Laboratory and field studies of Hydrotaea aenescens were undertaken to provide information on this predator's biology and ecology under differing conditions, and to promote its use as a biocontrol agent for controlling house flies in poultry houses. Taxonomic diversity of arthropods in a new high-rise cage layer poultry house was determined from manure samples collected from different manure moisture categories. Stabilization of taxonomic diversity and taxonomic evenness of manure-inhabiting arthropods occurred after the house had been operating for eight months. In contrast, overall diversity in high-rise poultry houses at a well managed, longer established farm was significantly higher than that observed at the new farm, even after 1 1/2 years of operation. When the densities of selected manure-inhabiting predatory arthropods collected from the manure samples (including Carcinops pumilio, pseudoscorpions, a dermapteran species, an anthocorid species, and H. aenescens) were correlated with percent manure moisture, the results showed that, of these predators, only H. aenescens was positively correlated with both manure moisture and with densities of house fly larvae. This information emphasized that although predators such as C. pumilio may exhibit high predation rates on house fly eggs and first instars, their effectiveness 1s reduced by their spatial separation from their supposed prey. This contrasts greatly with H. aenescens performance. Decreases in survival of house fly larvae occurred when the larvae were exposed to H. aenescens of higher larval stadia. This was dramatically demonstrated when 100 first instar house flies were exposed to 100 second instar H. aenescens. No house fly larvae survived. Developmental times were determined at constant temperatures for egg, and larval H. aenescens. Developmental times decreased as temperature increased. Median time for egg and larval development ranged from 1.3 and 14.6 days at 22.2°C to 0.5 and 8.3 days at 35.0°C, for the respective stages. All of this information, together with developmental times and mortality of H. aenescens immatures gathered in a study of temperature dependent development has enhanced understanding of the biotic interactions in accumulated poultry manure. These data will be invaluable in designing integrated pest management programs especially in the area of computer-aided decision making. / Ph. D.

The pine bark adelgid, Pineus strobi (Hemiptera: Adelgidae) is a native herbivore of eastern white pine, Pinus strobus (Pinales: Pinaceae), in eastern North America. It is a sessile insect that settles on P. strobus and inserts its stylet bundle to feed on the tree’s phloem. Although P. strobi is not considered a serious pest, it shares its range with the invasive hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae). Predators introduced as biological control agents of A. tsugae interact with P. strobi and its native predators, including Laricobius rubidus LeConte (Coleoptera: Derodontidae). Prior to this study, little work had been done to document the phenology or predators of P. strobi, particularly in its southern range. In the present study, the phenology of P. strobi is reported in southwestern Virginia. Patterns in overwintering population dynamics varied notably from those described from this species’ northern range. The number of annual generations could not be measured due to overlap following two distinct spring generations. Adult body size varied seasonally and was greatest in the spring. Variation between observations from the northern and southern ranges of P. strobi indicate phenological plasticity that informs biological control efforts and offers insight into implication of climatic effects on population dynamics of this and related species. Arthropod predators associated with P. strobi in forests of southwest Virginia were collected during a two-year survey. Morphology and DNA barcoding were used for identification. Species of predators found included: Laricobius rubidus (Coleoptera: Derodontidae), a native adelgid specialist, and two species from the dipteran family Chamaemyiidae, Leucopis piniperda Malloch and L. argenticollis Zetterstedt, which are adelgid specialists. Members of the families Cecidomyiidae, Coccinellidae, Chrysopidae, Hemerobiidae, and Syrphidae were also recovered. Most diverse were the Cecidomyiidae, with 15 different species inferred from their DNA barcodes. Additional work was performed to quantify supercooling points of L. rubidus collected from November – December 2016. These will be compared to those of other Laricobius species in a parallel study. Knowledge of this predator complex is beneficial to describing P. strobi ecology, and also with regard to potential biological control of invasive adelgids in the same region. / Ph. D.

