Roughly 79-100% of maize in the United States (US) is treated with a neonicotinoid seed treatment (NST), and transgenic (GMO) maize, Zea mays L. (Poaceae), that produces insecticidal toxins by way of genes derived from Bacillus thuringiensis (Bt), occupies more than 75% of maize acreage. Among a variety of secondary pests targeted by NSTs, the primary soil-dwelling pest targeted by Bt maize is the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae). Transgenic Bt technology has dramatically reduced insecticide use for WCR, and insect resistance poses the greatest threat to its utility. To delay resistance to Bt traits, in 2010 the US Environmental Protection Agency (EPA) approved a "refuge-in-a-bag" (RIB) insect resistance management (IRM) strategy, where 5% of seeds do not express Bt toxins (i.e., "refuge" maize). The RIB strategy is intended to preserve Bt trait effectiveness if mating between 'resistant' insects from Bt plants and 'susceptible' insects from refuge plants occurs at a high enough frequency. Investigations into the effectiveness of RIB for WCR have shown that beetles emerged from Bt plants tend to vastly outnumber beetles emerged from refuge plants, which contributes to low rates of mixed mating. Large proportions of Bt beetles is one of several factors that contributes to resistance development, and resistance to all currently-available WCR-Bt traits has been documented. I conducted field experiments in two regions (Indiana and Virginia) comparing refuge beetle proportions in NST-treated (NST+) and NST-untreated (NST-) 5% RIB maize, to determine whether NSTs may be limiting refuge beetle emergence. To assess advantages of combining use of Bt and NSTs, I compared stand, root injury rating, and yield between NST+, NST-, Bt and non-Bt maize in both states. I also measured neonicotinoid residues in soil, water, and stream sediment within and surrounding fields of maize, to study the off-site movement and soil residence time of these compounds. I found that 5% seed blends did not produce large populations of refuge beetles in any site-year, and that NSTs showed inconsistent effects on refuge beetle populations. Treatment comparisons showed inconsistent benefits of NSTs when combined with Bt traits. I detected neonicotinoid residues in soil matrices throughout the growing season (range: 0 – 417.42 ppb), including prior to planting, suggesting year-round presence of these compounds. My results suggest that, while the effects of NSTs on Bt IRM may be inconsistent, the benefits of universally applying NSTs to Bt maize for soil pests may not be worth the ecological costs of doing so in all cases. / Doctor of Philosophy / About 75% of maize planted in the United States is encoded with genetic traits allowing them to manufacture insecticidal proteins which are toxic to key pests. These insecticidal proteins, known as "Bt toxins," are highly specific to targeted insects, and are virtually non-toxic all other animals and non-target insects. One target insect is the western corn rootworm (WCR), which feeds on and damages maize roots. In the past, WCR was controlled by applying millions of kilograms of chemical insecticides every year, at ever-increasing rates, to compensate for reduced effectiveness due to pest resistance. "Bt" plants, were released for commercial use in the late 1990s; they reduced the need for growers to apply chemical insecticides for managing key pests. The Environmental Protection Agency established regulations aiming to maintain the effectiveness of Bt technology, which they declared have "provided substantial human health, environmental, and economic benefit." This Insect Resistance Management (IRM) strategy, also known as the "refuge" strategy, is meant to preserve Bt-susceptible insects so they can pass on their Bt-susceptible genes to successive generations. The refuge strategy works by incorporating a certain percentage of non-Bt plants per every field of Bt plants; if enough insects survive on these "refuge" plants to outnumber "resistant" insects, population-wide Bt-susceptibility may be maintained. While this strategy has been effective for other key maize pests, it is failing for WCR, evidenced by WCR populations that have developed Bt-resistance. We know current refuge configurations aren't producing enough refuge-WCR to mix sufficiently with resistant WCR, likely due to insufficient refuge sizes. I wanted to know whether the refuge strategy could be improved, if increasing refuge proportions is not an option. Considering that Bt toxins are only effective against a narrow range of insects, seed manufacturers always coat seeds with an insecticide to protect seedlings against other insects. These coatings, or neonicotinoid seed treatments (NSTs), are included on nearly all seed, including those reserved for Bt refuges. I conducted two years of field trials to investigate whether removing NSTs from refuge seeds would improve WCR-IRM by providing an insecticide-free "refuge." My results suggest that removing NSTs may increase refuge beetle proportions, however my results also show that refuge plant proportions are simply too small to support large enough refuge-WCR populations to delay resistance, regardless of whether NSTs are present or not. While NSTs may provide extra comfort to growers at little additional cost, questions regarding their necessity at current use patterns have been raised. Several studies have shown inconsistent benefits, and others have shown longer-than-expected persistence in the environment, movement into streams and groundwater, and even alterations to insect and non-insect animal communities associated with their use. I conducted research comparing their relative effectiveness against WCR and non-WCR insect pests in fields of Bt maize. Additionally, I scouted for their residues in soil collected in field margins, forests buffering streams, and in water collected from agricultural ditches and waterways neighboring fields. I found that, while NSTs produced higher plant populations, they rarely resulted in greater yields. I detected neonicotinoid residues in soil matrices throughout the growing season, including prior to planting, suggesting year-round presence of these compounds. My results suggest that, while the effects of NSTs on Bt IRM may be inconsistent, the benefits of universally applying NSTs to Bt maize for soil pests may not be worth the ecological costs of doing so in all cases.

