Agrilus planipennis, the emerald ash borer (EAB), is a destructive invasive forest pest decimating North American ash trees. Population-wide management of EAB focuses on biological control, with the introduction of four parasitic wasps; one egg parasitoid, Oobius agrili and three larval parasitoids- Spathius galinae, Spathius agrili and Tetrastichus planipennisi. This thesis examines some of the factors influencing the establishment of these larval biocontrol agents. Chapter 1 examines the relationship between woodpeckers and the parasitoids S. agrili and T. planipennisi. Both woodpeckers and these parasitoids attack the larval stage of EAB, which means their impacts overlap and potentially interact. To examine this relationship, I established parasitized larvae on ash trees and then used screening to exclude woodpeckers from some sections of the tree. Results show that while there is no evidence of discriminatory feeding for or against parasitized larvae, the presence of parasitized larvae changes woodpecker feeding behavior at a stand-level. I hypothesize that this change is due to these larval parasitoids being a low-food reward and that parasitism contributes to a change and decrease in patch quality, causing woodpeckers to quit foraging sooner than usual. My second chapter focuses on Spathius galinae, which was recently approved for release in the north central and northeastern US in 2015, to provide additional population control. Spathius galinae’s long ovipositor (4-5.3mm) is theoretically expected to help target EAB in ash with larger diameters and bark thicknesses. Using experimentally infested logs of varying thicknesses in the laboratory I tested the limits and preferences for oviposition of S. galinae, to understand its potential impact on EAB. My results demonstrated that although parasitism by S. galinae drops significantly when bark thickness reaches 8 mm, this prevents S. galinae only from reaching EAB larvae in my largest ash trees (S. galinae will play a vital role in providing additional control and in supporting ash regeneration in aftermath areas of EAB invasions.

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Full thesis document accompanied by all data files and R scripts to reproduce analyses in both data chapters (2 & 3). / Animals should favour breeding locations that maximize their lifetime reproductive output. Parents ought to rear young in sites that positively affect offspring but sometimes these same sites inflict costs on parents. How parents balance their own needs against those of their offspring when selecting a site for reproduction remains unclear, particularly in animals that provide extended parental care at one location. Further, few studies have linked variation in life history traits to reproductive site choices within a single species. In this thesis, I addressed both knowledge gaps by studying the plainfin midshipman (Porichthys notatus), a marine toadfish where fathers provide sole parental care to broods of young in intertidal and shallow subtidal nests for up to two months. In Chapter 2, I measured the costs incurred by caring males and the benefits conferred to offspring in nest sites along a tidal gradient. Males suffered similar rates of body condition deterioration in all nests across the intertidal gradient. Young developed more quickly in intertidal nests compared to subtidal nests, but broods in the highest intertidal nests suffered the highest mortality rates, despite receiving more parental care from the males at these nest sites. We found the most competitive males in lower intertidal nests, a trend that agrees with life history theory—in species with relatively slow offspring development, parents should accrue greater reproductive benefits from nest sites where offspring benefits are highest. In Chapter 3, I describe a laboratory experiment designed to examine how warm water and air exposure (two abiotic conditions that vary dramatically within the intertidal) affect development and survival of plainfin midshipman young, and how these effects vary with maternal traits. Exposure to warm water enhanced embryonic and larval development rates; this effect was attenuated by air exposure. Juveniles raised in warm water also exhibited superior swimming performance, while air-exposed young suffered higher mortality rates. Although larger juveniles emerged from larger eggs, development rates were similar across egg sizes. Offspring survival increased with maternal body condition in cold water but decreased with maternal body condition in warm water. Juvenile body sizes increased with maternal condition in cold water without emersion, and in warm water with emersion—the two ecologically relevant rearing environments. Thus, low condition mothers might accrue greater benefits by depositing their eggs in nests at higher tidal elevations—where development is more rapid—further supporting the idea that among-individual variation in the expression of life history traits might influence nest site preferences in these fish. In sum, my research (Chapters 2 & 3) elucidates the link between life history traits and the spatial component of animal reproductive strategies. / Thesis / Master of Science (MSc)

