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Prey-mediated effects of imidacloprid were evaluated for Laricobius nigrinus Fender and Sasajiscymnus tsugae Sasaji and McClure after feeding on hemlock woolly adelgid (HWA), Adelges tsugae Annand (Hemiptera: Adelgidae). Two methods were evaluated for detecting imidacloprid in hemlock tissues: a commercially available enzyme linked immunoassay (ELISA) kit and a high performance thin-layer chromatography technique for detecting and quantifying imidacloprid residues in hemlock wood and needle tissues. ELISA is advantageous because of its cost, sensitivity, and ease of use. However, matrix effects in the form of false positives and overestimated imidacloprid concentrations were evident in hemlock wood and needle tissue extracts. Matrix effects could be reduced by dilution with water, effectively raising the lower detection range of the kit from 0.2 to 200 ppb. High performance thin-layer chromatography was accurate, quick, easy to use, and matrix effects were not evident. However, the technique is sensitive in the lower ppm range and tissue samples from field-treated hemlocks are often in the ppb range, making this technique less desirable than more sensitive analytical methods. Lethal and sublethal effects on both predators were evident after eastern hemlock branches infested with HWA were spiked with imidacloprid in the laboratory. HWA mortality increased with dosage and time, and its 30 d LC50 was determined to be 242 ppb. Both predator species exhibited reduced survivorship and fitness parameters after feeding on HWA from the treated branches. In a topical application bio-assay, 6 d imidacloprid LD50 values for L. nigrinus and S. tsugae were 2.43 and 1.82 µg/g, respectively. Imidacloprid and its major metabolites in hemlock tissues were analyzed by liquid chromatography-tandem mass spectrometry. Imidacloprid recovery from beetle cadavers was correlated with beetle mortality from feeding on treated hemlock branches. Olefin was the primary imidacloprid metabolite recovered from hemlock wood tissues. When predators fed on HWA from field-treated trees, impacts on survivorship and fitness were variable. In 2005, significantly higher proportions of both species of beetles were affected by feeding on control branches compared with treated branches. In 2006, beetles feeding on HWA from some of the trees treated in the field exhibited longer fliptimes compared with beetles feeding on controls, although beetle mortality was not significant among treatements. In the field, imidacloprid controlled HWA populations 1-3 years post-treatment. Hemlock health improved in the highest dosage group, with significantly greater lengths of new shoots compared with shoots from control trees. Eastern hemlock trees primarily metabolized imidacloprid into the olefin metabolite, which can have increased insecticidal toxicity compared with imidacloprid. Imidacloprid was detected in beetle cadavers after feeding on HWA from treated branches, suggesting that prey-mediated impacts of systemic imidacloprid are possible on nontarget predators. However, because of HWA's sensitivity to imidacloprid, in field situations predators are more likely to be affected by reduced adelgid density and quality. / Ph. D.

Four to six separate inundative releases of ~30,000 to 50,000 T. ostriniae per 0.02 ha significantly reduced damage by O. nubilalis in bell pepper. Egg parasitization averaged 48.7% in T. ostriniae release plots, which was significantly higher than non-release plots (1.9%). Also, cumulative green pepper fruit damage averaged 8.7% in release plots, which was significantly less than non-release plots (27.3%). Pesticides tested against T. ostriniae were spinosad and methoxyfenozide. Spinosad adversely affected adult T. ostriniae producing 100% mortality at the field rate of 498 mg [AI]/L for both the preimaginal and adult toxicity tests. Use of T. ostriniae can provide effective control of O. nubilalis in pepper compared to conventional and organic spray regimes (Spinosad and methoxyfenozide respectively). Augmentative releases of T. ostriniae integrated with methoxyfenozide with its limited toxicity to both preimaginal and adult stages indicate that it is a superior candidate for control O. nubilalis. To determine the behavioral differences of T. ostriniae and its response to O. nubilalis in green bell peppers, experiments were carried out to classify likely areas of O. nubilalis oviposition within the green pepper plant canopy. A total of 426 O. nubilalis egg masses were found on pepper plants during our study. Over 92% of egg masses were found on the lower surface of the leaf compared with the upper surface indicating a significant ovipositional preference for the undersides of leaves in 2002 ( x2 = 9.68; df = 1; P < 0.05) followed by similar results in 2004 (x2 = 4.34; df = 1; P < 0.05). No significant differences were found in the observed spatial distribution of egg masses among the three vertical strata in either 2002 or 2004 (x2 = 1.75; df = 2; P < 0.05 and x2 = 5.69; df = 2; P < 0.05 respectively). Field release rates of 17.0 foraging T. ostriniae females can achieve 80.0% parasitism of O. nubilalis egg masses distributed throughout the pepper plant canopy found primarily on the undersides of leaves. These data demonstrate that T. ostriniae has potential as a biocontrol agent for O. nubilalis in solanaceous crops. / Ph. D.

