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Theses and Dissertations--Entomology

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Practical Challenges In Delivering Dsrna For Woody Plant Protection, Zachary Bragg Jan 2024

Practical Challenges In Delivering Dsrna For Woody Plant Protection, Zachary Bragg

Theses and Dissertations--Entomology

Unprecedented weather events associated with climate change, coupled with extensive anthropogenic stresses, have reduced the effectiveness of traditional forest pest management approaches, which can no longer keep pace with invasive species overwhelming naïve landscapes and native pests experiencing eruptive and expansive outbreaks. Double-stranded RNA-(dsRNA) mediated gene silencing, a type of RNA interference (RNAi), has been coopted for use as a biopesticide against a multitude of agricultural and horticultural pests and could serve as a powerful tool for woody plant protection. While effective initiation of gene silencing and subsequent mortality have been demonstrated in multiple tree pests, effective and efficient methods …


Polymorphism And Plasticity In The Termite Caste System, Austin Merchant Jan 2024

Polymorphism And Plasticity In The Termite Caste System, Austin Merchant

Theses and Dissertations--Entomology

Developmental plasticity, manifested as a caste system, is integral to the success of eusocial insects because of the way in which it decouples the various tasks that a single, solitary organism would normally be responsible for, allowing individuals to specialize towards their given roles while keeping vulnerable members of the colony, such as those responsible for reproduction, safe from harm. Termites exhibit particularly complex caste systems that can be divided into three types of castes: workers perform manual labor, such as foraging and brood care; soldiers defend the nest; and reproductives lay eggs. Each of these castes, as well as …


Indoor Pests And Human Health: Characterizing Contaminants And Improving Mitigation, Johnalyn Gordon Jan 2024

Indoor Pests And Human Health: Characterizing Contaminants And Improving Mitigation, Johnalyn Gordon

Theses and Dissertations--Entomology

Much of the United States population spends the majority of their time indoors and within the home. Not only are indoor arthropod pests an unsightly nuisance in our homes, but they can have serious impacts on human health, especially with chronic exposure. In addition to negative health impacts, costs associated with these medical impacts can be tremendous. For these reasons, it is critical to develop comprehensive strategies for pest reduction and mitigation of associated health impacts. The work outlined in this dissertation represent examination of the distribution of a hypothesized bed bug allergen within the indoor environment, identification of a …


Addressing The Trade-Offs Of Pest Protective Row Covers For Integrated Pest Management In Vegetable Production, Kathleen Fiske Jan 2024

Addressing The Trade-Offs Of Pest Protective Row Covers For Integrated Pest Management In Vegetable Production, Kathleen Fiske

Theses and Dissertations--Entomology

The success of many integrated pest management (IPM) strategies is limited by trade-offs with economic, cultural, and environmental factors. While some IPM strategies reduce pests, they may harm pollinators, reduce the efficiency of weed or disease management, or may increase costs relative to revenue. Pest protective row covers, in the form of fine-mesh netting (referred to as mesotunnels), are an effective alternative to pesticide use, but they are not without their challenges. While row covers exclude insect pests and the pathogens they vector, it is unknown how well non-vectored diseases can be managed underneath the row cover. The exclusion of …


Ecological Risk Assessment Of Transgenic Conditional Lethality Systems For Genetic Biocontrol Strategies, Fernan Rodrigo Perez-Galvez Jan 2023

Ecological Risk Assessment Of Transgenic Conditional Lethality Systems For Genetic Biocontrol Strategies, Fernan Rodrigo Perez-Galvez

Theses and Dissertations--Entomology

Genetic strategies of insect pest control are receiving increased interest due to recent advances in genetic engineering. By introducing alleles that make males functionally sterile, genetically modified (GM) insects can be used in area-wide pest management programs similar to the Sterile Insect Technique (SIT). Genetic control strategies carry several potential benefits, including improved efficacy and specificity, lower operation costs, and reduced dependence on chemical insecticides, but their use is still controversial despite extensive technical documentation indicating their innocuity to humans and the environment. This hesitation is likely due to the novel nature of the approach.