Successive releases of male German cockroaches heterozygous for the translocation T(8;9) and the recessive deleterious gene sty were made into laboratory populations of Blattella germanica to study the effect of this genetic load on population growth. Five experimental populations and one control were followed by monthly census for eight months. Individual progeny groups were held in separate containers. Various aspects of reproductive behavior of different genotypes were evaluated in separate experiments including fecundity, competitiveness and assortative mating. Linkage between T(8;9) and sty was also measured. The translocation T(8;9) and sty were successfully introduced into the experimental populations by the introduction of heterozygous males. Experimental populations were only 50% the size of the control at the F₁ generation and only 25% as large by the F₂ generation. At the F₃ generation, experimental populations were only 15.4% as large as the control. Cross connected contingency table analyses of population data showed a significant retardation of population growth from releases of T(8;9) sty males. T(8;9) provided a successful mechanism for driving the deleterious gene, sty, into the normal segment of the population. The frequency of sty/sty females in F₂ groups was predicted at 0.20. Observed frequencies approached the expected frequency at 0.11 to 0.22. This deleterious gene provided additional genetic load by reducing possible F₃ progeny by approximately 15%. Linkage tests of T(8;9) with sty showed cross-over frequencies of less than 1% for both males and females. Results were less than optimum due to the following: a possible reduction in competitiveness of released males; higher than expected fecundity in the sty· homozygote female; possible shift in the frequency of alternate disjunction in T(8;9) sty heterozygote males; possible assortative mating between non-translocation bearing wildtype males and T+/+sty females in the population as well as T+/+sty males and non-translocation bearing wild-type females; fewer T+/+sty males and more sty males released than planned due to phenotyping errors in larger releases. Laboratory studies of the reproductive behavior of three genotypes involved in the experimental populations evaluated competitiveness among males, initial productivity, maximum number of matings per male, and assessed the prospect of assortative mating among the genotypes. Competitiveness of the T+/+sty males in small number tests was consistently better than wild-type males but in later assessments by mass mating and population studies, they demonstrated equal competitiveness with wild-type males. A statistically significant tendency toward assortative mating between VPI wild-type males and T+/+sty females was observed. The feasibility of techniques for mass rearing of semi-sterile males for releases of up to 10,000 males per month was demonstrated. / Ph. D.

Significant mortality of the invasive tree of heaven (TOH), Ailanthus altissima (Mill.) Swingle, was first observed in Pennsylvania in 2002 to be caused by an apparently host-specific strain of Verticillium albo-atrum Reinke & Berthold, a soil-borne, vascular wilt fungus. A limited survey conducted in Virginia revealed two sites where TOH stands were infected with V. albo-atrum. A virulence test confirmed that fungal isolates from both states were found to be highly pathogenic on TOH, killing all inoculated seedlings in 9 weeks. After overwintering, 11% (n = 37) of TOH root sections tested positive for V. albo-atrum, although the origin of the colonies was not identified. The pathogenicity of this pathogen suggests that it could be used together with host-specific insects for the biological control of TOH. A host-specific herbivorous weevil from China, Eucryptorrhynchus brandti Harold (Coleoptera: Curculionidae) that has been extensively studied as another potential biological control agent for TOH is currently pending quarantine release. Quarantine experiments were conducted to test different forms of transmission with E. brandti and V. albo-atrum simultaneously. In one experiment, 75% (n = 32) of adult E. brandti transmitted V. albo-atrum to TOH seedlings after walking on an actively growing culture and feeding on infected plant material. In another study after feeding on infected TOH stems for 24, 48 and 72 h, respectively, 16.7% (n = 120), 15.0% and 12.5% of adult E. brandti ingested and passed viable V. albo-atrum propagules into feces. Surviving weevils (83%, n = 20) overwintering in infested potting mix carried viable V. albo-atrum propagules externally. In addition, all weevil progeny that emerged from infected TOH billets appeared to be as healthy as weevils reared from non-infected billets and wild parents from China. Results from these laboratory studies indicate E. brandti has the ability to spread V. albo-atrum from tree to tree in a laboratory setting. / Master of Science in Life Sciences

