Small creatures, Big problems, Biological solutions
Research Experiences & Projects
Effects of arboviruses on blood-feeding behaviors of mosquitoes and biting midges
A western encephalitis mosquito, Culex tarsalis (top), a southern house mosquito, Culex quinquefasciatus (middle), and a biting midge, Culicoides sonorensis (bottom), all wired for EPG.
Small Creatures: Mosquitoes, biting midges, and arboviruses
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Big Problems: Transmission of arboviruses to livestock and humans causes disease, and thus suffering, loss of life, yield losses, and food insecurity.
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Biological Solutions: Identify physiological factors that influence arbovirus transmission that can be targeted for vector and disease control.
Many pathogens are known to manipulate the behavior of insect vectors to enhance disease transmission; however, it is still unknown if or how many of the arthropod-borne viruses (aka arboviruses) influence the blood-feeding behaviors of vector insects. As a postdoctoral fellow, Dr. Cooper is currently investigating the effects of arboviruses, specifically bluetongue virus, vesicular stomatitis virus, Japanese encephalitis virus, and Rift Valley fever virus, on the blood-feeding behavior of Culex mosquitoes and Culicoides biting midges. For this investigation, Dr. Cooper is using a technique called electropenetrography (EPG) to visualize the unseen mouthpart movements that occur inside opaque host tissues by visualizing changes in electrical signals, called waveforms, that occur during feeding. Her initial task is to create a collection of waveforms (called a waveform library) for each insect species feeding on human hands and deduce the biological meaning of the different waveforms. Then she will investigate if and how the waveforms change when infected and uninfected insects feed on animal models or artificial feeding systems. Differences observed in feeding behaviors may lead to new hypotheses and methods for disrupting the ability of these insect vectors to transmit diseases to livestock.
EPG with a wired mosquito on a human hand. The insect is attached to the head stage amplifier by a thin gold wire. When the insect feeds on the human host holding an electrode it creates a complete circuit. Salivation, blood feeding, and mouthpart movements change the flow of electricity and appear as waveforms on a computer.
The EPG set-up used by Dr. Cooper at KSU. Recordings of blood-feeding behavior take place inside a faraday cage to reduce electrical noise (top). Adjacent to the faraday cage is the EPG machine, which consists of a power supply, a control box, a DATAQ interface adapter, and an analog-to-digital converter, all connected to a laptop computer (bottom).
The NO/sGC/cGMP signaling pathway as a novel target for insect pest management
A red flour beetle, Tribolium castaneum.
Insecticides and ovicides (i.e., pesticides that kill eggs) with novel modes of action are needed to overcome pesticide resistance and combat a wide array of pre- and post-harvest insect pests. Nitric oxide (NO) is a signaling molecule that activates soluble guanylyl cyclase (sGC) to generate cyclic GMP (cGMP) as a second messenger in target cells, which regulates a variety of physiological processes in insects. High concentrations of NO were previously shown to kill insects under ultralow oxygen conditions, and Zhu’s group found that RNA interference-mediated suppression of sGC subunits completely inhibited egg hatching in red flour beetles. Dr. Cooper briefly worked on this project during the end of her Ph.D. and for a few months after graduation. She also submitted a proposal for a USDA APHIS Postdoctoral Fellowship to investigate the disruption of sGC subunits as a new mode of action for ovicides, but the proposal was not funded, and she moved on to other opportunities.
Small Creature: Red flour beetles
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Big Problems: Stored product pests consume and damage grain after harvest.