While the first field applications …


Surveillance For Ticks And Tick-Borne Pathogens In Kentucky, Anna Rosalee Pasternak Jan 2023

Surveillance For Ticks And Tick-Borne Pathogens In Kentucky, Anna Rosalee Pasternak

Theses and Dissertations--Entomology

Tick-borne diseases are an emerging threat to human and animal health. In Kentucky, tick-borne disease surveillance has identified rising incidences of spotted fever rickettsiosis, ehrlichiosis, and Lyme disease. Since these diseases occur through the bites of infected ticks, effective prevention efforts are reliant upon knowing where the risk of exposure to tick bites exists. Historical data on tick distribution in Kentucky is variable, with very little reported on a statewide scale, leaving vector control workers, public health personnel, physicians, veterinarians, and others to rely on outdated, intermittent, or out-of-state information. In my dissertation, I surveyed ticks and select tick-borne pathogens …


Managing Death In Termites, Jizhe Shi Jan 2023

Managing Death In Termites, Jizhe Shi

Theses and Dissertations--Entomology

Death of individuals from the same species represents potential risks from disease, predation or competition for animals. Diverse responses associated with death have evolved ranging from simply avoiding or being attracted to the corpses in solitary animals to complicated undertaking behavioral repertoire in eusocial insects. A systematic review in chapter 1 suggested cannibalism is an ancestral and widespread death-related behavior in all non-human animals. Termites are suggested to switch their undertaking behavioral responses from cannibalism to burial based on interactions between chemicals associated with death to balance risks and benefits associated with decomposition. In eusocial animals like termites with caste …


Landscape Influences And Preventive Management For Drosophila Suzukii In Small Fruit Crops, Ryan William Kuesel Jan 2023

Landscape Influences And Preventive Management For Drosophila Suzukii In Small Fruit Crops, Ryan William Kuesel

Theses and Dissertations--Entomology

Small fruit production has been greatly challenged by the repeated invasion of non-native arthropod pests, impacting economic feasibility, and increasing the need for insecticides. Spotted wing drosophila (Drosophila suzukii (Matsumura)) is the most recent invasive pest of small fruits. It causes damage by ovipositing beneath the skin of ripening fruits. Its wide-spread distribution and development of resistance to insecticides has resulted in the need for improved alternative management strategies for small fruit growers.

My surveys of D. suzukii at thirteen blackberry (Rubus fruticosus L.) and raspberry (Rubus idaeus L.) fields found that while D. suzukii was present …


Rnai-Mediated Gene Silencing In The Exotic Redbay Ambrosia Beetle, Xyleborus Glabratus, And Insect-Fungal Interactions Within The Laurel Wilt Complex, Morgan Christine Knutsen Jan 2023

Rnai-Mediated Gene Silencing In The Exotic Redbay Ambrosia Beetle, Xyleborus Glabratus, And Insect-Fungal Interactions Within The Laurel Wilt Complex, Morgan Christine Knutsen

Theses and Dissertations--Entomology

Laurel wilt disease (LWD) is a lethal vascular disease impacting lauraceous hosts caused by Harringtonia lauricola, the fungal symbiont of the redbay ambrosia beetle (Xyleborus glabratus Eichoff) (RAB) (Coleoptera: Curculionidae). LWD has caused tree mortality throughout the southeastern United States and is continuing to spread into new regions. Current management methods have not been successful in preventing spread, warranting investigation into innovative techniques including RNA interference (RNAi).

Elongation factor-1 alpha (ef1a) and actin (act) were established as stably expressed reference genes after exposing beetles to different photoperiod, temperature, and dsRNA exposure. After RAB oral …


Costs And Benefits Of Integrating Poultry Into Cover Crop - Vegetable Rotations, Viktor Halmos Jan 2023

Costs And Benefits Of Integrating Poultry Into Cover Crop - Vegetable Rotations, Viktor Halmos

Theses and Dissertations--Entomology

Integrated livestock systems were once common leading up to the 20th century where livestock generated manure that was then used to fertilizer crop production in a closed- or semi-closed system. However, with the industrialization of agriculture in the mid to late 20th century, specialization drove simplified systems that segregated livestock from cropping systems. This segregation led to nutrient surpluses occurring in livestock operations, and nutrient demands in cropping systems creating a plethora of environmental issues. With the increased awareness of environmental issues, a renewed interest in integration has driven producers and researchers to experiment with integration again. For research, the …


Linking Previous Experiences To Behavior And Health In The Honey Bee (Apis Mellifera), Rebecca R. Westwick Jan 2023

Linking Previous Experiences To Behavior And Health In The Honey Bee (Apis Mellifera), Rebecca R. Westwick

Theses and Dissertations--Entomology

An organism’s ability to respond to changing conditions can be vital to its success. Indeed, plasticity is a common feature of living organisms. Much of the research in this area, though, has focused on effects caused by environmental conditions. What has received relatively less attention is how social experiences and broader features of an organism’s social environment can lead to long-lasting changes in health and behavior. This knowledge gap exists despite the well-documented existence of health and behavioral effects after social interactions in certain taxa such as humans.

