This thesis is composed of two chapters. Chapter one reviews what is known about adaptive plasticity in response to predators, describes the physiological systems involved in such plasticity, and outlines the evolutionary consequences of adaptive plasticity. Chapter two describes a scientific experiment that investigates how malathion may impact adaptive plasticity in the Sierran Treefrog, Pseudacris sierra. Anuran tadpoles suffer high mortality rates due to predation. In response to strong selective forces relating to these high predation rates, tadpoles evolved the ability to adaptively respond to predators through morphological and behavioral plasticity. The morphological and behavioral responses are varied and depend on the hunting strategy of the predator, and the adaptive responses may be influenced by other biotic and abiotic factors. Tadpoles detect alarm cues released from tadpoles being eaten and kairomones that are released by predators. Tadpoles respond to these signals by changing tail and body shape along with a reduction of activity level, which enables tadpoles to escape predators more effectively. These changes in morphology can occur within a week, and behavioral changes can occur within 15 minutes. The adaptive responses are critical for increasing survival rates of tadpoles to metamorphosis and may have important evolutionary consequences for anurans. Amphibians are in decline worldwide, and pollutants are considered to be a major contributor to these declines. Every year 5.2 billion pounds of active ingredients of pesticides are applied worldwide, and these application rates have led to ubiquitous low-level contamination of aquatic ecosystems. How low-level contamination of pesticides directly and indirectly affect how tadpoles respond to their predators is poorly understood. One potential indirect effect of pesticides is the inhibition of adaptive plasticity. Pesticides have been shown to modulate corticosterone levels in tadpoles. Corticosterone is the most likely mediator of the physiological response that results in adaptive morphological change. If the physiological system of tadpoles relies on corticosterone as the mediator of adaptive response, and pesticides can modulate corticosteone levels, then pesticides may inhibit or negatively impact adaptive responses to important biotic factors, like predators. Pesticides have been shown to weaken immune systems, affect developmental and physiological pathways that lead to malformations, and cause direct mortality in anurans. Little research has investigated the effect of pesticides on adaptive morphological and behavioral plasticity in response to predators. Adaptive phenotypic responses to predators increase survival rates to metamorphosis and are important in stabilizing amphibian populations through time. If pesticides influence the ecological interactions of tadpoles and their predators, this could play a part in amphibian declines. In the experiment explained in Chapter two, I tested the hypothesis that malathion at a concentration of 0.1 mg/L inhibits anti-predator morphological and behavioral responses of Pseudacris sierra to the predatory dragonfly larvae Anax junius. The results of this experiment show that malathion alone caused the tail muscle depth to increase to the same magnitude as tadpoles that only experienced a predator’s presence. Malathion also caused a significant increase in tail depth, demonstrating that malathion directly causes morphological change. The experiment did not support the hypothesis that malathion inhibits adaptive plasticity, and malathion had no impact on behavioral plasticity. The results from this experiment give evidence that an ecologically relevant concentration of malathion can influence morphological components that are critical in escaping depredation events, which could affect predator-prey interactions.

Understanding how organisms respond to climatic variability and novel conditions is becoming an increasingly important task for ecologists. For ectotherms in the northern hemisphere, the response to cold is of special interest, considering that poleward range expansion events and increasing variability of temperatures during winter are already being observed as consequences of a warming planet. Though direction of change in physiological variables in response to cold is well studied in ectotherms, the extent to which traits can change and the rate at which they can change is not. We compared the extent and rate of change in cold tolerance (CTmin) between two long-term captive populations of the Italian wall lizard (Podarcis siculus) during a lab cold-acclimation treatment. Heat tolerance (CTmax), thermal preference (Tpref), temperature dependent rates of oxygen consumption (SMRO2), and temperature dependent rates of water loss (EWL) were also compared between Italian wall lizards previously introduced to Long Island, NY and San Pedro, CA before and after the lab cold acclimation treatment. Because our study coincided with a cold snap during the spring 2018 season for the San Pedro, CA population, we also studied the effects of cold acclimatization on wild lizards from the CA population. After initial lab acclimation of the lizards to laboratory conditions, SMRO2 at 15°C and EWL at 10°C were higher in NY lizards compared to CA lizards. Lizards from the two populations did not differ in any other variables measured before the cold acclimation treatment. We found that lizards from the NY population experienced an 80% decrease in CTmin following a switch from 20°C:18°C to 17.5°C:16°C (12h light:12h dark) acclimation treatment. Lizards from the CA population did not decrease CTmin in response to the same cold acclimation treatment. Overall, NY lizards decreased CTmin, CTmax, and Tpref following cold acclimation, whereas CA lizards decreased CTmax only. Wild CA lizards decreased CTmax following the cold spring 2018 season in a manner similar to that of lab acclimated NY and CA lizards, suggesting that these lizards do not maintain a high CTmax when the environment is unlikely to expose them to high temperatures. Thermal sensitivity (Q10) of SMRO2 and EWL was lower in NY lizards, suggesting physiological adaptation to fluctuation in diurnal temperatures. The ratio of CO2 produced to O2 consumed (respiratory exchange ratio, RER) measured at 15°C increased in NY lizards following cold acclimation suggesting an increased use of carbohydrates and/or an increased production of lipids in the colder conditions. These responses in combination with the higher observed plasticity in NY lizards are in accordance with the climatic variability hypothesis, which predicts that organisms from more variable climates will be better adapted to physiologically respond to variable conditions. The higher capacity for physiological plasticity may explain the relatively high success of P. siculus in NY and other northern U.S. states. By describing the rate of change of CTmin during cold acclimation we hope to better understand how these lizards minimize the risk of low temperature exposure during winter. We ultimately hope to incorporate the rate at which cold tolerance can change into predictions of species distributions and hypothesis tests investigating the relationship between climatic variability and the rate at which animals can exhibit plasticity.