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Biological Solutions: Develop new pesticides that attack novel biological targets, such as components of the NO/sGC/cGMP signaling pathway
Mechanisms influencing RNA interference in insects
A European corn borer, Ostrinia nubilalis, caterpillar (top), a western corn rootworm, Diabrotica virgifera virgifera (upper middle; image from agfax.com), an Asian corn borer, Ostrinia furnacalis (lower middle; image from cabi.org), and a migratory locust, Locusta migratoria (bottom; image from ozanimals.com)
Small Creatures: Caterpillars, rootworms, and locusts
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Big Problems: Insect pests consume and damage crops, reduce yields, and contribute to food insecurity
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Biological Solutions: Develop species-specific pesticides, such as RNAi-based biopesticides, that use natural molecules to silence essential genes
RNA interference (RNAi) is a gene silencing mechanism that helps regulate gene expression and defend against viruses and transposons. RNAi-mediated gene silencing is triggered by complementary double-stranded RNA (dsRNA) and can be exploited by experimentally introducing dsRNA to silence specific genes. RNAi-based biopesticides are currently being developed to control various insect pests; however, not all insects are equally susceptible to RNAi. For her dissertation, Dr. Cooper investigated mechanisms influencing RNAi efficiency (i.e., susceptibility) in the European corn borer (ECB) and tested strategies for enhancing RNAi efficiency in ECB caterpillars. Her investigation revealed that ECB has all the correct machinery for RNAi, although some of those components may not work very effectively. In addition, dsRNA was rapidly degraded in ECB gut contents and hemolymph (i.e., blood), likely due to the activity of nucleases (i.e., PAC-MAN-like proteins) called dsRNases and REases. In contrast, dsRNA was stable in western corn rootworm tissues. Furthermore, some genes in ECB are more susceptible than others to RNAi-mediated silencing. To overcome these issues, Anastasia tested a variety of RNAi efficiency-enhancing reagents, including transfection reagents, nanoparticles, and nuclease inhibitors, that are designed to improve dsRNA stability and/or cellular uptake of dsRNA. While some of the reagents did enhance dsRNA stability in ECB gut contents and hemolymph, none consistently improved RNAi efficiency. Together, these findings indicate that multiple mechanisms likely contribute to low RNAi efficiency in ECB. During this investigation, Anastasia developed a qPCR method for quantitative measurement of dsRNA stability, and a tissue culture system for quick screening of target genes and investigation of potential factors affecting RNAi efficiency. The results of this investigation will facilitate the development of novel approaches that simultaneously overcome multiple RNAi efficiency limitations in lepidopteran insects.
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As a doctoral student in Insect Molecular Toxicology, Anastasia and her advisor collaborated with a group at Shanxi University in China to investigate mechanisms influencing RNAi efficiency in the migratory locust and Asian corn borer. The group also collaborated on several review articles related to chitin formation and degradation in the exoskeleton, and to the peritrophic matrix that lines the insect midgut.
Anastasia using a nanoinjector to deliver dsRNA to European corn borer caterpillars for one of her RNAi experiments at KSU.
Newly hatched European corn borer caterpillars feeding on artificial diet treated with dsRNA.
Excised gut tissue from European corn borer caterpillars maintained in tissue culture media inside a 96 well plate.
The role of plant volatiles in plant-plant defense signaling in response to a novel herbivore
A South American cactus borer, Cactoblastis cactorum (top), and a North American cactus borer, Melitara prodenialis (bottom). Photographs were taken by T. Marsico.
Small Creatures: Native and invasive cactus-boring moths
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Big Problems: Ecological damage and agricultural losses; Stem borers are hard to detect at early stages and/or control because they feed internally
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Biological Solutions: Use airborne signals to trigger defenses in naive host plants; Exploit easily observable plant defenses for early detection
Plants have evolved a variety of defenses to combat insect herbivory. Defense priming is one type of plant defense that involves the release of airborne chemical signals, called plant volatiles, in response to herbivory that activates defense pathways in other parts of the same plant and in neighboring plants, so undamaged plants are “primed” and ready to defend before an attack occurs. As a graduate student, Anastasia worked to develop plant volatiles as a potential control strategy and monitoring technique for the invasive South American cactus borer. She performed experiments to characterize defense transfer between prickly pear cacti, collected and identified plant volatiles released from defending and non-defending prickly pear cacti, and created hairy root cultures from prickly pear cacti to use as a bioproduction platform for mass production of cactus volatiles. The results of this investigation confirm that plant volatiles do mediate the transfer of defenses between cacti infested with the native North American cactus borer to cacti infested with the newly-associated, invasive South American cactus borer. During this investigation, Anastasia developed a continuous rearing procedure for North American cactus borers. Although largely unpublished, this investigation may facilitate the use of plant volatiles as a tool to monitor and combat insect pests that are invasive due to the lack of bottom-up control associated with co-evolutionary naïve host plants.
Anastasia in the laboratory with a potted prickly pear cactus at ASU.
The dynamic headspace sampling apparatus at Penn State that Anastasia used to collect plant volatiles released from prickly pear cacti undergoing herbivory from native and invasive cactus borers.
Anastasia eluting plant volatiles from collection traps for analysis and identification with gas chromatography-mass spectrometry and gas chromatography-flame ionization detection.
Hairy root cultures generated from cactus cuttings by Anastasia using Agrobacterium rhizogenes.
Undergraduate Research in Plant Ecology, Disease Ecology, and Parasitology
South American cactus borers, Cactoblastis cactorum (top), and plant defenses associated with feeding by North American cactus borers, Melitara prodenialis (middle & bottom). Upon feeding by the co-evolved North American cactus borers, plants gush thick yellow mucilage (middle) that prevents the caterpillars from boring into the plant and/or the entire segment turns black (bottom) and detaches from the plant thus preventing the caterpillars from consuming the entire plant. Photographs were taken by T. Marsico. The lower two are published in Woodard et al. 2012 Ecol Evol.