Social insects such as honey bees provide an excellent opportunity to better …


Mechanisms Of Rnai In Insects, Jin Mo Koo Jan 2023

Mechanisms Of Rnai In Insects, Jin Mo Koo

Theses and Dissertations--Entomology

RNA interference (RNAi) mechanism is found in many eukaryotes and animal cells, with its endogenous functions as defense against transposable elements and viruses, as well as post transcriptional regulation of gene expression. Since its first discovery, RNAi has contributed enormously as a reverse genetic tool for functional genomic studies. In application, RNAi is used in therapeutics, as well as agricultural crop and livestock protection. Yet, for the wider application of RNAi, improvement of its potency and delivery technologies is needed. Deeper mechanistic understanding of every step of RNAi, from cellular uptake of RNAi trigger molecule to targeted mRNA degradation is …


Understanding The Effects Of Age, Environmental Conditions, And Placement On Cockroach Gel Bait Performance, Isabelle Lucero Jan 2023

Understanding The Effects Of Age, Environmental Conditions, And Placement On Cockroach Gel Bait Performance, Isabelle Lucero

Theses and Dissertations--Entomology

Cockroach baits are one of the most effective tools used for German cockroach (Blattella germanica L.) control. This is due in part to the number of cockroach baits on the market with various matrix compositions and active ingredients, aiding in the control of resistant (physiologically and behaviorally) cockroach populations through bait rotation. However, it remains unclear how cockroach gel baits perform over time and under different environmental conditions. Therefore, we aged six cockroach gel baits for varying times (24 hours, one month, three months, six months) and at three relative humidities (15%, 40%, and 80%), and tested their performance …


Gene Silencing Provides Insights Into Bark Beetle Biology And Creates Potential For Broad Scale Forest Protection, Beth R. Kyre Jan 2022

Gene Silencing Provides Insights Into Bark Beetle Biology And Creates Potential For Broad Scale Forest Protection, Beth R. Kyre

Theses and Dissertations--Entomology

Dendroctonus bark beetles are among the most economically and ecologically significant forest pests in North America and play a critical role in the overall health of conifer forest ecosystems. Dendroctonus bark beetles influence ecosystem benefits and biodiversity and drive forest succession, and adversely affect timber production, forest management, and recreation. As temperatures surge and climatic fluctuations become more extreme, catastrophic bark beetle outbreaks are increasing in frequency, escalating pressures on highly vulnerable conifer forests already compromised by heat and drought. Eruptive outbreaks of Dendroctonus beetles are largely unhindered by traditional silvicultural management; these practices further disrupt forest ecosystem services, including …


Net Effects Of Wild And Managed Birds In Agroecosystems, Karina Garcia Jan 2022

Net Effects Of Wild And Managed Birds In Agroecosystems, Karina Garcia

Theses and Dissertations--Entomology

Agricultural intensification and expansion have reduced biodiversity in agroecosystems, jeopardizing the ecosystem services that nature provides to humans in these landscapes including wildlife-mediated pest control. Among these purveyors of ecosystem services are birds, who can provide services to agroecosystems by consuming arthropod pests. Yet some bird species also act as pests by consuming crops. Herein, I use molecular diet analysis through high-throughput sequencing on DNA extracted from bird fecal samples to examine birds’ diet and classify bird species as consumers of major strawberry pests (Lygus spp.). Additionally, I use targeted PCR approaches to determine which bird species consumed strawberries. …


Rna Interference For Emerald Ash Borer Suppression: Ecotoxicological Assessment And Delivery Methods, Flavia Pampolini Jan 2022

Rna Interference For Emerald Ash Borer Suppression: Ecotoxicological Assessment And Delivery Methods, Flavia Pampolini