Molting in arthropods is a complex process governed by regulatory mechanisms that have evolved and adapted over millennia to allow these animals to grow, despite being confined by a hardened exoskeleton. We isolated the molt-regulating Y-organs (YO) from the common shore crab Carcinus maenas at molt stages B, C1-3, C4, and D0 to assess how changes in protein abundances might underline the unique physiology of each of these stages. We found that changes in protein abundance were most notable in the postmolt stages (B and C1-3), where an increase in energy metabolism and the reactive oxygen species stress (ROS) response proteins was observed. An increase in triosephosphate isomerase and transketolase suggest that the postmolt YO is participating in triglycerides storage and is also actively recycling excess ribose sugars manufactured during the YO’s previously activated state. We also propose as mechanism through which ROS-induced release of cyclophilin A may contribute to YO atrophy during postmolt through the remodeling of structural proteins such as collagen. We support the standing observation of YO atrophy during postmolt by drawing attention to hemolymph protein abundances, especially those of cryptocyanin isoforms, which dropped precipitously in intermolt (C4) and remained at low abundances into early premolt (D0). Finally, though our evidence is preliminary, we propose that future investigations into the YO proteome address the significance of the protein glutamate dehydrogenase. Glutamate dehydrogenase, a key enzyme involved in the formation of glutamate, represents a potential nutrient-sensing checkpoint that might be involved in YO activation. Historically, most attention has gone to the acute molt stages, where signaling mechanisms involved in the activation of the YO have been the focus. Here, we present data suggesting that other regulatory mechanism may be governing the atrophy the postmolt YO. A better understanding of crustacean physiology has the potential to benefit ecosystems and economies worldwide.

This thesis successfully integrates laminate microfluidic devices with an analytic Surface Plasmon Resonance (SPR) instrument. Integration was accomplished at low-cost using materials such as polydimethylsiloxane (PDMS), Poly(methyl methacrylate) (PMMA), Tygon tubing, and a 3-way stopcock. The main components of this thesis are the design and fabrication of the low-cost, in-house fluidics that can integrate with upstream microfluidics and the validation of the in-house fluidics using the Biosensing Instruments BI-2000 SPR instrument. The low-cost fluidics was designed and fabricated “in-house” using a novel investment casting technique that required the use of laser cutting technology to make a master cast, and candle wax to make the fluidic flow gasket. Integration of upstream microfluidic devices is the next step towards fully integrated point-of-care (POC) diagnostics. Development of low-cost POC diagnostics will enable physicians to diagnosis patients outside of clinical settings, granting treatment access to a much wider population. Surface Plasmon Resonance is used for its detection abilities combined with its ability to perform real-time sample analysis. Validation of the in-house fluidics was accomplished by conducting (2) experiments: (1) to compare the angular shift elicited by ethanol solutions between in-house fluidics, factory fluidics, and the literature, and (2) to compare the angular shift between in-house fluidics and factory fluidics caused by the cleaving of fibroblasts from the SPR sensor chip. Successful comparisons made in both experiments proved successful development of low-cost fluidics that could integrate upstream microfluidic devices.

