<p dir="ltr">Sepidiini is a large and morphologically diverse tribe of darkling beetles (Tenebrionidae) in the subfamily Pimeliinae, containing ~1,000 species and subspecies. Despite the presence of many large and charismatic species and the cultural significance of some of its members, the toktokkie beetles, this tribe has been lacking revision at all taxonomic levels. To develop a framework for taxonomic revisionary work, in Chapter 1, I reconstructed a phylogeny of the tribe using targeted enrichment sequencing data. I also scored a comprehensive suite of diagnostic characters for the tribe to test in a phylogenetic context. Based on the results of the study, I proposed revising the subtribe Oxurina Koch, 1955, <b>sens. nov. </b>(now containing the genera <i>Oxura </i>Kirby, 1918, and <i>Miripronotum </i>Louw, 1979) and moving the genera <i>Decoriplus </i>Louw, 1979, <i>Pterostichula </i>Koch, 1952, <i>Stenethmus </i>Gebien, 1937b, and <i>Synhimba </i>Koch, 1952 to a new subtribe Stenethina <b>subtr. nov. </b>The tree topology also supports revising or synonymizing the genera <i>Dichtha </i>Haag Rutenberg, 1871, and <i>Amiantus </i>Fåhraeus, 1870,<i> </i>and revising the genus <i>Somaticus </i>Hope, 1840.</p><p dir="ltr">In Chapter 2, I performed a partial revision of the subtribe Hypomelina Koch 1955. A new genus <i>Bufoniopsis </i><b>gen. nov. </b>is erected containing one newly described species <i>Bufoniopsis hypnosis</i> <b>sp. nov. </b>The genus <i>Hypomelus </i>Solier, 1843, and all of its species are redescribed, and three new species are described: <i>Hypomelus johnprinei</i> <b>sp. nov</b>., <i>Hypomelus lorettalynnae</i> <b>sp. nov.</b>, and <i>Hypomelus tomhalli</i> <b>sp. nov</b>. One species was moved from <i>Hypomelus </i>to <i>Triangulipenna</i><i> </i>Louw 1979<i> </i>and redescribed, <i>Triangulipenna vulipnus </i>(Haag Rutenberg, 1873)<i> </i><b>comb. nov. </b><i>Triangulipenna </i>was redescribed and three new species were described, <i>Triangulipenna tylerchildersi </i><b>sp. nov.</b>, <i>Triangulipenna dollypartonae</i> <b>sp. nov</b>., and <i>Triangulipenna ralphstanleyi</i> <b>sp. nov</b>. A revised key to the genera of Hypomelina is provided as well as keys to the species of <i>Hypomelus </i>and <i>Triangulipenna</i>.</p><p dir="ltr">Toktokkie beetles—members of the subtribe Molurina Solier, 1843—are known for their substrate tapping behavior which is a form of sexual communication in which beetles tap their abdomens on the ground to locate mates. Despite the fact that this behavior is well-known among people in southern Africa, very little research has been done on the topic. In Chapter 3, I document variation in tapping patterns across the subtribe Molurina. Three characters of tap trains were found to vary: length of tap trains, tapping rate, and tapping pattern. Ancestral state reconstruction was performed for each of the characters. Characters were found to be only partially linked to ancestry. Additionally, in any given locality sampled, no two species had the same “song”. This suggests that signal partitioning could be influencing species “songs”.</p>