La plupart des toxines de Bacillus thuringiensis perméabilisent la membrane intestinale des insectes sensibles en formant des pores qui abolissent le potentiel électrique et les gradients ioniques. Plusieurs toxines ont été étudiées avec des vésicules purifiées de la bordure en brosse intestinale des insectes. Malheureusement, la membrane intestinale de beaucoup d’insectes ne forme pas des vésicules suffisamment étanches pour les expériences de perméabilisation. Une nouvelle technique utilisant des liposomes géants et une sonde de perméabilité membranaire a été développée pour caractériser deux nouvelles toxines particulièrement prometteuses pour le biocontrôle d’un des principaux ravageurs du maïs, la chrysomèle des racines du maïs (Diabrotica virgifera virgifera LeConte), Cry6Aa1 et la toxine binaire DS10/DS11. Les deux toxines perméabilisent efficacement les liposomes. La toxine binaire forme des pores qui sont légèrement sélectifs pour les cations, comme la plupart des toxines de B. thuringiensis. Bien que la Cry6Aa1 puisse former des pores sélectifs pour les anions, les résultats suggèrent aussi qu’elle pourrait, contrairement aux autres toxines de cette bactérie, ne former des pores qu’en présence d’une force ionique élevée. La formation des pores par ces deux toxines semble être sensible à la courbure de la membrane cible étant donné qu’elle est beaucoup plus efficace dans des liposomes géants que dans des liposomes de même composition, mais plus petits. Ce travail jette les bases de la mise au point d’une technique qui permettrait l’étude des toxines dans des liposomes géants enrichis avec des protéines et des lipides provenant de la membrane intestinale des insectes cibles. / Most Bacillus thuringiensis toxins permeabilize the intestinal membrane of susceptible insects by forming pores that abolish transmembrane electrical potentials and ionic gradients. Several toxins have been studied using brush border membrane vesicles purified from the insect midgut. Unfortunately, the intestinal membrane from many insects does not form vesicles that are tight enough to be used in permeabilisation experiments. A new technique using giant liposomes and a membrane permeability probe was developed to evaluate the pore-forming ability of two particularly promising toxins for the biocontrol of a major corn pest, the Western corn rootworm (Diabrotica virgifera virgifera LeConte), Cry6Aa1 and the binary toxin DS10/DS11. Both toxins permeabilized the liposomes efficiently. However, analysis of the permeabilisation rates under different experimental conditions indicates that these toxins differ in their biophysical properties. The binary toxin forms pores which are slightly selective for cations, like most B. thuringiensis toxins. On the other hand, although the results suggest that Cry6Aa1 could form anion-selective pores, they could also indicate that, in contrast with other toxins produced by this bacterium, it could form pores only under high ionic strength conditions. Pore formation by both toxins appears to be sensitive to membrane curvature since it is much more efficient in giant liposomes than in liposomes with identical composition, but smaller in size. This study sets the bases for the development of a technique that would allow the toxins to be studied in giant liposomes enriched with proteins and lipids from the intestinal membrane of target insects.