Project : USDA ARS

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

Location: EUROPEAN BIOLOGICAL CONTROL LABORATORY

2019 Annual Report

Objectives
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.

Approach
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: exploration for bagrada bug (BAGRADA HILARIS) and its natural enemies was pursued in Kenya at the Agricultural and Livestock Research Organization (KALRO) and was initiated in South Africa at the University of Stellenbosch where malaise traps and sentinel egg exposures were set up. Hymenopteran parasitoids (TRISSOLCUS SP., GRYON SP.) were collected and shipped to U.S. cooperators for morphological and genetic analyses. Over 17,300 adults of the South African strain of the parasitoid PSYTTALIA LOUNSBURYI were mass-reared and shipped to California for release to control the olive fruit fly (BACTROCERA OLEAE). Over 1,900 adults of the parasitoid, PSYTTALIA PONEROPHAGA, which is a prospective biocontrol agent for olive fruit fly, were sent to cooperators in California for host specificity studies. Exploration for natural enemies of the allium leaf miner PHYTOMYZA GYMNOSTOMA began in its native range in France and Italy in cultivated leek ALLIUM PORRUM fields and in natural sites containing wild ALLIUM SPP., resulting in the collection of an unknown pupal parasitoid PTEROMALUS SP and ca. 100 specimens of the allium leaf miner which will form the basis of a future integrative taxonomy study. Exploration for natural enemies of the viburnum leaf beetle PYRRHALTA VIBURNI was done in Western and Central Europe resulting in the collection of > 1,000 egg masses, ca. 2,000 larvae, and ca. 200 egg parasitoids belonging to the genus APROSTOCETUS. Expositions of laboratory-reared sentinel cattle ticks (RHIPICEPHALUS ANNULATUS) to investigate the presence of and potentially collect natural enemies were initiated in different locations in Vietnam, in South Vietnam (Dak Lak) and North Vietnam (Hanoi and Ha Giang) by EBCL cooperators at the National Institute of Veterinarian Research (NIVR), and were pursued in Chania in Crete and in Xanthi in NW Greece. Engorged ticks were held for the emergence of possible parasitoids and entomopathogenic nematodes. Objective 2A, genetic comparisons were made between APROSTOCETUS CELTIDIS and APROSTOCETUS SUEVIUS, two egg parasitoids of the viburnum leaf beetle PYRRHALTA VIBURNI. Analyses of 168 APROSTOCETUS specimens collected from populations of PYRRHALTA VIBURNI throughout Europe revealed that they all belong to a unique species, that was confirmed by proteomic analyses using a MALDI-TOF method and morphometric measurements of the length of the ovipositor. Taken as a whole, these data strongly suggested that there is only one egg parasitoid of PYRRHALTA VIBURNI present in Europe, which provided important clues regarding the assemblage of natural enemies attacking PYRRHALTA VIBURNI in its native range. Genetic barcoding of egg parasitoids accidentally recovered from sentinel eggs of the brown marmorated stink bug (HALYOMORPHA HALYS) exposed in Italy revealed that these parasitoids belong to TRISSOLCUS JAPONICUS and to TRISSOLCUS MITSUKURII. This was the first discovery of the accidental presence of TRISSOLCUS MITSUKURII in Europe suggesting that it might be occurring in North America too. Objective 2B: genetic barcoding of olive psyllid EUPHYLLURA OLIVINA individuals collected in Spain, France and California revealed that the Californian population is closely related to one population in Southern Spain and one population in Southern France, indicating the potential origin of the Californian invasive populations and suggesting where to look for promising natural enemies. Genetic fingerprinting of adventive populations of the egg parasitoid of the brown marmorated stink bug (TRISSOLCUS JAPONICUS) was pursued on populations recovered in 2017 and 2018 in British Columbia in Canada, in Utah and Ohio in the USA, in Italy, and in Switzerland. First results showed that the adventive populations of the parasitoid recovered in Europe were different from those recovered in North America, suggesting that the parasitoid present in Europe has been directly introduced from Asia. Objective 3A, life history studies were pursued on the most promising egg parasitoid (GRYON GONIKOPALENSE) of bagrada bug BAGRADA HILARIS. The rearing of quarantine colonies of this parasitoid was optimized to facilitate the maintenance and to help the design of host specificity experiments. As a result, GRYON GONIKOPALENSE foraging behavior was analyzed and determined to be 75% successful in finding buried bagrada eggs. This behavior should be relevant against bagrada bug which lays over 80% of its eggs in the soil under laboratory conditions. The olfactory preferences of PSYTALLIA PONEROPHAGA, a prospective biological control agent of the olive fruit fly BACTROCERA OLEAE were investigated using olfactometer and videotracking devices. Female parasitoids did not prefer olive odors to those of other fruits but did spend significantly more time inspecting infested fruits than intact. The