The brown marmorated stink bug, Halyomorpha halys (Stål), is an invasive species that has spread throughout the United States and become a serious agricultural pest. Studies were conducted in Virginia on the survival of H. halys nymphs on different vegetable hosts and on the oviposition preference of H. halys on common tree hosts in southwestern Virginia. To determine the effects of vegetable host on H. halys survival, nymphs were placed on one of five vegetable host plants and their corresponding vegetable fruit. Survival was highest on sweet corn, followed by bell pepper and snap bean. Eggplant and tomato were poor compared to other vegetable hosts tested, potentially due to secondary compounds released by the plant to reduce feeding. Of six common host trees sampled, H. halys laid the most egg masses on catalpa, Catalpa bignonioides, while aggregation lure had no effect on the number of eggs laid. Across host trees, egg masses were more commonly found in trees with fruiting structures present. An additional study was conducted on a red-sternum phenotype of H. halys. Mortality was recorded by sternum phenotype for each sex. There was no difference in survival between groups at two time points. Biochemical tests were used to determine if the red-sternum was associated with the potentially pathogenic red bacteria Serratia marcescens. Only one stink bug's gut contents had a confirmed infection of S. marcescens. Results from these studies offer insight into H. halys development on vegetable hosts and oviposition preferences on different host trees. / MSLFS / The brown marmorated stink bug (BMSB), is an invasive insect that has spread throughout the United States and become a serious pest. Current research is focused on reducing their populations without increasing insecticide use. Studies were conducted to determine if vegetable host type influenced survival and development of this insect. Nymphs were placed on one of five vegetable plants with the corresponding vegetable fruit. Overall, survival was highest on sweet corn, followed by bell pepper and snap bean. Eggplant and tomato were poor hosts, potentially due to chemicals released by the plant to reduce insect feeding. Six common host trees of BMSB were sampled to determine where females lay the most egg masses and the effect of using an attraction lure. Catalpa trees had the most egg masses throughout the season. Use of a lure had no effect on the number of eggs laid on any tree. A red colored abdomen was observed in some adult insects throughout these studies. To determine if this was associated with higher mortality, male and female insects were observed for two weeks. There were no mortality differences at either seven or 14 days between any of the groups. Tests were used to determine if the red abdomen was caused by a red-colored bacterium. Of the insects sampled, there was only one stink bug with a confirmed infection. Results from these studies provides information that increases knowledge of this pest’s ecology in Virginia.

Field studies were conducted in southwestern Virginia to determine the bionomics and ovipositional preferences of Culex restuans Theobald and Culex pipiens Linnaeus using ovitraps and gravid traps. Both species have been implicated as enzootic and epizootic vectors of West Nile virus (WNV) and these studies provide information on the relative abundance of gravid mosquitoes. Ovitraps were used in the summers of 2002 and 2003 to measure the oviposition activity of Culex mosquitoes, mainly Cx. restuans and Cx. pipiens. In 2002, 1,345 egg rafts were collected from 5 traps set at different locations in the New River Valley (NRV). Cx. restuans constituted 93.2% of the catch; the remainder was Cx. pipiens (6.7%) and Cx. salinarius (<1%). In 2003, 4 ovitraps were placed at each of 6 locations in the NRV. Of 9,794 egg rafts collected, Cx. restuans comprised 92.8%, Cx. pipiens 6.5%, and Cx. salinarius <1%. Oviposition patterns were similar in both years. Cx. restuans oviposition was detected about mid-May, and raft numbers were highest in late June and the middle of July and then showed a steady decline throughout the remainder of each season. Cx. pipiens oviposition activity began later in the season and gradually increased, reaching its peak in August. Although the number of egg rafts of Cx. restuans decreased in August and September while the number of Cx. pipiens egg rafts increased, a crossover in the relative abundance of the two species never occurred. In 2003, the attractiveness of four infusions (cow manure, straw, grass, and rabbit chow) were compared in oviposition traps. For Cx. restuans, the manure infusions collected more egg rafts than the other three infusions for the first four weeks, with two of the weeks showing significance. During week 1, the manure infusions collected significantly more egg rafts than straw (P<0.01), grass (P<0.01), and rabbit (P<0.001). During week 2, manure collected more than grass (P<0.05) and rabbit (P<0.01). The straw and grass infusions yielded the most egg rafts after week 2, and only three weeks showed any significance. When traps began to collect Cx. pipiens, the majority were collected in the straw and grass infusions. During week 6, the hay infusions had significantly more egg rafts when compared to manure (P<0.001) and rabbit infusions (P<0.001) and the grass infusions had significantly more when compared to manure (P<0.05) and rabbit (P<0.01). Week 9 also showed significance when the hay infusions collected significantly more egg rafts than manure and rabbit (P<0.01 and P<0.01, respectively). The attractiveness of the cow manure and straw infusions were also compared in gravid traps. Because it is difficult to accurately discriminate between Cx. pipiens and