The biology of Laricobius nigrinus Fender (Coleoptera: Derodontidae) and its potential for control of hemlock woolly adelgid (HWA), Adelges tsugae Annand (Homoptera: Adelgidae) in the eastern United States were investigated. Laricobius nigrinus completed development on HWA in laboratory studies. There are four larval instars. Mean larval consumption was 225.9 and 252.3 HWA eggs at 12 and 18 °C, respectively. Post-aestivation activity period was 36.6 and 30.8 weeks for males and females at 13 °C, respectively. Mean lifetime fecundity was 100.8 eggs over a mean ovipositional period of 13.2 weeks. Laricobius nigrinus is host specific. In paired-choice and no-choice oviposition tests, L. nigrinus laid more eggs in HWA ovisacs than other test species. Laricobius nigrinus consumed more eggs of HWA than eggs of Adelges piceae (Ratzeburg) and Pineus strobi (Hartig) but not of Adelges abietis (L.). In larval development tests, L. nigrinus only completed development on HWA. A two-year field study conducted in British Columbia showed that: 1) the life cycle of HWA in British Columbia is similar to that previously reported in Virginia and Connecticut; 2) L. nigrinus adults undergo an aestival diapause that coincides with the diapause of the first instar HWA sistens. Adult activity beginning in autumn coincides with resumption of development of the sistens generation; 3) oviposition and subsequent larval development of L. nigrinus coincide with oviposition by HWA sistens adults. Temperature-dependent development was determined for egg, larval, pre-pupal and pupal stages of L. nigrinus at five constant temperatures (9, 12, 15, 18 and 21 °C). Development time was inversely proportional to temperature between 9 and 18 °C. Laricobius nigrinus did not complete development at 21 °C. Minimum developmental temperatures for eggs (6.5 °C), larvae (5.1 °C), pre-pupae (3.8 °C), and pupae (3.1 °C) were determined by extrapolation of linear regression equations to the x-intercept. Degree-days calculated for eggs, larvae, pre-pupae, pupae, and egg to adult were 54.6, 161.3, 196.1, 212.8, and 666.7, respectively. The laboratory derived degree-day model for predicting egg hatch was validated with field data collected in British Columbia. Observed median egg hatch at three of four sites over two years was within 15% of the predicted DD value (54.6). / Ph. D.