Theses and Dissertations--Entomology

The emerald ash borer (EAB), Agrilus planipennis (Coleoptera: Buprestidae), is a highly invasive phloem-feeding insect native to Asia. It has killed millions of ash trees (Fraxinus spp.) since its accidental introduction into North America, causing profound economic and ecological impacts. RNA interference (RNAi) or double-stranded RNA (dsRNA)-mediated gene silencing is an emerging biotechnology that is developing as an innovative tool for pest management. Exogenous dsRNA triggers the RNAi pathway, silences genes, and disrupts protein function, causing insect mortality. RNAi has proven effective in reducing target gene expression and causing mortality in EAB; however, two aspects stand as barriers …


A Functional Genomics Approach To Overwintering Mechanisms In Insects, Emily Allison Wheeler Nadeau Jan 2022

A Functional Genomics Approach To Overwintering Mechanisms In Insects, Emily Allison Wheeler Nadeau

Theses and Dissertations--Entomology

Insects living in temperate and polar environments have developed numerous adaptations to increase survival at low temperatures. A majority of insects are freeze-intolerant and die from internal ice formation, but some are freeze-tolerant and can survive ice formation in extracellular spaces. Both categories of insects prepare for winter with a combination of seasonal and rapid acclimation responses, which differ both in time course and in underlying mechanisms. In this dissertation, I examine adaptations for winter survival in several insect species with a specific focus on molecular mechanisms. To better understand the underpinning mechanisms of these adaptations, I leverage functional genomics …


Effects Of Physiological Stage And Diet On Drosophila Suzukii Olfaction, Aerianna Shindindowi Littler Jan 2022

Effects Of Physiological Stage And Diet On Drosophila Suzukii Olfaction, Aerianna Shindindowi Littler

Theses and Dissertations--Entomology

Massive economic damage by spotted-wing drosophila (SWD), Drosophila suzukii, results from their unique egg laying behavior. Gravid SWD flies employ a complex suite of behaviors to find fruit with the optimum firmness and chemistry. We tested for SWD attraction to various fruits and found overripe raspberries to be most attractive to gravid flies which was unexpected since they oviposit in ripe fruit. We next tested the same choice assay in a T-maze and found that the results were comparable. We also found discrete odor profiles among the ripening stages. When testing for sugars and CO2 concentrations from field-collections, overripe …


Distribution And Diversity Of Heliothine And Other Lepidopteran Nudiviruses, Emrah Ozel Jan 2021

Distribution And Diversity Of Heliothine And Other Lepidopteran Nudiviruses, Emrah Ozel

Theses and Dissertations--Entomology

Helicoverpa zea nudivirus 2 (HzNV-2) is the only known sterilizing and sexually-transmitted insect virus and causes pathological symptoms in H. zea reproductive tissues. HzNV-2 has features that make it a candidate as a H. zea (corn earworm) control agent, such as the ability to cause asymptomatic (latent) and symptomatic (lytic) infections and the ability to influence mating behavior of its host to favor virus spread. HzNV pathology has been studied and its genome sequenced, however, its prevalence in natural populations is largely unknown. In this study, we developed and used a low-cost PCR-based molecular survey to investigate HzNV-2 prevalence and …


Southern Pine Beetle-Specific Rna Interference Demonstrates No Effects On Nontarget Insects, Hannah Marie Hollowell Jan 2021

Southern Pine Beetle-Specific Rna Interference Demonstrates No Effects On Nontarget Insects, Hannah Marie Hollowell

Theses and Dissertations--Entomology

The endemic southern pine beetle (SPB), Dendroctonus frontalis Zimmermann, is a highly destructive bark beetle that has devastated coniferous forests of the southern United States for decades. Traditional management strategies have been unable to respond effectively to largescale SPB outbreaks, prompting exploration of novel techniques including RNA interference (RNAi). Double-stranded RNAs (dsRNAs) are effective at triggering gene silencing and inducing mortality in SPB, but determining delivery methods and demonstrating specificity of the treatments is necessary before this technology may progress to deployment.