Cx. restuans that have been collected as adults in gravid traps, these collections were combined into Culex. More Culex mosquitoes were collected in the manure infusions for the first two weeks (P<0.05). No significant differences were found between the numbers of mosquitoes captured in the traps baited with the different infusions after the second week. The shift observed in oviposition preference for both types of traps may have been due to cooler temperatures in the early part of the season. The straw infusions were aged outdoors for 3 days prior to use, and a sufficient incubation temperature to generate the bacteria producing the attractant chemicals may not have been attained. In addition, the manure lagoon had been drawn down and the consistency of the remaining manure became much drier by this time. No chemicals were ever added to the lagoon, but the draw down may have affected the attractiveness of the manure. / Master of Science

White grubs (Coleoptera: Scarabaeidae) are important turfgrass pests in Virginia. Insecticides such as the neonicotinoid imidacloprid are commonly applied to turfgrass in order to control these pests. As an alternative to synthetic insecticides, entomopathogenic fungi (EPF), including Metarhizium brunneum (Petch) and Beauveria bassiana (Balsamo) Vuillemin may also be used for white grub control. The interaction of combining these two control tactics for white grubs in Virginia merits further investigation as does their effects on other soil organisms such as earthworms, which cohabitate with white grubs in turfgrass soil ecosystems. Herein, I investigate the following: 1) the efficacy of combined applications of the EPF, M. brunneum and B. bassiana with lower rates of imidacloprid or the diamide insecticide, chlorantraniliprole against white grubs; 2) interactions of earthworms with white grubs and EPF; and 3) the effect of white grub control products on earthworms. In the laboratory, a combined application of one half the recommended rate of chlorantraniliprole plus the full recommended rate of B. bassiana caused significantly higher mortality of third instar Cyclocephala spp. grubs than the untreated control. In the field, imidacloprid applied at lower rates as a single treatment or as part of a combined treatment with EPF resulted in significantly fewer grubs when applications were made in June. In the greenhouse, Japanese beetle, Popillia japonica Newman females laid a significantly reduced number of eggs in turf treated with lower rate of imidacloprid either applied as a single treatment or as part of a combined treatment compared with untreated control. In an earthworm-white grub interaction study, the earthworms Eisenia fetida (Savingy) and E. hortenis (Michaelsen) were shown to transfer B. bassiana spores from fungus-infected soil to uninfected soil in the laboratory. However, the presence of earthworms in fungal infected soil did not enhance the mortality of Cyclocephala spp. grubs. In bioassays conducted in the laboratory, only two neonicotinoids, dinotefuran and imidacloprid, caused significantly higher mortality to adult Lumbricus terrestris L. earthworms than untreated control consistently. When applied as a drench to turfgrass in spring, summer, and fall, none of the insecticides significantly reduced the earthworm densities compared with a water control. / Ph. D. / White grubs cause serious damage to turfgrass in Virginia and as a part of turf management programs, insecticides such as the neonicotinoid imidacloprid are routinely applied to eliminate these pests. However, there are environmental concerns over the excessive use of neonicotinoids. In addition, these insecticides are typically only effective when they are applied in the summer to target small grubs. Herein, I investigated if combining reduced rates of imidacloprid or another insecticide, chlorantraniliprole, with commercially-available insect-killing fungi <i>Beauveria bassiana</i> or <i>Metarhizium brunneum</i>, could enhance white grub control. Neither the insecticides alone, nor the aforementioned fungal biopesticides provided effective control of larger white grubs in lab bioassays and field experiments. Additional research was aimed at understanding the interactions of insecticides and fungi on both white grubs and earthworms, which cohabitate frequently with white grubs in turfgrass soil ecosystems. In a greenhouse experiment, Japanese beetle females laid fewer eggs in turf treated with imidacloprid, but that chlorantraniliprole or the insect disease causing fungi did not affect beetle egg laying. In another experiment, earthworms were shown to transfer insect disease causing fungi in the soil in the laboratory. However, the presence of earthworms did not increase white grub fungal infection in fungi-infested soil in the lab. Additional experiments showed that two neonicotinoids, dinotefuran and imidacloprid, killed more earthworms than other insecticides when applied to soil in the lab. However, when applied as a drench to turfgrass in spring, summer, or fall, neither of these insecticides nor several others registered for use on turfgrass resulted in fewer earthworms compared with a water control. The complex interactions between turf-damaging white grubs, the insecticides used to control them, insect disease-causing fungi in the soil and non-target beneficial organisms such as earthworms warrant further investigation to help us move toward more sustainable pest management approaches in the future.