<p dir="ltr">Rapid ʻōhiʻa death (ROD) is a disease complex caused by two <i>Ceratocystis fungi</i>, <i>C. lukuohia</i> and <i>C. huliohia</i>, that is devastating the keystone tree of the Hawaiian Islands, ʻōhiʻa lehua (<i>Metrosideros polymoropha</i>). The causal agents of ROD were identified in 2015 and I began researching entomological aspects of the complex in 2016. Much like other <i>Ceratocystis</i> diseases, my colleagues and I suspected that beetles and frass might be involved in the system. Together, we identified four species of invasive ambrosia beetles (Coleoptera: Curculionidae) that contribute to the spread of ROD: <i>Xyleborinus saxesenii, Xyleborus affinis, Xyleborus ferrugineus</i>, and <i>Xyleborus perforans</i>. Both ROD-<i>Ceratocystis</i> fungi and the ambrosia beetles inhabit the xylem of ʻōhiʻa. When these beetles create their home galleries, they produce frass particles infested with resting chlamydospores that can be transported in the environment through the soil, wind, and water. Secondly, the beetles are capable of vectoring the fungi directly to stressed trees via viable propagules attached to their exoskeleton. The natural progression of this research was to investigate the chemical ecology of the system, therefore building the foundations for management strategies to reduce the spread of ROD. In addition, I satisfied my curiosity to explore the fungal mutualisms of these beetles through the use of phylogenetics.</p><p dir="ltr">In Chapter 1, I review the literature describing ROD and the four ROD-associated ambrosia beetle species. I report all of the research to date regarding ROD, including current monitoring and management strategies. Then, I introduce ambrosia beetles and the Xyleborini tribe, focusing on the life history of the ROD-associated beetles and current literature describing the use of semiochemicals to control them.</p><p dir="ltr">In Chapter 2, I determine the volatile organic compounds associated with the ROD <i>Ceratocystis</i> – ʻōhiʻa pathosystem and the response of the associated beetles to those compounds. I investigated the volatiles produced by <i>C. lukuohia</i> and <i>C. hulihia</i> in culture in addition to when inoculated into ʻōhiʻa seedlings. Then, I describe olfactometer assays to determine if the ROD-associated beetles are attracted to the volatiles emitted from ROD-<i>Ceratocysti</i>s in culture.</p><p dir="ltr">In Chapter 3, I investigate semiochemicals for attracting and repelling ambrosia beetles in ʻōhiʻa forests. I describe separate trapping experiments, first, testing the attraction of beetles to 100% ethanol and 1:1 methanol ethanol. Second, we investigate the use of two beetle repellent products, one with verbenone and the other with verbenone + methyl salicylate active ingredients.</p><p dir="ltr">In Chapter 4, I describe the testing of the repellent, verbenone, in the SPLAT^® Verb formulation, to deter ambrosia beetle attack from both healthy ʻōhiʻa trees and trees infested with ROD-<i>Ceratocystis</i>. Over two field seasons, we monitored ambrosia beetle attacks on trees treated with verbenone and measured the abundance of verbenone released from the repellents over time during the first season.</p><p dir="ltr">In Chapter 5, I investigate the ambrosia fungi of the ROD-associated beetles and native Hawaiian ambrosia beetles on the Island of Hawaiʻi. We isolated a dozen fungal symbionts from the mycetangia of ambrosia beetles, most of which are first reports in Hawaiʻi, and use phylogenetics to investigate putative new species of <i>Raffaelea</i> and <i>Ambrosiozyma.</i></p><p dir="ltr">Finally, in Chapter 6, I synthesize the results and future directions of the aforementioned chapters. Together, these dissertation chapters provide insights into ambrosia beetle monitoring and management strategies in Hawaiʻi and beyond. I describe the groundwork for understanding the pathosystem from a chemical ecology perspective and touch on the understudied world of Hawaiʻi fungi and potential pathogens.</p>

<p dir="ltr"><i>Phormia regina</i> Meigen is a forensically relevant species of blow fly, common in North America and used to estimate the minimum postmortem interval in forensic casework. It is also possible to use blow flies to survey the environment for biotic and abiotic information drawn from both larval and adult stages. There are both forensic and environmental uses for genetic analysis of blow flies. Blow fly kinship is especially useful for detecting postmortem movement of a corpse or to assess abundance of carrion in a given location. To test genetic relationships between individuals, discriminatory loci such as microsatellites, or polymorphic tandemly repeated sequences of DNA are necessary. Here, we characterize novel microsatellites generated from the genome of <i>P. regina</i>. Thirty-four candidate polymorphic loci with conserved flanking regions, have been isolated. To date, seven are heterozygous and polymorphic testing in two lab populations and one wild population. The simple sequence repeats characterized here complement existing loci (N = 6) for greater discrimination for testing relationships between individual flies.</p>