The predatory beetle, Laricobius nigrinus Fender (Coleoptera: Derodontidae), has been released in the eastern U.S. since 2003 for the management of hemlock woolly adelgid (HWA), Adelges tsugae Annand (Hemiptera: Adelgidae). The establishment of L. nigrinus at release sites in Virginia was assessed in 2017 and 2018. Sampling was performed in both years to determine presence or absence L. nigrinus. Stand level HWA densities, tree health, predator-prey ratios, and Laricobius spp. identifications were also assessed at each site. Laricobius nigrinus established at 82% of sites and was the primary species recovered. HWA densities appeared to decline, and tree health appeared to improve in year two. Predator-prey ratios were lower than those indicated in the native range, however much is left to be understood about the dynamics of this system in its introduced range. A separate two-year study to assess the impact of L. nigrinus on HWA was initiated in 2014 (Phase One) at nine sites in the eastern U.S. Significant predation of HWA sistens ovisacs was demonstrated during this period, therefore, it was continued from 2016-2018 (Phase Two) to provide longer-term evaluations. Predator exclusion cages were used to monitor predator and prey populations. In Phase Two of the study, mean ovisac disturbance rates on no-cage branches were significantly greater than caged branches and were as high as 80%, suggesting that L. nigrinus can have a significant impact on the sistens generation. Microsatellite analysis of Laricobius larvae indicated that L. nigrinus was the primary species recovered at study sites. / Master of Science in Life Sciences / Hemlock woolly adelgid (HWA), an invasive insect native to Asia and western North America, is a significant threat to two native hemlock species in the eastern U.S. Since 2003, a predatory beetle, Laricobius nigrinus, has been released for management of HWA. In biological control programs such as this one, it is important to evaluate the ability of predators to establish and impact prey in areas where they are introduced, as this helps guide future management decisions. As such, a study was conducted to examine the ability of L. nigrinus to establish after being released at 26 locations in Virginia. In those investigations we found that L. nigrinus established at 82% of the sites. Although promising, longer-term studies are needed to understand if the predation of HWA by L. nigrinus helps improve the health of our native hemlocks. A separate two-phase study was carried out from 2014-2016 and 2016-2018 evaluating the impact of L. nigrinus on HWA at nine release sites in the eastern U.S. In Phase Two reported here, cages were used to exclude L. nigrinus on some HWA infested branches while on others, no cage was applied to allow free access to HWA. Comparisons between branches were made to determine the level of predation by L. nigrinus. These studies showed a significant impact by L. nigrinus on the winter generation of HWA with as many as 80% of those insects being attacked on study branches which indicates that this species has potential as an effective predator.

A computer simulation model written in PL/I of the flowering phenology of C. thoermeri and the seasonal population dynamics of R. conicus was constructed based on a synthesis of the literature, and on laboratory experiments which determined developmental periods, mortality for immature stages and the ovipositional pattern of R. conicus. The model successfully simulated the ovipositional pattern of R. conicus and the flowering phenology of C. thoermeri. Larval population dynamics of R. conicus, in the model however, did not fit the validation data. Though the model was temporally synchronized with the field data, the model results were one degree of magnitude less. The poor fit was attributed to sampling errors in the validation data. Studies of the overwintering mortalities of both organisms were performed. R. conicus was found to overwinter with lowest mortality on musk thistle rosettes; mortality was 73.6% in 1981, higher than previously reported but possibly more representative of field conditions. No overwintering mortality was observed for established rosettes of C. thoermeri. Studies of seed dispersal of C. thoermeri were performed to determine the percentage of seeds removed from the vicinity of the plant where they originated. Seed dispersal observed for different wind velocities demonstrated that less than 1% of seeds are blown further than 100m and most seeds are deposited within 50m of the release site. A Gaussian model of seed dispersal based on experimentally determined parameters and literature values corroborated these results. / Master of Science