Fire may alter both aquatic and terrestrial habitat used by all amphibian life stages, yet, our knowledge of its effects on amphibians is primarily limited to adult responses. I present an integrated approach to test the response of Cope’s Gray Treefrog (Hyla chrysoscelis) to fire by examining responses in tadpole performance and survivorship, adult abundance, and oviposition. Tadpoles raised with burned leaf litter had similar survival, but total mass and total length were 440% and 170% greater, respectively, for tadpoles raised in unburned litter. I assessed terrestrial and aquatic oviposition cues by embedding burned and unburned litter treatments within burned and unburned terrestrial plots. Oviposition was an order of magnitude higher in unburned plots, regardless of the litter treatment. This difference was not statistically significant or driven by adult abundance. My results indicate the need to explore the dynamic effects forest management practices can have on amphibians across life stages.

Increasing temperatures resulting from climate change have within recent years been shown to advance phenological events in a large number of species worldwide. Species can differ in their response to increasing temperatures, and understanding the mechanisms that determine the response is therefore of great importance in order to understand and predict how a warming climate can influence both individual species, but also their interactions with each other and the environment. Understanding the mechanisms behind responses to increasing temperatures are however largely unexplored. The selected study system consisting of host plant species of the Brassicaceae family and their herbivore Anthocharis cardamines, is assumed to be especially vulnerable to climatic variations. Through the use of this study system, the aim of this thesis is to study differences in the effect of temperature on development to start of flowering within host plant species from different latitudinal regions (study I), and among host plant species (study II). We also investigate whether different developmental phases leading up to flowering differ in sensitivity to temperature (study II), and if small-scale climatic variation in spring temperature influence flowering phenology and interactions with A. cardamines (study III). Finally, we investigate if differences in the timing of A. cardamines relative to its host plants influence host species use and the selection of host individuals differing in phenology within populations (study IV). Our results showed that thermal reaction norms differ among regions along a latitudinal gradient, with the host plant species showing a mixture of co-, counter- and mixed gradient patterns (study I). We also showed that observed differences in the host plant species order of flowering among regions and years might be caused by both differences in the distribution of warm days during development and differences in the sensitivity to temperature in different phases of development (study II). In addition, we showed that small-scale variations in temperature led to variation in flowering phenology among and within populations of C. pratensis, impacting the interactions with the butterfly herbivore A. cardamines. Another result was that the less the mean plant development stage of a given plant species in the field deviated from the stage preferred by the butterfly for oviposition, the more used was the species as a host by the butterfly (study IV). Finally, we showed that the later seasonal appearance of the butterflies relative to their host plants, the higher butterfly preference for host plant individuals with a later phenology, corresponding to a preference for host plants in earlier development stages (study IV). For our study system, this thesis suggest that climate change will lead to changes in the interactions between host plants and herbivore, but that differences in phenology among host plants combined with changes in host species use of the herbivore might buffer the herbivore against negative effects of climate change. Our work highlights the need to understand the mechanisms behind differences in the responses of developmental rates to temperature between interacting species, as well as the need to account for differences in temperature response for interacting organisms from different latitudinal origins and during different developmental phases in order to understand and predict the consequences of climate change. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript. Paper 4: Manuscript.</p>