In this work, a novel microfluidic cell culture platform capable of automated electrical impedance measurements and immunofluorescence and brightfield microscopy was developed for further in-vitro cellular research intended to optimize cell culture conditions. The microfluidic system design, fabrication, automation, and design verification testing are described. Electrical and optical measurements of the 16 parallel cell culture chambers were automated via a custom LabView interface. A proposed design change will enable gas diffusion, removing the need for an environmental enclosure and allow long-term cell culture experiments. This "lab on a chip" system miniaturizes and automates experiments improving testing throughput and accuracy while creating a highly controllable microenvironment for cell culture. Such a system can be applied to drug development, bioassays, diagnostics, and animal testing alternatives. This work is part of a collaborative effort to define protocols for the electrical and optical characterization of cell culture within a novel microfluidic device with the intent of optimizing microenvironment conditions.

Endocrine disrupting chemicals (EDCs) are compounds that can interfere with hormone signaling pathways and are now recognized as pervasive in estuarine and marine waters. One prevalent EDC in California’s coastal waters is the xenoestrogen 4-nonylphenol (4-NP), which has been shown to impair reproduction, development, growth, and in some cases immune function of marine invertebrates. To further investigate effects of 4-NP on marine invertebrate immune function we measured total hemocyte counts (THC), relative transcript abundance of immune-relevant genes, and lysozyme activity in Pacific oysters (Crassostrea gigas) following bacterial infection. To quantify these effects we exposed oysters to dissolved phase 4-NP at high (100 μg l-1), low (2 μg l-1), or control (100 μl ethanol) concentrations for 7 days, and then experimentally infected (via injection into the adductor muscle) the oysters with the marine bacterium Vibrio campbellii. 4-NP significantly altered the effects of bacterial infection had on THC. Oysters exposed to both high and low 4-NP did not experience a bacteria-induced increase in THC, as seen in control oysters. We also determined that V. campbellii infection induced differential expression of a subset of immune-related genes tested (Cg-bigdef2, Cg-bpi1, Cg-lys1, Cg-timp) in some, but not all, tissues; 4-NP exposure altered expression patterns in two of these genes (Cg-bpi1 and Cg-tgase). Exposure to 4-NP alone also caused differential expression in some genes (Cg-bpi1, Cg-galectin1, Cg-clec2). Lastly, low levels of 4-NP significantly increased lysozyme activity 24 h post-infection. These results suggest that exposure to 4-NP can alter both cellular and humoral immune responses to bacterial infection in C. gigas.

BACKGROUND: All human societies have developed ways of maintaining health, dealing with illness and injury in ways that conform to their culture and environment. People worldwide draw upon a variety of healing systems, therapeutic methods, practices, and products, often referred to as Traditional and Complementary Medicine (T&CM). The World Health Organization (WHO) has encouraged member states to develop national policies that advance the integration of T&CM in national healthcare systems to harness their potential contribution to health, wellness, and people-centered health care. Yet, no global guidelines have been developed for assisting countries in structuring and developing such policies, and little is known about the characteristics of existing T&CM policies in Latin America and their level of implementation. METHODS: A qualitative research design was used to conduct a landscape analysis characterizing existing therapeutic pluralism policies in Latin American countries through a comprehensive literature review, a policy focused qualitative content analysis, key informant interviews and a case study analyzing the formulation process of Brazil's National Policy for Integrative and Complementary Health Practices (PNPICS). A technical cooperation tool to guide T&CM policy development and revision for Latin American countries was refined through face validation and an expert consensus method (Delphi process). Data categorization and analysis were performed in MS Excel and NVivo, using deductive and inductive coding. RESULTS: A total of 74 T&CM policy documents from the 16 Latin American countries were identified and characterized according to policy mechanisms. A typology of Latin American policy approaches identified policies as: health services-centered, model of care-based, participatory, and indigenous people-focused. Selection of T&CM practices for policy inclusion vary across countries; criteria change over time in Brazil might have compromised PNPICS’ political status. Lack of PNPICS financing jeopardized its implementation. A technical cooperation tool for T&CM policy development was structured on the policy cycle: national situation analysis, policy formulation, policy implementation, policy monitoring and evaluation, and policy re-formulation, re-prioritization for incremental policy developments. CONCLUSION: With a few exceptions, therapeutic pluralism policy implementation in Latin America seems to be a real challenge, often reducing policies to cultural and political symbols. Continued research is needed on assessing the various stages of the policy process in T&CM. / 2023-09-10T00:00:00Z

The current gold standard treatment for bone defects is autologous cancellous bone graft, which involves increased surgery time and donor site morbidity, and limited supply of bone and cells for regeneration. Bioreactors may aid in the generation of mechanically conditioned bone grafts with more cells compared to traditional grafts. However, the specific parameters of fluid flow and mechanical loading which contribute to osteogenesis and cell viability in bioreactors are not fully characterized. Here, a perfusion-compression bioreactor system was developed to study osteogenesis in porcine trabecular bone explants. Loading accuracy was over 88% across six bioreactors at a 0.1 s-1 strain rate and 20 N target force, akin to running. A flow rate of 0.2 mL/min appeared to be more favorable for cell viability than 1 mL/min. Overall, this work offers a foundation for future efforts to enhance cell viability and osteogenesis in bone explants.