Small Creatures: Native and invasive cactus-boring moths
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Big Problems: Invasive species, like the South American cactus borer, cause ecological damage and agricultural losses
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Biological Solutions: Understand what factors facilitate biological invasions so new control, detection, and prediction strategies can be developed
Small Creature: Leprosy bacteria
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Big Problems: Leprosy is a disfiguring infectious disease that causes human suffering and disability. Environmental and animal reservoirs allow diseases, such as leprosy, to persist and circulate in nature
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Biological Solutions: Understand what factors are responsible for the patchy distribution of leprosy among armadillos in the southeastern US so that new control strategies and guidelines to minimize exposure and transmission can be developed
Small Creatures: Myxozoa and trematode parasites of fish
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Big Problems: Parasites that infect catfish cause economic losses for commercial catfish production
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Biological Solutions: Understand which fresh-water snails, fish, and fish-eating birds transmit these parasites so they can be excluded from commercial catfish operations
Plant Ecology
Biological invasions, facilitated by globalization, are one of many anthropogenic concerns affecting the world today. As an undergraduate student worker in Plant Ecology, Anastasia helped investigate factors and mechanisms that allowed the South American cactus borer to invade North America successfully. Specifically, she helped survey prickly pear cacti in the field for host plant defenses associated with cactus borer infestations, and she ran a differential defense experiment to completion. Together, she and her supervisor discovered that the North American prickly pear cacti fail to defend against the newly-associated South American cactus borer to the same extent that they do against the co-evolved North American cactus borer, and showed that North American prickly pear could be induced to defend against the invasive cactus borer when reared in the presence of plants actively defending against the native cactus borer. These findings indicate that defense-free space and lack of bottom-up control associated with co-evolutionarily naïve host plants likely facilitate the invasion of the South American cactus moth in North America, and that defense limitations associated with novel herbivorous insects can be overcome by airborne chemical signals which trigger defenses in neighboring plants. This investigation advances the understanding of mechanisms that enable certain species to establish populations in new locations and may aid in predicting and controlling invasive species.
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As a student worker in Plant Ecology, Anastasia also helped maintain cacti in the greenhouse, mounted and cataloged herbarium specimens, and assisted with the field portion of a river cane restoration study.
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Anastasia surveying a prickly-pear cactus in Florida for the presence of cactus-boring moths.
Disease Ecology
As human populations grow, encroachment into natural habitats is increasing. Thus, human contact with wildlife that carries zoonotic diseases (diseases that spread between animals and people) is ever more critical for human health. To understand the risk that wildlife diseases pose to humans, the physiological constraints and ecological interactions that govern the range limits of hosts and pathogens must be understood. As an undergraduate student worker in Disease Ecology, Anastasia helped investigate the effects of environmental and host genetic variation on the patchy distribution of leprosy infection in nine-banded armadillos in the southeastern U.S.. Specifically, she netted armadillos, collected blood and tissue samples, helped design and use microsatellite markers to investigate genetic factors responsible for the susceptibility of armadillos to leprosy, and created ArcGIS files to explore the role of environmental factors on the distribution of leprosy in armadillos. Ultimately, the group documented little genetic variation among armadillo populations in the Southeastern U.S. and found that local adaptation did not influence the geographic distribution of leprosy in armadillo populations. In addition, no correlation was found to exist between temperature, moisture, and leprosy prevalence in armadillos, indicating that either environmental factors are not a strong driver behind leprosy distribution, or the model used was lacking essential components. Together, these results provide a foundation for further study of genetic, ecological, and environmental factors contributing to variations in leprosy prevalence in armadillos and possible armadillo zoonosis in the southeastern U.S.. A better understanding of factors contributing to the prevalence of potentially zoonotic diseases will aid in developing new strategies to mitigate disease transmission to humans.
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As a student worker in Disease Ecology, Anastasia also helped survey cacti for cactus moth infestations in Florida for an invasion modeling project, and inspected fish gills for the presence of encysted mussel larvae, called glochidia, for a project investigating changes in glochidia prevalence after river modifications.
Anastasia and graduate student, Leah Chinchilla, with a baby nine-banded armadillo that they caught and sampled in Mississippi.
Parasitology
As a student worker in Parasitology, Anastasia collected parasites from the intestinal contents of pelicans, and helped maintained snail and catfish colonies for studies on the transmission of catfish parasites in aquaculture.