<p dir="ltr">Blow flies are a family of carrion insects that are among the first to arrive in the decomposition process. Blow flies are known to ingest carrion, feces, water, and occasionally nectar to meet nutritional requirements. These behaviors make blow flies a unique organism potentially containing genetic material from a variety of sources within one environment. Their global distribution and ease of capture makes them a strong candidate for resource monitoring and identification. While previous studies have evaluated the suitability of blow flies for vertebrate biodiversity estimates, no work has been done looking at their ability to ingest and store genetic material from plants and microbes present in water. It is also not known how long these DNA signals persist in the gut. Through DNA analysis of the blow fly gut, researchers can identify vertebrates that have recently died in an environment, what plant species are present, and what water source the insect utilized. Through lab colony (Phormia regina) feeding experiments, it was determined that at 25 ˚C and 50 % relative humidity, vertebrate and plant DNA persist in the gut for over 120 hours post-ingestion. Wild sample analysis of flies collected from Yellowstone National Park was performed to identify plant species ingested by P. regina in the wild. Following Sanger sequencing, top hits on BLASTn included Brassicales, Juglans cathyensis, and uncultured Candida. This is the first application of environmental DNA analysis techniques to insects for the purpose of plant identification. This work also attempts to characterize microbial profiles of the gut of P. regina for the purpose of water resource identification. Over a two-month collection period, samples were collected from different water resources across Indianapolis. Flies were exposed to these samples in a controlled feeding experiment, followed by sampling at 0- and 72-hours post-exposure. Gut samples were sequenced using Illumina and Operational Taxonomic Unit clustering grouped reads by sequence similarity for identification. Bacteria classes identified included Gammaproteobacteria, Bacteroidia, Flavobacteria, Alphaproteobacteria, Bacilli, Clostridia, Actinobacteria, Betaproteobacteria, and Fusobacteria. Many bacteria classes were common across water samples, although the abundance of each class changed between samples and across time. These unique microbial profiles can be used to identify water resources for potential contamination and chemical dumping. Further work is necessary to generate microbial profiles from the original water sources themselves and for generation of alpha and beta diversities. Overall, this work spans multiple fields. Species identification is important for biodiversity monitoring and environmental surveys. Utilizing blow fly derived DNA allows for detection of living and deceased vertebrates in an environment, plant life, and water quality within one sample. This work also has implications in forensic science, specifically wildlife forensics and chemical detection of clandestine laboratories and chemical weapon compounds.</p>

Traditionally, hypoxia has been defined as the situation where DO levels have fallen below 2.0 mg O2 L-1, but increasing evidence suggests that this low level of DO is inadequate to describe the onset of hypoxia impacts for many organisms. Consequently, there is a need for a greater understanding of how ‘moderate’ alterations in DO levels will affect ecosystem processes and functionality, specifically through behavioural and physiological alterations at the organism and community level. This thesis reports on mesocosm experiments which were conducted to examine the effects of moderate ( > 3.0 mg O2 L-1) hypoxia on firstly, a key ecosystem engineer, the brittlestar Amphiura filiformis, and secondly, on the Station L4 infaunal macrobenthic community. Station L4 is a longstanding marine biodiversity and MSFD reference site and forms part of the Western Channel Observatory. At the organism level, short-term (14 d) exposure to moderate hypoxia significantly reduced oxygen uptake rates, oocyte diameter and oocyte development in A. filiformis. However, these physiological affects occurred irrespective of brittlestar population density. Additionally, moderate hypoxia reduced brittlestar activity, in terms of bioturbation behaviour, consequentially having an effect on ammonium and silicate fluxes. These observations were only detected when brittlestar population density was high. It was concluded that denser populations of A. filiformis may therefore exhibit the greatest changes in behaviour and shifts in ecosystem function as competition for resources and oxygen heightens. The benthic community at Station L4, displayed considerable tolerance to medium-term (6 wk.) exposure to moderate hypoxia, in terms of structure, diversity and bioturbatory behaviour, but these results may be different if exposure was longer or more severe. Alterations in nutrient fluxes were detected, but there was little evidence to suggest these changes were due to macrofaunal behavioural alterations. Additionally, results from this study revealed that bringing complex natural communities into the mesocosm caused a substantial loss of individuals and species, mainly due to translocation and disturbance effects. This important insight into the effects of bringing community assemblages into the mesocosm confirms that even with a loss of diversity, the L4 community maintained functionality and was resilient to alterations in DO. This suggests that the L4 benthic community does not depend on any one specific species for the provision of important ecosystem processes, resulting in considerable functional resilience within the L4 system. However, vulnerability to benthic systems may increase if functionality is dominated by species such as A. filiformis. Consequently, moderate hypoxia may not immediately affect benthic communities in terms of structure and diversity, but the physiological effects on individuals, especially to reproductive development, may cause alterations in the quality and quantity of planktonic propagules supplied by benthic species to the pelagic environment. This could affect benthic community diversity and functionality in the long term if repeated hypoxic events occur.