The alfalfa weevil, Hypera postica (Gyllenhal) (Coleoptera: Curculionidae), remains a serious pest in Virginia despite being under complete biological control in the northeastern U.S. In 1996, a survey of 187 alfalfa fields in Virginia was initiated to determine the current pest status of alfalfa weevil and incidence of natural enemies. Fields located in the Piedmont region of the state had significantly higher alfalfa weevil pressure than those in the Shenandoah Valley and southwestern region. The dominant natural enemy of alfalfa weevil larvae was the parasitoid, Bathyplectes anurus (Thomson). Larval parasitization was lower in the Piedmont compared with the other regions. The entomopathogenic fungus, Zoophthora phytonomi, also killed weevil larvae in all regions. A more intensive ecological investigation of alfalfa weevil population dynamics and mortality was initiated in 1997 to determine why H. postica remains a pest in Virginia, particularly in the Piedmont region. Nine alfalfa weevil populations from three geographic locations were sampled and measured over a period of two years. Results showed that warmer winter temperatures in the Piedmont region contributed to a higher rate of alfalfa weevil oviposition compared with the Shenandoah Valley and southwestern mountains. Parasitization of adult weevils by Microctonus aethiopoides (Loan) was low in all regions of Virginia relative to rates reported in the northeastern U.S. This also contributes to relatively high alfalfa weevil fecundity in Virginia because a greater percentage of adults reproduce. Phenological asynchrony between M. aethiopoides and the alfalfa weevil may explain the low parasitism. The 1st generation of M. aethiopoides adults did not emerge until most of the overwintering alfalfa weevil adults had reproduced and died. A majority of alfalfa weevil eggs were laid before January at all locations in Virginia. Approximately half of the egg population survived to contribute to spring larval infestations. In the northern U.S., very few alfalfa weevil eggs survive the winter, and larval populations result primarily from spring-laid eggs. Mortality of larvae was high in Virginia and comparable to that reported in other states. Bathyplectes anurus was well-synchronized with alfalfa weevil and killed a high percentage of larvae. / Ph. D.

Bacterial secretion systems have been playing essential roles in modulating the microbiota of most ecological niches. Among a variety of secretion systems, the Type VI Secretion System (T6SS), a nanomachine widely distributed in Gram-negative bacteria, is gaining increasing attention due to its involvement in microbe-microbe and microbe-host interactions through secreting toxins into host cells, microbial competitors, and the extracellular milieu. Most secreted toxins, also known as T6SS effectors, have bacteriostatic effects upon delivery into competing bacteria, and therefore bacteria with potent T6SS may acquire competition advantage and represent promising biological control agents (BCAs). The main body of this dissertation will focus on the characterization of the T6SS of a phytopathogen, Acidovorax citrulli (strain AAC00-1), and the secreted T6 effectors, and will also discuss the possible application of AAC00-1 as a BCA. The seed-borne, gram-negative A. citrulli is able to cause bacterial fruit blotch (BFB) disease and then result in devastating decrease in yields of important cucurbits including watermelon, melon, squash and cucumber. Our inter-microbial competition assays demonstrate that AAC00-1 contains an active T6SS and presents a dramatic antimicrobial activity against a variety of microbes, including Gram-negative bacteria, Gram-positive bacteria, and yeast, dependent upon its T6SS. A group of novel non-enzymatic effectors, Hyde1 proteins, delivered into prey cells through the T6SS, are responsible for this broad-spectrum antimicrobial activity. Expressing Hyde1 or its N-terminal transmembrane domain shows significant toxicity in both E. coli and AAC00-1, and the toxicity of Hyde1 can be counteracted by its immunity protein, Hyde2. A non-pathogenic AAC00-1 strain suppresses the growth of multiple deleterious phytopathogens in