I evaluated three SPB-specific dsRNAs on model nontarget insects to assess potential lethal effects, sublethal effects such as …


A Tangled Web: The Dynamics Of Endosymbiotic Infections In A Linyphiid Spider, Laura Cecilia Rosenwald Jan 2020

A Tangled Web: The Dynamics Of Endosymbiotic Infections In A Linyphiid Spider, Laura Cecilia Rosenwald

Theses and Dissertations--Entomology

Many arthropods are infected with bacterial endosymbionts that manipulate host reproduction, but few bacterial taxa have been shown to cause such manipulations. Mermessus fradeorum (Linyphiidae) is a sheet-weaving spider that displays both feminization and cytoplasmic incompatibility (CI). To correlate reproductive manipulations to endosymbionts, I surveyed the bacterial community of M. fradeorum using high throughput sequencing and found that individuals can be infected with up to five different strains of bacteria from the genera Wolbachia, Rickettsia, and Rickettsiella. Rickettsiella was found in all 23 tested spider matrilines. I used antibiotic curing to generate uninfected matrilines that I reciprocally crossed with …


Dynamic Balancing Of Foraging And Defensive Effort Contribute To The Optimality Of The Honey Bee Robbing Strategy, Grayson James Grume Jan 2020

Dynamic Balancing Of Foraging And Defensive Effort Contribute To The Optimality Of The Honey Bee Robbing Strategy, Grayson James Grume

Theses and Dissertations--Entomology

The optimality of a foraging strategy shifts in response to dynamic ecological conditions and the need to devote effort to other tasks. Nest defense and foraging effort in the honey bee may trade off as both tasks are performed by a shared workforce of physiologically-specialized individuals in exclusive roles. Honey robbing is a foraging strategy predicted to benefit from simultaneous increases in foraging and defensive effort, but may be constrained by workforce specialization. We developed a methodology to induce robbing behaviors with uninhabited bait hives. We used this methodology to evaluate foraging and defensive effort before and during robbing by …


Optimizing Monarch Butterfly And Bee Conservation Efforts In The Urban Landscape, Adam M. Baker Jan 2020

Optimizing Monarch Butterfly And Bee Conservation Efforts In The Urban Landscape, Adam M. Baker

Theses and Dissertations--Entomology

The eastern migratory population of monarch butterflies (Danaus plexippus) is in serious decline. Most of the efforts to conserve this iconic insect focus on habitat restoration in the US Midwest. Often overlooked are small butterfly-centric gardens that can act as stepping stones between urban and rural areas. These studies aim to optimize the conservation value of such gardens.

Eight milkweed (Asclepias) species varying in height, form, and leaf shape were compared over two years in a common-garden experiment. I measured milkweed growth, rhizome spread, and bloom periods, conducted bi-weekly counts of monarch eggs and larvae, evaluated …


Physiological Ecology Of Overwintering And Cold-Adapted Arthropods, Leslie Jean Potts Jan 2020

Physiological Ecology Of Overwintering And Cold-Adapted Arthropods, Leslie Jean Potts

Theses and Dissertations--Entomology

Given their abundance and diversity, arthropods are an excellent system to investigate biological responses to winter. Winter conditions are being majorly impacted by climate change, and therefore understanding the overwintering biology of arthropods is critical for predicting ecological responses to climate change. In Chapters 2 and 3, I investigate the winter biology of a winter-active wolf spider. I show that winter-active spiders can take advantage of periodic prey resources and grown in the winter, which may allow them to get a jumpstart on spring reproduction. I also investigate spiders’ ability to track changes in their environment by quantifying low temperature …


Identification And Characterization Of Epigenetic Regulators In The Yellow Fever Mosquito, Aedes Aegypti, Shankar C. R. R. Chereddy Jan 2020

Identification And Characterization Of Epigenetic Regulators In The Yellow Fever Mosquito, Aedes Aegypti, Shankar C. R. R. Chereddy

Theses and Dissertations--Entomology

Due to the increased interactions between the sylvatic and urban cycles of Aedes aegypti life cycle, there has been an increase in the incidence of vector-borne diseases. The risk involved through the transmission of viruses like Dengue, Zika, Yellow-fever and Chikungunya calls for an effective control method for the deadly vector. The possibility of pesticide resistance and the costs involved in producing new pesticides have pushed Sterile Insect Technique (SIT) into prominence. However, current methods used to sterize insects for SIT are not highly efficient. This study tackles current issues in mosquito control by identifying and characterizing epigenetic regulators that …


Developing Alternative Practices For Management Of Flea Beetles Attacking Eggplant And Leafy Brassicaceous Greens, Robert Brockman Jan 2020