Increased biodiversity generally enhances terrestrial ecosystem productivity. While niche-use efficiency is thought to drive the biodiversity-productivity relationship, the mechanisms within niche-use efficiency are not well understood. A potential mechanism for niche-use efficiency is nutrient-use efficiency. To measure nutrient-use efficiency, we calculated nitrogen-resorption efficiencies (NRE) because nitrogen is an important growth limiting nutrient for forest productivity. We used a plantation implemented as a full factorial design that included two levels of competition, implemented as different planting densities (one- and two-meter planting densities), and three diversity levels (monocultures, two-, and three-species plantings) that included three hardwood tree species (northern red oak (<i>Quercus rubra</i>), black cherry (<i>Prunus serotin</i><i>a</i>), and American chestnut (<i>Castanea dentata</i>). For our nitrogen-resorption efficiency data, we found that NRE increased as diversity and planting density decreased, but the magnitude of the response varied among species. This outcome suggests that while increased diversity likely provides a release from intra-specific competition, different combinations of species will play a critical role in shaping biodiversity-productivity relationships. Forest nutrient cycling can also be influenced by herbivory. To address the effects of forest diversity on herbivory rates, we monitored rates of foliar damage along with foliar nitrogen content. To measure foliar nitrogen content, we collected spectral data from early, midseason, and late season foliar samples. To assess foliar damage, we collected and imaged leaves from two canopy positions in order to measure late season foliar area and estimate pre damaged foliar area. We found that diversity and foliar nitrogen content have a positive relationship, and diversity does influence canopy damage but the effects vary among species and density. Upon further analysis, we found that foliar nitrogen content and canopy damage are correlated. Meaning individual trees showed a release from intraspecific competition, which lead to an increase in available nutrients and higher canopy quality, showing that stands with higher canopy quality experienced higher levels of damage.<br>

The main object of this study was to investigate the effects of predation by starlings (Sturnus vulgaris) on populations of the grass grub (Costelytra zealandica) in Canterbury, with the secondary objective of investigating the feasibility of life table studies of grass grub populations under Canterbury conditions. Study plots were established in four areas, two on non-irrigated pastureland at Lincoln and Weka Pass and two on irrigated pastureland at Winchmore. A third study plot was established at Winchmore in the second year of field work.

<p>Despite being distributed worldwide and easily collected, the biology, ecology, and taxonomy of Ptiliidae Heer, 1843, or featherwing beetles, have not been well studied. In a study from 2007 to 2009, Ptiliidae were extracted from various polypore fungi collected throughout Costa Rica in an effort to expand biogeographic knowledge of Ptiliidae. Fungi and Ptiliidae were identified to genera and collection sites mapped. Beetle genera are able to inhabit different polypore genera and were found at a higher rate of co-occurrence than reported in previous studies. We identified <i>Cylindrosella costariciensis </i><b>sp. n.</b>, with the potential of two more new species to be described.</p>

