Entomology
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Item GENETIC DIVERSIFICATION, SAPROPHYTIC COMPETENCE AND GENETIC ENHANCEMENT OF THE ENTOMOPATHOGENIC FUNGUS METARHIZIUM(2009) Pava-Ripoll, Monica Patricia; St. Leger, Raymond J; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Entomopathogenic fungi are being investigated as alternatives to chemical insecticides. This study explored the versatility of the entomopathogenic fungus Metarhizium anisopliae by examining its diversification, saprophytic competence and potential for genetic enhancement. M. anisopliae is a radiating species containing both generalist and specialized lineages with broad and narrow host ranges and as such provides an excellent model system to study the evolution of pathogenesis. Using 18S RNA and protease sequences, I demonstrated that strains can be selected representing evolutionary distances ranging from <1 to 8 MY and their natural molecular variation allows analysis of processes of adaptive change. M. anisopliae is particularly abundant in the rhizosphere. Germination of M. anisopliae strain 2575 was >96% in 1 mg/ml root exudate (RE) and growth in RE resulted in 29 (58%) genes being up-regulated and 21 (42%) down-regulated. The identity of these genes is helping to define the physiological requirements for rhizosphere competence. Hypothetical and orphans proteins (41.4%) were also actively expressed indicating that many previously uncharacterized genes may have functions related to survival at the soil-root interphase. Using the fungus as a delivery vehicle for foreign toxins presents a powerful approach for increasing virulence. M. anisopliae was modified to express a scorpion toxin (AaIT) in insect haemolymph and bioassayed against the coffee berry borer Hypothenemus hampei. AaIT increased mortality up to 56.6%, and reduced the medial lethal concentration (LC 50 ) by 15.7-fold and the average survival time (AST) by 20.1%. The AaIT gene and the M. anisopliae esterase gene (Mest1) were inserted into three strains of Beauveria bassiana (ARSEF 252, 8998 and 9184) with high, medium and low mortality, respectively, against the Colorado potato beetle (CPB) Leptinotarsa decemlineata. Mortality rates were strain- and dose- dependant and increased from 16.1 to 36.7% in single transformants (AaIT or Mest1) and from 7.1 to 33.5% in double transformants (AaIT-Mest1). The AST was reduced up to 33% and the LC50 up to 5.9-fold. Although singly both AaIT and Mest1 increased the killing power of B. bassiana against second instar CPB, combining AaIT and Mest1 together did not produce synergistic effects.Item Metarhizium anisopliae's persistence as a saprophyte, genetic basis of adaptation and role as a plant symbiont(2008-11-17) O'Brien, Tammatha Rose; St Leger, Raymond; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Metarhizium anisopliae is a model for elucidating the basis of entomopathogenicity. However, many aspects of the saprophytic life of M. anisopliae remain unclear, including: 1) how the rhizosphere maintains populations of M. anisopliae; 2) the potential for microevolution; 3) the genetic factors that allow M. anisopliae to adapt to a saprophytic life and 4) the extent to which plant ecology is impacted by the rhizospheric competence of M. anisopliae. To extend our knowledge of plant-insect-fungal interactions, a field trial was conducted with M. anisopliae applied to turf. The specific objectives were to: 1) investigate the genetic basis of adaptation and strain stability of M. anisopliae; 2) monitor long-term survival of M. anisopliae, 3) compare the winter survival of M. anisopliae ΔMcl1 (disrupted in an immune evasion gene and nonpathogenic) with M. anisopliae ΔMad2 (adhesin-deficient and unable to adhere to plant epidermis) and 4) investigate root colonization and its impact on plant growth of Triticum aestivum seeds coated with conidia. Results showed that M. anisopliae ΔMad2 had a linear decrease in population and did not colonize roots, while M. anisopliae and M. anisopliae ΔMcl1 cycled with seasons in the soil and colonized roots. Microarray analyses were used to assay for any mutations affecting gene expression during survival in the field. After 3.5 years, field recovered Metarhizium isolates differed in gene expression by an average of 0.26 % for the 1,749 expressed sequence tags. Mutations disproportionately affected cell wall and stress responses, while genes coding for pathogenicity determinants such as adhesins and toxins were highly conserved. Triticum seeds inoculated with M. anisopliae ΔMcl1 and M. anisopliae ΔMad2 prior to planting in the field produced a 14.92%, 4% and 0% increase in seed weight respectively. M. anisopliae increased growth rates of winter wheat in microcosms and may act as a biofertilizer by making insoluble phosphate available to plants. This study showed Metarhizium benefits plants by protecting them from insect pests and by potentially solubilizing otherwise unavailable nutrients. Metarhizium, therefore, may be implemented in an integrated pest management (IPM) approach to reduce the use of chemical insecticides and fertilizers.Item Genetic diversity and persistence of mayfly populations in disturbed headwater streams(2007-04-25) Alexander, Laurie Constance; Lamp, William O.; Hawthorne, David J.; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Movements of individuals shape the spatial structure of populations and play an important role in their persistence. For aquatic insects with winged adult stages, properties of the terrestrial landscape influence in-stream habitat quality and, in naturally patchy habitats such as dendritic stream networks, connectivity among habitat patches. Connectivity here refers to the population dynamics dependent on migration and gene flow among insect populations in semi-isolated stream segments. When populations are spatially connected, effects of local disturbance (e.g., habitat loss or degradation) can have a ripple effect, ultimately altering regional processes that reflect back to the local patch. But since regional and local population dynamics occur at different rates, detrimental effects of local disturbance are often not detected by biomonitoring efforts at the patch level until they have rippled through regional processes, by which time large-scale population extinction risk may have become unacceptably high. My dissertation examines the effect of local and regional disturbance on the population density, genetic structure, genetic diversity, and persistence of mayfly populations living in forested and deforested headwater streams in the Central Piedmont region of Maryland and Virginia. I sampled populations of the mayfly Ephemerella invaria (Walker) in 24 first-order streams across 9 headwater stream networks. The sampling period (2001-2004) spanned a regional drought during which some of the streams went dry. Thus I was able to look at the interaction of local deforestation and stochastic regional disturbance in my study system. In summary, my results indicate that in these mayfly populations: 1. Historically, long-range dispersal of Ephemerella occurred at levels sufficient to maintain gene flow across major watersheds, indicating excellent passive or active dispersal capability in these insects. 