planta and protects plant host. Transgenic plants expressing either full-length Hyde1 or its transmembrane domain demonstrate improved resistance against both bacterial and oomycete pathogens. Altogether, we characterize the T6SS killing of AAC00-1, identify the determinant effectors and discuss the application of both AAC00-1 and its T6SS effector in plant disease management. Additionally, in order to develop molecular tools better serving our T6SS-related studies, we successfully generate a series of salicylic acid (SA)-inducible vectors, functioning in A. citrulli, that can be used for inducible gene expression, protein purification and other applications. The core regulatory component that we employ, is a transcriptional regulator, Sal7AR-V295F, due to its responsiveness to salicylate. By cloning this fragment to a broad-host-range plasmid, in this study, we establish multiple SA-inducible vectors that may be used in most Gram-negative bacteria. When using the E. coli strain C41(DE3) as the expression host, protein purification can be conducted routinely, upon the addition of affinity tags to our vectors, such as the maltose-binding protein (MBP) tag. Combining the modified vectors with the robust NanoLuc binary Technology (NanoBiT), we are able to devise a novel bacteria two-hybrid system as an effective method to detect protein-protein interaction. Two complementary fragments of the NanoLuc protein, LgBiT and SmBiT, with extremely low affinity, are fused to potential interactors, and they will be brought into proximity and reconstitute NanoLuc bioluminescence upon the occurrence of interaction. This system is used in our T6SS study to validate the interaction between Hyde1 toxin and its cognate immunity protein. Another fragment, HiBiT, which automatically interacts with LgBiT and reconstitutes NanoLuc, is cloned to the SA-inducible vector as well, enabling us to generate a split-NanoLuc-based method, for the purpose of detecting secretion of tagged T6 toxins into the prey bacterial cells expressing LgBiT. Overall, our SA-inducible vectors and their further modifications enrich the molecular tool repertoire for T6SS-related studies. / Doctor of Philosophy / Effective crop disease management is critical for agricultural production. Chemical spray has been practiced as one major approach to control plant diseases for more than a decade. However, increase of pesticide application could threaten public health and the environment. Biological control has been considered as one of the effective and environmental-friendly alternative approaches for disease control. In this dissertation, we identify that, Acidovorax citrulli (strain AAC00-1), a Gram-negative pathogen causing bacterial fruit blotch (BFB) disease in Cucurbitaceae, could be a potential biological control agent (BCA), because it carries an active Type VI Secretion System (T6SS), and the T6SS has been shown to contribute to the protective effects of many plant-associated BCAs. T6S is believed to mediate inter-bacterial competition through secreting toxins into microbial competitors. Most secreted toxins, also known as T6SS effectors, have bacteriostatic effects upon delivery into competing bacteria, and therefore bacteria with potent T6SS may acquire competition advantage and represent promising BCAs. We demonstrate that AAC00-1 suppresses the growth of multiple phytopathogens, depending upon its T6SS. Expressing ten out of eleven microbial toxins, encoded by Hyde1 genes, in E. coli shows significant toxicity. The wild type AAC00-1 strain inhibits the growth of multiple Arabidopsis leaf bacterial isolates, while an AAC00-1 Hyde1 mutant loses this capacity. The antimicrobial activity of AAC00-1 is proven to be broad-spectrum since this strain also shows inhibitory effect on the growth of Gram-positive bacteria and yeast. In planta disease assay suggests that a non-pathogenic AAC00-1 mutant defective in Type III secretion system (T3SS) maintains its capacity to suppress disease symptoms and pathogen growth on plants infected with different phytopathogens. Our study demonstrates the viability of the employment of non-pathogenic A. citrulli as an effective BCA in plant disease management.