<p>  </p> <p>Vector-borne diseases (VBDs) represent a significant health burden worldwide, threatening approximately 80% of the global population. Insecticide-based vector control is the most effective method to manage many VBDs, but its efficacy has been declining due to high levels of resistance in vector populations to the main insecticide classes which operate via limited modes of action. Therefore, the discovery of new chemistries from non-conventional chemical classes and with novel modes of action is a priority for the control of vectors and VBDs. Natural products (NPs) are diverse in chemical structures and, potentially, modes of action. They have been used as insecticides for many decades and have inspired the development of multiple synthetic insecticides, suggesting the discovery of novel NPs could lead to the development of highly effective insecticides. </p> <p><br></p> <p>In this thesis, I report two studies with a main goal to identify novel mosquito-active insecticide leads that operate via modes of action distinct from existing insecticides. First, I tested the hypothesis that new mosquito-active insecticide leads with novel chemical structures, possibly operating via novel modes of action, can be identified by high-content larval phenotypic screening against a natural product collection and using novel phenotypic endpoints in addition to mortality endpoints. Here, I performed a high-content larval phenotypic screen using first instar (L1) larvae of <em>Aedes aegypti</em> (Linnaeus, 1762) against 3,680 compounds from the AnalytiCon MEGx Natural Product Libraries and a screening platform developed by Murgia et al., (2022). Compounds were screened in a 384-well plate format using the Perkin Elmer Opera Phenix and larvae were scored for lethal and novel phenotypic endpoints. Screening revealed five chemistries that caused larval mortality, including rotenone and a new NP chemistry, NP-4. The identification of rotenone confirmed the ability of the screen to detect mosquito-active NP chemistries. NP-4 caused high levels of larval mortality in the screen, and toxicity was confirmed in a subsequent concentration-response assay against third instar (L3) larvae of <em>Ae. aegypti</em>. 140 chemistries that caused atypical larval phenotypes, including cuticular pigmentation and morphometric changes relative to negative controls, were also identified by the screen. Some of these chemistries may operate by disruption of pathways regulating melanization, growth and development, and novel targets in the insect nervous systems, thus representing potential leads for further insecticide toxicity and mode of action studies. To facilitate quantitative analyses of atypical phenotypes, an attempt was made to assess the morphometrics of the thorax in larvae exposed to test chemistry, relative to control larvae. However, assessment was limited by the number of larvae images of suitable quality for measurements. </p> <p><br></p> <p>In the second study, I tested the hypothesis that metergoline (Murgia et al., 2022) and NP-4 (this study), two chemistries identified by the HTP phenotypic screen described in this project, operate via disruption of targets in the insect nervous systems that are distinct from the current insecticidal modes of products used in mosquito control programs. Specifically, I explored the hypothesis that metergoline operates via one or more insect orthologs of the mammalian G protein-coupled serotonin and dopamine receptors. An electrophysiology study was performed using the suction electrode technique and ganglia of the German cockroach, <em>Blattella germanica </em>(Linnaeus, 1767). To facilitate the investigation of metergoline agonism/antagonism and disruption of invertebrate GPCR signaling, 5-hydroxytryptamine (5-HT; serotonin) was included as a chemical probe. Electrophysiological recordings showed 5-HT (10µM and 1mM) and metergoline (10µM) caused no significant neurological activity at the tested concentrations in comparison to the saline control. However, a consistent neuro-inhibitory trend was observed, suggesting possible agonism of a 5-HT1-like receptor ortholog and antagonism of a putative 5-HT7-like receptor ortholog in the cockroach, respectively.  NP-4 caused significant neuro-inhibition at the tested concentration of 20µM, in comparison to the negative saline control. Given the demonstration of rapid contact toxicity to <em>Ae. aegypti</em> larvae and neurological inhibition in <em>B. germanica</em>, we propose NP-4 may act at one or more conserved targets in the insect nervous systems, which remain to be elucidated. </p> <p><br></p> <p>The significance of the present study is three-fold. First, this study reports the first high-content phenotypic screen of mosquito larvae against a NP collection and identification of 145 mosquito-active chemistries associated with lethal and phenotypic endpoints. These data confirm that the screening platform provided an innovative and effective system to rapidly identify mosquito-active small molecules with potential novel modes of action. Second, metergoline and NP-4 represent potential novel chemical leads for the development of new insecticides that can be incorporated into vector control programs targeting insecticide-resistant populations. Lastly, the study describes the first electrophysiology study of 5-HT, metergoline, and NP-4 via the suction electrode technique in an insect system and contributes new knowledge to the study of the insect serotonergic system, which represents an expanding area of vector biology research given its roles in feeding regulation.  </p> <p><br></p> <p>Future studies resulting from this thesis might include: (1) development of a set of morphometric criteria for quantitative analyses of atypical larval phenotypes, (2) incorporation of new phenotypic endpoints to expand the capacity of the screen to identify novel mode of action chemistries for insecticide discovery, and (3) identification of chemistry candidates suitable for further development from the 140 chemistries associated with atypical larval phenotypes in the primary screen using chemo-informatic and toxicological studies. In addition, studies using reverse transcription-polymerase chain reaction (RT-PCR), cell-based expression systems, mutant/insecticide resistant strains, and patch clamp electrophysiology could be pursued to further investigate the molecular mode of action of metergoline and NP-4, and potential for vector control.</p>

<p dir="ltr">The Asiatic garden beetle (AGB), in its larval stage, has become an increasingly detrimental to the health of commercially grown mint in northern Indiana by feeding on their roots. In my research, I have optimized a method to quantify populations by determining the proper sample size to maintain accurate and precise estimates, determining the best sampling method and sampling scheme. Additionally, I established the relationship between AGB larval feeding and mint performance and have established a threshold of the average number of larvae required to cause significant yield loss. </p>