested with Pyrrhalta viburni adults and non-infested plants were successfully collected using SuperQ and Haysep Q adsorbents under laboratory conditions with cut twigs. Results have to be compared to the ones obtained in 2020 with two other host plants of Pyrrhalta viburni , Viburnum opulus and Viburnum lantana. The next step will be to test the compounds consistently associated with infested leaves as attractants to Pyrrhalta viburni adults under laboratory conditions in order to develop lures that could be used to trap and monitor the pest in the field.
11. Under 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) the most suitable irradiation dose for the use of the Sterile Insect Technique (SIT) against bagrada bug eggs was examined in an effort to increase the period of exposure of sterile sentinel eggs in the field used to collect natural enemies. A total of 9,000 eggs were irradiated using four different doses (16, 24, 32 and 40 Grays). Irradiated eggs stored under 22°C or 5°C were exposed to Gryon gonikopalense for different durations (1, 5, 12, 18, and 22 days). Results still need to be analyzed, but the irradiation process is now optimized and will bring new insights into the use of sterile sentinel eggs.
12. Under 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) a molecular method using real-time PCR and species-specific primers for detecting sand fly larvae in soil was tested under field conditions by collecting soil samples from a multi-species animal farm. Sand fly DNA directly from field-collected soil samples were successfully detected and quantified. Work is currently ongoing to determine the abiotic and biotic characteristics of soil samples (e.g., the mechanical composition of soil, pH, soil humidity, and soil microbes) associated with sand fly abundance. Knowledge of the breeding habitat of this vector is vital for implementing appropriate interventions. In addition, West Nile Virus (WNV) surveillance research continued. WNV infected mosquito pools and sentinel chickens were examined bi-weekly, and data was shared with public health authorities in an effort to apply timely and targeted vector control treatments. EBCL contributed to the development VectorMap-GR: a local scale operational management tool for entomological monitoring to support vector control activities in Greece and the Mediterranean Region. The tool in pilot form was implemented in South Greece in collaboration with public health authorities and is available for use at a national scale.
13. Exploiting biting arthropods as animal feed may be a powerful incentive for farmers as well as the pest control industry to transform vector control strategies from insecticide-based solutions to mechanical interventions (Objective 4b, Evaluate novel and optimize existing vector control strategies under field conditions including but not limited to Mosquitoes, sand flies and ticks). The potential of biting arthropods produced in high numbers in agricultural landscapes to be used as animal feed has never been investigated. The nutritional value and safety of three different mosquito species produced in massive numbers in rice-field ecosystems is being evaluated. Based on preliminary findings wild-harvested adult mosquitoes seem to be superior to some of the most widely used edible insects and, thus, show high potential to be used as animal feed. For example, their calcium and phosphorus content is 12 and 3 times higher, respectively, than that of Acheta domesticus (the house cricket) (one of the most common edible insect worldwide).
Accomplishments
1. An online operational tool for monitoring vector borne disease. EBCL scientists in Greece contributed to the design and implementation of VectorMap-Gr: an online, operational tool for entomological and epidemiological data monitoring to support timely and targeted vector control decisions. Using VectorMap-Gr, end users can obtain information on vector spatiotemporal dynamics, insecticide resistance status, occurrence of vector-borne diseases (pathogens/infections in vectors, sentinel animals and humans), and operationally relevant physical feature georeferenced data set (i.e., mosquito breeding sites). The tool, already implemented in Greece, may be readily adapted to other local vector-borne disease settings and vector surveillance/management programs.
Review Publications
Chen, M.N., Santander, R.D., Talamas, E.J., Jentsch, P.J., Bon, M., Acimovic, S.G. 2021. Molecular Identification of Trissolcus japonicus, Parasitoid of the Brown Marmorated Stink Bug, by Species-Specific PCR. Insects. 12,467-482. https://doi.org/10.3390/insects12050467.
Desurmont, G., Kerdellant, E., Lambin, N. 2020. Between a rock and an egg-crushing place: selection pressure from natural enemies and plant defenses on eggs of the viburnum leaf beetle in its native range. Ecological Entomology. 2021. https://doi.org/10.1111/een.12936.
Ganjisaffar, F., Talamas, E., Bon, M., Perring, T. 2020. First report and integrated analysis of two native Trissolcus species utilizing Bagrada hilaris eggs in California. Journal of Hymenoptera Research. 80: 49-70. https://doi.org/10.3897/jhr.80.57024.
Giantsis, I.A., Beleri, S., Balatsos, G., Karras, V., Patsoula, E., Papchristos, D., Michaelakis, A., Chaskopoulou, A. 2020. Sand fly (Diptera: Psychodidae: Phlebotominae) population dynamics and natural Leishmania infections in Attica Region, Greece. Journal of Medical Entomology. XX(X), 1-6.
Kniha, E., Dvorak, V., Milchram, M., Obwaller, A.G., Koehsler, M., Poeppl, W., Antoniou, M., Chaskopoulou, A., Paronyan, L. 2021. Phlebotomus (Adlerius) simici NITZULESCU, 1931: first record in Austria and the phylogenetic relationship with other Adlerius species.. Parasites & Vectors. 14:20. https://doi.org/10.1186/s13071-020-04482-8.
Martel, G., Sforza, R. 2021. Catch me if you can: novel foraging behavior of an egg parasitoid, Gryon gonikopalense (Hym. Scelionidae), against the stinkbug pest, Bagrada hilaris (Hem. Pentatomidae). Journal of Pest Science. https://doi.org/10.1007/s10340-020-01325-4.
Wang, X., Ramualde, N., Aparicio, E.M., Maspero, M., Duan, J.J., Smith, L. 2021. Optimal conditions for diapause survival of Aprostocetus fukutai, an egg parasitoid for biological control of Anoplophora chinensis. Insects. 12(6). https://doi.org/10.3390/insects12060535.