olfactory preferences of the olive fruit fly were also investigated in an olfactometer, testing the attractiveness of 17 compounds produced by olive trees in the south of France. Results showed a significant attraction towards several compounds. The attractiveness of these compounds is currently being tested in the field in baited traps. Modifications to current rearing methods for the egg parasitoid APROSTOCETUS FUKUTAI of ANOPLOPHORA CHINENSIS were implemented with diapausing eggs being kept at 100% humidity levels in incubators inside quarantine, representing significant progress for the rearing this particularly challenging parasitoid. Field and laboratory experiments started in 2016 and ended in November 2018 showed that mites inhabiting the egg masses of PYRRHALTA VIBURNI in France do not pose a predation threat to its eggs. A combination of manipulative experiments and genetic analyses showed that the most abundant mite species, TRICHORIBATES TRIMACULATUS, consumed the “egg cap” covering the egg masses but not the eggs themselves, suggesting that this mite has no potential as a biological control agent Objective 3B: Laboratory experiments to evaluate the virulence of entomopathogenic fungi (EPF) on the olive fruit fly BACTROCERA OLEAE were pursued. Levels of virulence of one strain of METARHIZIUM ANISOPLIAE, four strains of BEAUVERIA BASSIANA isolated from soil in olive orchards, and one commercial strain (GHA) of BEAUVERIA BASSIANA, all maintained in the EBCL fungal collection, were re-evaluated using the same protocol as last year in order to confirm results obtained in previous years. Results confirmed that the METARHIZIUM ANISOPLIAE strain 02068 is more virulent than the others, confirming its prospective use as an effective microbial biocontrol agent for BACTROCERA OLEAE. Objective 3C, WOLBACHIA infection status was assessed in populations of PSYTTALIA LOUNSBURYI recovered in 2015 in California. These recovered populations primarily originated from a South African colony in which WOLBACHIA type II was present in up to 50% of individuals. WOLBACHIA was detected in 98% of the populations recovered in California of which 58% were singly infected by WOLBACHIA type II. The present infection status is indicative of the spread of the WOLBACHIA in the Californian populations. The presence of an opportunistic pathogenic bacteria SERRATIA SP. was evidenced using next generation sequencing in the quarantine colonies of PSYTTALIA LOUNSBURYI and PSYTTALIA PONEROPHAGA. The full genome of the two SERRATIA strains isolated from these two species was sequenced by ARS researchers at Beltsville. Genome analyses revealed that their genomes are identical, and that this species is new to Science. Pathogenicity testing suggested that the two strains are likely non-pathogenic under laboratory conditions. Objective 4, ARS-EBCL scientists in Greece studied bio-efficacy of larvicides against mosquitoes breeding in rice fields. The impact of a chitin synthesis inhibitor, diflubenzuron, applied by air, was investigated on these important disease vectors. Emergence inhibition and larval population densities were compared between treated and un-treated (organic) fields and the persistence of diflubenzuron was assessed through Gas Chromatography (GC) coupled with Electron Capture Detector (ECD) techniques. The larvicidal effects of diflubenzuron and its degradation rate under field conditions were determined and will be used to optimize the application guidelines for this product under field conditions. Some important mosquito vectors, such as AEDES ALBOPICTUS (ZIKA virus vector) lay their eggs in a variety of microhabitats that are almost impossible to spray with conventional larviciding methods. The efficacy of four larvicide formulations, diflubenzuron, pyriproxyfen, methoprene, and Bti, applied as Ultra-low volume space sprays using a London Fog Colt-4 ULV sprayer, was tested against AEDES ALBOPICTUS and CULEX PIPIENS. Findings indicated a range of efficacy across environmental exposure times, formulations, and target species, exemplifying the difficulty of controlling these species with traditional spray techniques. Trials to control sand fly (Leishmania vectors) and mosquito (West Nile virus and malaria vectors) with novel methodologies were pursued. The capability of a spatial repellent, transfluthrin, to reduce vector and biting/nuisance insects was tested in a variety of environments in Greece (temperate climate). One single treatment of transfluthrin was effective in repelling both sand flies and mosquitoes for up to 8 weeks post-treatment. These data were used to support the US Environment Protection Agency registration of a novel insecticidal product, the Bayothrin (Bayer) which is now available to be used against arthropod vectors in the US. The novel molecular method developed by EBCL in Greece to detect and quantify sand fly larvae under laboratory conditions was further validated in the field. The method successfully detected and quantified sand fly larvae from different types of soil sampled from a variety of animal shelters.