2. Deforestation of small watersheds decreases the rate of stream re-colonization and the recovery of prior population densities following a major disturbance. 3. Deforestation is correlated with loss of population genetic diversity. 4. Highly differentiated migrants represent a disproportionate share of the diversity in some mayfly populations. 5. Stochastic regional disturbance (e.g., drought) interacting with local disturbance (e.g., small scale watershed deforestation) can increase population extinction risk.Item Phylogeography of an introduced insect pest and consequences of an insect introduction(2007-03-06) Ahern, Robert; Raupp, Micheal J; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Adelges cooleyi (Hemiptera: Adelgidae), a host-alternating gall-making insect pest native to the Rocky Mountains and Pacific Northwest and introduced into the eastern United States during the mid-19th century, was studied to address questions about phylogeography, to determine effects of introduction on genotypic and phenotypic variation, and to compare genetic variation associated with host use in native and introduced ranges. In Chapter One, sequence data from two mitochondrial (mtDNA) genes and amplified fragment length polymorphisms (AFLPs) were used to quantify the structure of genetic variation in the insect's native range. Several well-supported, divergent mtDNA lineages were identified. The structure of genetic variation among sampled locations is consistent with patterns shaped by glaciations. Samples from the southern edge of the insect's distribution are genetically isolated from the rest of the species, and hybridization of divergent mtDNA lineages via secondary contact was inferred from AFLP data. Changes in genetic and phenotypic variation associated with introduction were quantified in Chapter Two. Introduced populations had decreased genetic variation relative to native populations. Variation in an ecologically important trait, host preference, was also significantly lower in introduced populations than in native populations. An association between mtDNA haplotypes and host preference was identified. Adelges cooleyi in the eastern US have low genetic and phenotypic variation but appear to be sufficiently adapted for persistence. My results call into question the utility of neutral genetic variation to assess the probability of persistence in new environments by introduced species. Host-plants that A. cooleyi requires to complete its lifecycle are not native to the eastern US and occur together in patches that are often widely separated. In Chapter Three, analyses of mtDNA and AFLP genetic variation were conducted to determine the distribution of genetic variation within and among host plants in the native range and identify discrepancies that may be consistent with an incomplete lifecycle in the introduced range. Distribution of genetic variation within and among host-plants in the introduced range was not significantly different than that in the native range, as indicated by fixation indices, and I found no evidence for asexual populations in the introduced range.Item The Role of Host-Plant Species in the Differentiation of Sympatric Populations of Hymenopteran Parasitoids(2005-11-02) Medina, Raul Francisco; Barbosa, Pedro; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)ABSTRACT Title of Document: THE ROLE OF HOST-PLANT SPECIES IN THE DIFFERENTIATION OF SYMPATRIC POPULATIONS OF HYMENOPTERAN PARASITOIDS. Raul F. Medina, PhD, 2005 Directed By: Professor, Pedro Barbosa, Department of Entomology The biology and ecology of insect parasitoids is strongly influenced by the host-plant species on which their herbivorous hosts occur. Hymenopteran parasitoids in particular, present a series of characteristics that made them good candidates for phenotypic and genotypic differentiation. Thus, parasitoid adaptation to plant traits may promote significant phenotypic and genotypic differences among sympatric populations of parasitoids associated with different host-plant species. The present study assessed phenotypic and genotypic differentiation in two braconid parasitoids ovipositing on the same host species, the green cloverworm, Plathypena scabra Fabricius (Lepidoptera: Noctuidae) feeding on alfalfa and soybean. Developmental time, adult weight, percent parasitism and preference for host-plant odors of the generalist parasitoid Cotesia marginiventris Cresson (Hymenoptera: Braconidae) and of the specialist parasitoid Aleiodes nolophanae Ashmead (Hymenoptera: Braconidae) were compared among individuals ovipositing in green cloverworm larvae feeding on alfalfa and soybean. In addition, amplified fragment length polymorphisms (AFLP) were used to assess if genotypic differentiation between parasitoids ovipositing on green cloverworm larvae feeding on different host-plant species (i.e., alfalfa or soybean) was present. Phenotypic differentiation in adult mass, adult longevity and percent parasitism between parasitoids ovipositing green cloverworm larvae on alfalfa and on soybean were found. These phenotypic differences between parasitoids associated with different host-plant species were observed in both the generalist and the specialist parasitoids. No evidence of parasitoids showing preferences for the host-plant species from which their host fed was found in the generalist nor in the specialist parasitoid. Contrary to the expectations and predictions from the literature, these parasitoid species did not show evidence of reproductive isolation when associated with different host-plant species (i.e., alfalfa or soybean), as evidenced by the lack of genetic differentiation in AFLP profiles between parasitoids associated with alfalfa and soybean. In order to ensure that the number of wasps and the number of AFLP bands used were enough to provide an accurate assessment of genetic differentiation among wasps ovipositing hosts on different host-plant species, a method for determining the minimum number of individuals and AFLP bands to include to obtain accurate genetic profiles of hypothesized populations was proposed.