Bathyplectes anurus (Thomson) and B. curculionis (Thomson) (Hymenoptera: Ichneumonidae) are specialist parasitoids introduced to the United States for classical biological control of alfalfa weevil, Hypera postica (Coleoptera: Curculionidae). Adults of both species are morphologically similar, generally described as wasps ≈3-4 mm long with black, robust bodies. The Bathyplectes spp. occupy sympatric ranges throughout much of the United States and often coexist within alfalfa fields. In Virginia, B. anurus is the primary biological control agent, accounting for more than 90% of alfalfa weevil parasitization. A previous study, however, found that the levels of control by B. anurus varied among the three alfalfa-growing regions of Virginia, and noted that little is known about the mechanisms or factors responsible for the differential levels of parasitization. Of the factors that may affect host parasitization, three were addressed in this study: population density and spatial distribution of host and parasitoid, the effectiveness of parasitoids in response to host density as measured by the population functional response, and genetic variation among and between populations of parasitoids. GPS-referenced quadrat sampling was conducted in alfalfa fields in the Piedmont and southwestern region to assess alfalfa weevil population levels and spatial distribution, and the level and distribution of parasitization by B. anurus and B. curculionis. Results showed that there was no significant difference in alfalfa weevil densities among alfalfa fields in the Piedmont and southwestern region, and also no significant difference between regions in the level of parasitization. Mean alfalfa weevil density per 0.2-m2 quadrat per sampling date was 11.0 in the Piedmont and 8.0 in the southwestern region; mean percent parasitization per 0.2-m2 quadrat per sampling date was 9.1% in the Piedmont and 9.9% in the southwestern region. The spatial distributions of alfalfa weevil larvae and parasitization were found to be aggregated in all alfalfa fields sampled. The population functional response of B. anurus attack on alfalfa weevil was determined qualitatively to be Type II in all alfalfa fields sampled, except the field located at Blacksburg in the southwestern region. The Type II functional response suggests that, in general, the parasitoid has an inverse density dependent attack response to the density of alfalfa weevil larvae in the two regions of Virginia. Molecular analyses were conducted to 1) derive genetic sequences for B. anurus and B. curculionis, which could be used to identify and differentiate the two species, and 2) test for genetic variation within and between regional populations of Bathyplectes. Sequences for each species were obtained for the D2–D3 region of the 28S ribosomal DNA (28S rDNA) gene; sequence identity between B. anurus and B. curculionis was 96.6%. The 28S rDNA sequences were also obtained for another Bathyplectes species, possibly B. stenostigma, and for four taxa of Hymenoptera that hyperparasitize B. anurus and B. curculionis cocoons. Sequences were obtained for B. anurus and B. curculionis for a portion of the mitochondrial gene cytochrome oxidase subunit 1 (COI); sequence identity between B. anurus and B. curculionis was 89.7%. Genetic variation in the COI gene was observed for both species. One variation of the COI sequence expressed by 25% of B. anurus specimens (Haplotype 2) might indicate regional genetic variation correlated to temperature, in particular the average number of days per year that a region experiences temperatures above 32°C. Two major COI sequence haplotypes found in B. curculionis specimens might be indicative of incipient speciation. / Ph. D.

Sclerotinia homoeocarpa F.T. Bennett, causal agent of dollar spot on creeping bentgrass (Agrostis palustris Huds.); and Rhizoctonia solani Kihn, causal agent of brown patch on tall fescue (Festuca arundinacea Schreb.), are important pathogens of turfgrass. This research evaluated the ability of twenty bacterial strains of the genus Pseudomonas as potential biological agents for the control of these diseases. Year 1 dollar spot field trials resulted in the identification of five strains performing statistically as well as the recommended fungicide, chlorothalonil. Year 2 trials, using the top strains from Year 1, employed different application schedules and inclusion of a commercial spray adjuvant, Agri-Dex™, Results from Year 2 dollar spot trials indicated that eight strains performed statistically better than the non-treated control. Results from Year 1 and Year 2 of brown patch field trials provided three strains that performed statistically better than the nontreated control during Year 1, but only one strain during Year 2. Laboratory tests performed with all strains and both pathogens showed that demonstration of agar-based inhibition is more difficult to obtain with R. solani. Six strains evaluated using AgriDex™ and S. homoeocarpa in laboratory and greenhouse tests, determined that application with Agri-Dex™ can be more effective than with bacteria alone. Supporting greenhouse tests using the top strains from the S. homoeocarpa field trials to evaluate application timing in controlling dollar spot, indicated less disease with earlier and more frequent applications. With further research, several strains have the potential for disease control on turf, particularly dollar spot, and may reduce the reliance on chemical fungicides. / Master of Science