Accomplishments
1. Determination of the egg density of bagrada bug (BAGRADA HILARIS) and the foraging behavior of its potential biocontrol agent, GRYON GONIKOPALENSE. 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. Experiments were conducted to measure the longevity, fecundity, and ability to forage bagrada bug eggs buried in the soil. The results provided a basis for maintaining laboratory colonies and for designing host specificity experiments. Furthermore, it was determined that on average 80% of bagrada eggs are buried in the soil in laboratory conditions. Thus, it was shown that GRYON GONIKOPALENSE can find, trace, and successfully parasitize bagrada eggs in the soil that cannot be visually detected. Successful development of this classical biocontrol agent will benefit organic farmers, who have no effective methods to control bagrada bug on cole crops, as well as consumers of these products.

2. Assessment of health status of colonies of biocontrol agents maintained in quarantine prior to release for control of olive fruit fly (BACTROCERA OLEAE). Olive fruit fly (BACTROCERA OLEAE) is the most serious pest of cultivated olives worldwide. It invaded California in 2002 and has become a major pest. ARS researchers at the European Biological Control Laboratory in France (EBCL) developed methods to mass rear two species of parasitoids, PSYTTALIA LOUNSBURYI from Africa and PSYTTALIA PONEROPHAGA from Pakistan. Large numbers of PSYTTALIA LOUNSBURYI were shipped from EBCL to cooperators in California who mass released them in olive groves. This species has become established in California and is multiplying and spreading, providing hope for sustainable control of olive fruit fly. The other species, PSYTTALIA PONEROPHAGA, is currently under evaluation until it is approved for release. As a part of monitoring the sanitary status of the PSYTTALIA colonies maintained in quarantine, their microbial diversity was assessed using Next generation sequencing technology. Results made evident the presence of the bacteria SERRATIA SP. in all colonies. The full genome of this SERRATIA strain sequenced and analyzed by ARS researchers in Montpellier, France, and Beltsville, Maryland, suggested that SERRATIA SP. in PSYTTALIA parasitoids might be a new species to Science. Outcomes of pathogenicity tests indicated this SERRATIA is non-pathogenic. These results provide useful information regarding the risk of disease transmission within the context of a biological control program against the olive fruit fly and the introduction of a new exotic species into the U.S.

3. Species identification of Asian egg parasitoids of stink bugs including HALYOMORPHA HALYS. The brown marmorated stink bug HALYOMORPHA HALYS emerged as a very damaging invasive insect pest in North America in the 1990s. Native to eastern Asia, this highly polyphagous bug (>170 different host plants) is spreading rapidly worldwide. In the mid-Atlantic region of the USA, this bug has become one of the most significant pests in apple production, causing >$37 million in losses in 2010. ARS researchers began searching in 2005 across Asia for natural enemies of HALYOMORPHA HALYS and other stink bugs. From collections over the years, ARS researchers established colonies of egg parasitoids in ARS quarantine facilities. Although these egg parasitoids were known to belong to the genus TRISSOLCUS, a critical question remained unanswered regarding their species names. Concurrent with efforts to address the morphological taxonomy of these TRISSOLCUS, sequencing analyses of five regions in their genomes was conducted by ARS researchers at the European Biological Control Laboratory in France. This approach helped to correctly delimit and identify 17 Asian species of TRISSOLCUS, for which voucher specimens have been deposited in National Insect collection housed at the National Museum of National History (NMNH). These results will be useful to researchers and biocontrol managers who need to know precisely which species they are going to release because USDA Animal Plant Health Inspection Service (APHIS), which Agency regulates the release of non-native biocontrol agents, requires highly accurate identification of the agent to be released.

4. A novel method to detect and quantify soil-dwelling immature stages of sand flies PHLEBOTOMUS SP. in the field. Sand flies (PHLEBOTOMUS SP. are responsible for the transmission of LEISHMANIA parasitic protozoans. At least 20 LEISHMANIA species cause visceral and cutaneous leishmaniasis in humans worldwide. Approximately 1.3 million new cases of leishmaniasis and 20,000 – 30,000 deaths occur annually. Even though leishmaniasis is not common in the United States (occasional cases have been acquired in Oklahoma and Texas) the disease constitutes a major threat to the US military during their overseas operations. In the process of developing an efficient strategy to control PHLEBOTOMUS SP. a critical and unanswered question remained as to where immature stages of the PHLEBOTOMUS SP breed. Until now the only approach for such monitoring was to collect adults through costly trapping techniques requiring significant field manpower and employing a long and tedious morphological identification process requiring highly specialized entomological skills. ARS researchers at EBCL Greece developed a direct molecular method with potential to revolutionize the way surveillance of arthropod vectors whose immature stages breed in soil is accomplished. The method successfully detected and quantified PHLEBOTOMUS SP. larvae from different types of soil sampled from a variety of animal shelters (chicken, horse, goat, sheep, rabbit, goose, pig) and can be adapted to detect other arthropods of medical and veterinary importance, whose immature stages breed in soil, such as Culicoides mosquito (i.e. bluetongue vector) and ticks (Lyme disease, cattle fever vectors). This method will increase our knowledge on insect ecology for designing targeted control methods against immature insect stages. Importantly it also has the potential to be used as a surveillance and risk assessment tool for determining the threat of vector borne diseases affecting wildlife, livestock, and public health.