The squash bug, Anasa tristis De Geer (Hemiptera: Coreidae), is a serious pest of cucurbit crops across the US, especially within summer squash (Cucurbita pepo L.) systems. Using its piercing sucking mouthparts, squash bug feeds on both leaf tissue and fruits, potentially leading to leaf necrosis, seedling death, and yield loss. Currently, A. tristis management strategies in summer squash focus exclusively on insecticide applications. Given that continuous use of insecticides imposes negative side effects on many non-target species, the major objective of this dissertation was to identify new and understudied management strategies for minimizing A. tristis damage in Virginia summer squash. To accomplish this goal, we first performed experiments to test the effects of colored mulches on squash bug density. A two-year field replicated study conducted in 2019 and 2020 in southwest Virginia found that black, white, and reflective plastic mulch colors had no effect on squash bug adult, egg mass, or nymphal densities. Overall, we observed a broad plastic mulch effect, as summer squash plants grown in any plastic mulch color tended to harbor greater densities of squash bug life stages than plants grown on bare ground. Next, the potential of augmentative releases of egg parasitoid, Hadronotus pennsylvanicus Ashmead (Hymenoptera: Scelionidae) was assessed by deploying lab-reared parasitoids on organic farms growing summer squash in southeastern Virginia in 2020 and 2021. In both years, we found higher levels of A. tristis egg parasitism at H. pennsylvanicus release sites compared to sites where the parasitoids were not released. Further, higher egg parasitism at the release sites was observed within two weeks of the field deployment date. The third goal of this dissertation research was to characterize the relationship between squash bug life stages and marketable summer squash yield. In 2020 and 2021, summer squash fields were established in Whitethorne, Virginia and individual plants were labeled as either 'managed' or 'infested'. Managed plants were subject to weekly manual removal of all squash bug egg masses and nymphs, while infested plants were left to have natural densities of squash bug life stages. We performed weekly A. tristis surveys on all infested plants in both years and added H. pennsylvanicus surveys on infested and managed plants in 2021. Also, we collected all fruit produced by each plant. Managed plants were found to produce more marketable fruit and fewer squash bug-damaged fruit than infested plants. Linear regression analyses of initial two-week accumulated squash bug life stage counts displayed adult and nymph densities to be significantly associated with marketable fruit yield per plant. However, nymphs showed a considerably stronger relationship with marketable yield than did adults. Hadronotus pennsylvanicus was visually sampled during each of the eight sampling weeks, but did not exhibit high egg parasitism until the latter half of the sampling interval. Lastly, we evaluated the toxicity of reduced risk insecticides on A. tristis nymphs and H. pennsylvanicus adults. To do so, we conducted laboratory bioassays exposing nymphs to insecticide treated summer squash fruit and parasitoids to treated filter paper, and recorded mortality at 24, 48, and 72 h following initial exposure. Only one reduced risk insecticide, flupyradifurone, was found to be highly toxic to squash bug nymphs and nontoxic to the parasitoids. Overall, the results of these studies will offer much needed information to improve the success and sustainability of squash bug management programs, as well as provide a fundamental basis and spark motivation for future research in squash bug biological control. / Doctor of Philosophy / The squash bug is a serious insect pest of summer squash across the United States. Squash bugs inflict damage by feeding on plant leaves, stems, and fruits, leading to significant reductions in fruit yield and overall economic losses for growers. To date, farmers have few useful tools to minimize squash bug damage in their summer squash crop, and resort to over-applying insecticides. While this use of insecticides helps lower squash bug numbers, it often has negative side effects on the environment and important beneficial insect species like pollinators. To better understand the squash bug as a summer squash pest and to promote nonchemical ways to reduce squash bug damage, this dissertation research pursued answers to questions regarding: (1) the effect of plastic mulch color on squash bug numbers, (2) the use of squash bug natural enemies to control squash bugs, (3) the relationship between numbers of squash bugs and damaged summer squash fruits, and (4) whether new types of insecticides can control squash bugs and also be safe for beneficial insects. The results of this research will provide new insights on current issues with squash bug pest management. In particular, this work will provide summer squash farmers with useful knowledge that can be applied to their future efforts to responsibly control squash bugs in their fields and maximize their crop yields. With more research-supported and sustainable squash bug control tools, farmers will be able to not only adopt more environmentally-friendly food production practices, but also lower their overhead farm management costs and ultimately provide higher quality squash to consumers.

The brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), is an invasive agricultural and nuisance pest in the United States and an increasing number of countries abroad. In the absence of other long-term solutions, biological control presents one of the most promising H. halys management options. An adventive population of Trissolcus japonicus (Ashmead) (Hymenoptera: Scelionidae), one of the most effective egg parasitoids of H. halys in their shared native range in Asia, was discovered in the Maryland in 2014 and in Frederick County, VA in 2015. Adventive populations of T. japonicus have also been detected recently in several other states and show strong indications of ongoing range expansion. Despite their importance, the foraging ecology of H. halys and especially of T. japonicus in the USA need further study. Small pyramid traps baited with a low-dose H. halys aggregation pheromone lure and deployed as vertical transects in the canopy of female tree of heaven (Ailanthus altissima (Mill.) Swingle), male tree of heaven, or hackberry (Celtis occidentalis L.), captured significantly more H. halys adults and nymphs near the top of the canopy than at mid- or lower canopy, regardless of host plant. The majority of H. halys egg masses collected via destructive sampling of felled tree of heaven were from mid-canopy, and the majority of those parasitized by T. japonicus were also from mid-canopy, with no T. japonicus emergence from egg masses collected from the lower canopy. A comparison of sentinel H. halys egg masses and yellow sticky traps deployed as vertical transects in tree of heaven revealed that the majority of T. japonicus detections occurred at mid-canopy and that while both methods proved to be equally effective, yellow sticky traps were more efficient for this purpose. This led to the development of a novel T. japonicus sampling method in which a yellow sticky trap is deployed atop a bamboo pole in the mid-canopy of wild host trees. This method was employed to compare the effect of habitat type on T. japonicus captures in female tree of heaven growing in spatially isolated patches, thin windbreaks, and at the edge of contiguous woodlots. Consistent habitat effects on T. japonicus detections were not found between sampling years, but provided the first documentation of the seasonal activity of T. japonicus in the USA. A study of host plant effects on T. japonicus detection in mid-canopy yellow sticky traps did not reveal consistent host plant effects on captures. These data provide important insights into the foraging ecology of H. halys and T. japonicus in the USA, as well as efficient sampling methods for future studies of T. japonicus. / Doctor of Philosophy / The brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), is an invasive agricultural and nuisance pest from Asia that is widely distributed in the United States and is present in an increasing number of countries abroad. In the absence of other long-term management solutions, biological control via the actions of natural enemies presents a promising brown marmorated stink bug management option. Trissolcus japonicus (Ashmead) (Hymenoptera: Scelionidae), also known as the samurai wasp, is one of the most effective egg parasitoids of brown marmorated stink bug in Asia. Samurai wasp arrived in the USA without deliberate introduction and was first detected in Maryland in 2014 and subsequently in Frederick County, VA in 2015. It has continued to expand its range in the USA and was recently detected in several eastern and western states. Despite their importance, little is known of where brown marmorated stink bug eggs, nymphs, and adults are most likely to be found in wild trees. Even less is known about where samurai wasp is likely to occur within its new range. To address these issues, traps containing a lure to attract brown marmorated stink bug were placed in the upper, middle, and lower canopy of female tree of heaven (Ailanthus altissima (Mill.) Swingle), male tree of heaven, and hackberry (Celtis occidentalis L.). Significantly more brown marmorated stink bug adults and nymphs were caught in traps near the top of trees than in those at other canopy locations, with no effect of host plant species observed. The majority of brown marmorated stink bug egg masses collected from felled tree of heaven were from the middle of the canopy. The majority of samurai wasps emerged from egg masses collected from the mid-canopy, while none emerged from those collected from the lower canopy. In a comparison of sentinel brown marmorated stink bug egg masses and yellow sticky traps placed in the upper, middle, and lower canopy of tree of heaven, the majority of samurai wasp detections occurred at mid-canopy. Both methods were found to be effective, but yellow sticky traps required less time and effort to use. This led to the development of a new sampling method for samurai wasp, in which a yellow sticky trap was deployed atop a bamboo pole in the mid-canopy of wild host trees. Using this technique, the effect of habitat type on samurai wasp captures was examined, revealing no consistent effects on detections. However, this study provided the first documentation of when samurai wasps are present and most abundant during the spring and summer. A study of host plant effects on samurai wasp captures, also using yellow sticky traps placed in the mid-canopy of trees, did not reveal consistent host plant effects on captures. These data provide important insights into the interactions of brown marmorated stink bug, samurai wasp, and their environment in the Mid-Atlantic region of the USA, as well as an efficient tool with which to study the samurai wasp.