Management of hemlock woolly adelgid (HWA), Adelges tsugae (Hemiptera: Adelgidae), is currently being implemented through several different methods including the release of host-specific predators such as Laricobius nigrinus Fender (Coleoptera: Derodontidae). Releases of this predator began in 2003. In 2014, an assessment of the efficacy of L. nigrinus at release sites from New Jersey to Georgia. Field sites were chosen based on the presence of moderate to high densities of HWA, that L. nigrinus was released at least four years prior to the start of the study, and that the predator was determined to be established at that site. Three treatments were set up at each of the sites: no cage, closed exclusion cage, and open cage. Three assessments were taken during key points throughout the season in order to monitor both HWA and L. nigrinus populations. Larval predator were recovered from most of the sites in year one and in higher numbers in year two. Many sites at which L. nigrinus were recovered showed high predation rates of HWA in uncaged samples ranging from 1.5 to 47.3% in year one and 0 to 66% in year 2. A survey for L. nigrinus establishment at previous release sites in VA was conducted. These sites date back as far as 2003 and as recently as 2015. Beat sheeting and branch clippings were conducted to recover Laricobius spp. adults and larvae, respectively. The recovered insects were then identified to species through genetic analysis. A mix of the introduced L. nigrinus and the native L. rubidus LeConte (Coleoptera: Derodontidae) were recovered at some sites, and only L. rubidus were recovered from others. Higher numbers of Laricobius were recovered in spring of year two. Overall, tree health in Virginia decreased from spring 2015 to 2016. / Master of Science in Life Sciences

The goals of this research were 1) to provide nutritional information that would be useful for the classification of Bacillus sphaericus, a heterogenous bacterial species, 2) to determine nutritional characteristics that would assist in the design of a selective medium for the mosquito pathogenic strains of B. sphaericus and 3) to determine the effect of carbon supplementation of complex media on sporulation and mosquito toxicity of B. sphaericus. In general, the pathogenic strains which constitute DNA homology group IIA of the species, grew with a greater variety of compounds as sole carbon sources than did the non-pathogenic strains of the other homology groups. Within homology group IIA, strains of phage group 3, which are the most toxic, grew with the largest number of compounds as sole carbon sources. However, the nutritional characteristics did not allow the unequivocal differentiation of any one DNA homology group or phage group from all of the other groups. It was possible to differentiate a DNA homology group or phage group from some of the other groups. A defined medium containing malate and acetate as carbon sources was found to enable the cells to produce 1.1x10⁸ spores/ml and thus would be useful for studying the physiology of sporulation. Eighteen of 22 pathogenic strains were capable of growth in a liquid, defined medium with arginine as the sole carbon source, while only 2 of 17 non-pathogenic strains were capable of growth under these conditions. Thus, arginine may be useful as the carbon source in a selective medium for the mosquito pathogenic strains. Of several compounds which could be used by B. sphaericus as sole carbon sources, acetate was the most useful in increasing sporulation and mosquito toxicity when added as a supplement to a nutrient broth-yeast extract-mineral salts medium. A glycerol supplement to this medium led to accumulation of acetic acid and suppression of sporulation and toxin formation. An acetate supplement and a mineral salts (Ca²⁺, Mg²⁺, Mn²⁺) supplement were found to increase sporulation in a fishmeal medium and thus may be useful for commercial production of mosquito larvicides from B. sphaericus. / M.S.