Developing Alternative Practices For Management Of Flea Beetles Attacking Eggplant And Leafy Brassicaceous Greens, Robert Brockman

Theses and Dissertations--Entomology

Flea beetles are a challenging pest for many producers of vegetable crops in Kentucky. Chewing numerous small holes in the leaves of their host plants, high flea beetle populations can quickly overwhelm unestablished crops. I tested the efficacy of several alternatives to insecticides within brassicaceous leafy greens and eggplant. Four field trials in 2019 and 2020 compared essential oil sprays, the woven-mesh row cover ProtekNet, the spunbonded row cover Agribon, and reflective silver mulch to an untreated control, a conventional insecticide rotation of dinotefuran and pyrethroids, and an organic insecticide rotation of spinosad and pyrethrins. The silver reflective mulch was …


Management Of Stink Bugs (Hemiptera: Pentatomidae) On Soybean In Kentucky, Yaziri Gonzalez Jan 2020

Management Of Stink Bugs (Hemiptera: Pentatomidae) On Soybean In Kentucky, Yaziri Gonzalez

Theses and Dissertations--Entomology

Phytophagous stink bugs (Hemiptera: Pentatomidae) are pests of significant importance to soybean, Glycine max (L.), because they can cause severe losses in seed quality, ultimately affecting yield. Damage to soybean is caused through a process called extra-oral digestion. This occurs when the adults and nymphs of stink bugs pierce plant tissues with their mandibular and maxillary stylets, insert digestive enzymes, break down tissue, and extract the digested plant fluid. Three endemic stink bug species of North America are present in Kentucky: green stink bug (Chinavia hilaris), brown stink bugs (Euschistus species complex), and red-shouldered stink bug (Thyanta custator) …


Epigenetic Regulation Of Hormone Action In The Red Flour Beetle, Tribolium Castaneum (Herbst), Smitha George Jan 2020

Epigenetic Regulation Of Hormone Action In The Red Flour Beetle, Tribolium Castaneum (Herbst), Smitha George

Theses and Dissertations--Entomology

Hormones are the chemical communication signaling molecules released into the body fluid to stimulate target cells of multicellular organisms. Two major hormones, ecdysteroids (20-hydroxyecdysone, 20E) and juvenile hormones (JH), regulate a wide variety of physiological and developmental processes in insects. Therefore, hormones have been extensively studied and are attractive targets for the development of target-specific insect control methods. Recent studies suggest that epigenetics adds another layer of regulation to explain multiple functions of the same circulating hormone in different tissues and at various time points. In my dissertation, I focused on a major post-translational modification, ‘acetylation,’ to elucidate the epigenetic …


Aggregation Behavior In The Bed Bug, Cimex Lectularius L., Paul D. Baker Jan 2020

Aggregation Behavior In The Bed Bug, Cimex Lectularius L., Paul D. Baker

Theses and Dissertations--Entomology

The bed bug, Cimex lectularius L., is a common household pest that feeds on the blood of its human hosts. Unlike many other hematophagic arthropods, bed bugs have not demonstrated the ability to vector disease; yet its presence in a household often leads to fear and anxiety amongst its human host victims. Bed bugs spend the majority of their time aggregating in enclosed and hidden locations, making it difficult to detect and eradicate them. One of the significant mediators of aggregation behavior is an aggregation pheromone. The pheromone has been identified to be six chemical compounds, five volatile compounds that …


Emerald Ash Borer Development Across A Latitudinal Gradient: Implications For Biocontrol, Sarah Pellecchia Jan 2020

Emerald Ash Borer Development Across A Latitudinal Gradient: Implications For Biocontrol, Sarah Pellecchia

Theses and Dissertations--Entomology

Emerald ash borer, EAB, (Agrilus planipennis Fairmaire) is an invasive beetle that kills ash trees. It was accidentally introduced from China, and has rapidly expanded across North America, now occupying much of the eastern US. Four classical biocontrol parasitoids have been released to help mitigate its spread and impact: Tetrastichus planipennisi Yang, Spathius agrili Yang, S. galinae Belokobylskij and Strazanac, and Oobius agrili Zhang and Huang. These parasitoids have been deployed throughout EAB’s invaded range, but there has been limited recovery of the parasitoids from their release sites in southern states. I evaluated whether this lack of establishment might …