Clay-based compounds are globally the most advanced and widely used method of direct suppression of marine harmful algal blooms, and are currently undergoing investigation as an option to control Karenia brevis blooms in Florida. Before clay may be accepted for widespread use, there are multiple concerns and challenges that must be addressed regarding the environmental safety of this method, such as effects on water quality, the fate of toxins, and potential impacts of clay treatment to non-target organisms. To contribute to ongoing assessments of clay as a potential control method for K. brevis blooms, we conducted experiments with a formulation of kaolinite clay modified with polyaluminum chloride known as Modified Clay II (MC II). In these experiments, we evaluated water chemistry dynamics and physiological responses in several bottom-dwelling marine species with ecological and economic significance, including blue crab (Callinectes sapidus), sea urchin (Lytechinus variegatus), and hard clam (Mercenaria campechiensis). First, we conducted an experiment with blue crabs in 20 L aquarium tanks (N = 48), exposing the animals to cultured K. brevis (1 x 106 cells L-1) and MC II (0.5 g L-1) and measuring mortality and reflexes over 192 hours. In our second experiment, K. brevis (1x106 cells L-1) and MC II (0.2 g L-1) were applied to 1,400 L mesocosms (N = 9) containing blue crabs, sea urchins, and hard clams, which were observed over 96 hours. In our final experiment, we modified the methods of the previous experiment to again examine K. brevis (1x106 cells L-1) and MC II (0.2 g L-1) with the same model species in 1,400 L mesocosms (N = 12) over 72 hours. In these two mesocosm experiments, we observed cell and toxin removal, changes in water quality characteristics including nutrients and carbonate chemistry, and measured mortality, respiration rate, reflexes, and internal toxin content. Our results were congruent across our three experiments. Treatment with MC II significantly reduced cell concentrations but did not reduce toxin concentrations in the water column. We found no notable impacts of clay treatment to reflexes, respiration rates, or internal toxin content for either of our three species. No significant differences in mortality were found for our three species, excluding crabs in the first mesocosm study, which were found to have pre-existing infections that confounded our results. Analyses of nutrients indicates MC II may remove dissolved phosphorus from the water column, and the potential to improve water quality which may make this formulation of clay desirable to managers. Overall, treatment with this formulation of clay did not appear to induce any significant measured effects on the model species within the observed time frames of these experiments. Clay appears to be a promising option to treat K. brevis blooms given its low cost, ease of application, and negligible impacts to the environment, and its use may relieve the damaging effects of K. brevis blooms by preventing mortalities that would otherwise occur were blooms allowed to persist. We therefore recommend that clays, including MC II, be considered for additional laboratory and field tests, with the goal of obtaining further information on potential ecological impacts so that managers and researchers can make informed decisions on the use of bloom control technologies in Florida waters.

The variation in the insect immune system is an important regulator of insect populations and the pathogens they carry. A central component of insect immunity is melanin, whose production creates cytotoxic intermediates that help to protect against a broad spectrum of pathogens. Melanin is also used in insect cuticle where it helps to improve thermoregulation and desiccation resistance, with insects having less melanized cuticles in warmer and more humid environments. Considering that cuticle melanin and immune melanin are formed by near identical biochemical pathways, they are pleiotropically linked (that is, one or more linked genes influence multiple traits). This has lead to the cuticle-dependent immune investment (CDII) hypothesis, which states that adaptive responses in the cuticle can lead to non-adaptive changes in immunity and could lead to an increase in transmission of insect vectored pathogens in warming climates, due to a weaker defense against the pathogen. However, the impact of CDII on cuticle melanin and immunity, as well as infection prevalence and intensity, under seasonal conditions in the field is still unclear. In this project, we study a population of Asian citrus psyllids, Diaphorina citri, in the field over four seasons. Diaphorina citri vectors a Gram-negative bacteria, Candidatus Liberbacter asiaticus (CLas), that is responsible for Huanglongbing, aka citrus greening disease, which has cost the Florida citrus industry several billion dollars. We assess pathogen load of CLas by quantitative PCR, and assess levels of phenoloxidase activity in the insect hemolymph to measure insect immune function. We assess levels of cuticle melanin. Our results show a significant correlation between temperature, cuticle melanin, and immune function. However, the affect of seasonality on infection prevalence and intensity remains unclear. THE EFFECT OF CLIMATE ON PHYSIOLOGY AND IMMUNE FUNCTION IN THE ASIAN CITRUS PSYLLID, DIAPHORINA CITRI