THE IMPACTS OF ENVIORNMENTAL PERTUBATIONS ON LIFE HISTORY TRAJECTORIES IN CAENORHABDITIS ELEGANS. by PRECIOUS ALEXANDRIA DE VERTEUIL A DISSERTATION Presented to the Department of Biology and the Graduate School of the University of Oregon in partial fulfillment of the requirements for the degree of Doctor of Philosophy June 2020 DISSERTATION APPROVAL PAGE Student: Precious Alexandria de Verteuil Title: The Impacts of Environmental Perturbations on Life-History Trajectories in Caenorhabditis elegans. This dissertation has been accepted and approved in partial fulfillment of the requirements for the Doctor of Philosophy degree in the Department of Biology by: Judith Eisen Chairperson Patrick C. Phillips Advisor William Cresko Core Member Carrie McCurdy Core Member Josh Snodgrass Institutional Representative and Kate Mondloch Interim Vice Provost and Dean of the Graduate School Original approval signatures are on file with the University of Oregon Graduate School. Degree awarded June 2020 ii © 2020 Precious Alexandria de Verteuil iii DISSERTATION ABSTRACT Precious Alexandria de Verteuil Doctor of Philosophy Department of Biology June 2020 Title: The Impacts of Environmental Perturbations on Life-History Trajectories in Caenorhabditis elegans. Environmental fluctuations are ubiquitous in nature and can serve to drive phenotypic differences among individuals in an environment-specific manner, a phenomenon known as phenotypic plasticity. Phenotypic plasticity can have implications for an organism’s fitness. Here, I address how two distinct environmental perturbations (acute nutrient deprivation and treatment with a compound known to extend lifespan) impact individual life-history traits within and across generations using the Caenorhabditis elegans nematode model system. To quantitively assess the impacts of acute maternal starvation, I engineered a novel microfluidic device to starve adults and simultaneously collect progeny with fine- scale temporal resolution. I found no evidence for changes in maternal provisioning of embryos (egg size) laid under acute maternal starvation, highlighting that, even in the face of limited nutrient availability, equal investment is provided to embryos in-utero. This consistency in provisioning is further evidenced by the fact that I also found no significant changes to life history traits such as lifespan and reproductive output in offspring produced by starved parents. To quantitively assess the role that timing of application of a compound iv previously identified lifespan extension (Thioflavin T) might play in shifting longevity responses, I contrasted early intervention responses from treatment early in development to those observed after treatment as adults. Here, I identify a novel, recoverable, developmental delay state induced by Thioflavin T treatment in a dose-dependent manner after larval treatment. These effects include disruption of normal development and increased early life mortality while on the compound, as well as decreased reproductive output and, importantly, increased longevity, after recovery following removal from the compound. Using mutants in known stress response pathways to assess specificity of response, it appears that developmental exposure results in a general hormetic stress response acting across multiple stress response systems. Overall my dissertation explores the way environmental perturbations affect life history trajectories within and across generations. Using a combination of novel experimental approaches and high throughput techniques, I find that the major drivers of phenotypic plasticity in my experiments to be the type of environmental stressor and the age of treatment onset. This work includes unpublished coauthored material. v CURRICULUM VITAE NAME OF AUTHOR: Precious Alexandria de Verteuil GRADUATE AND UNDERGRADUATE SCHOOLS ATTENDED: University of Oregon, Eugene Barry University, Miami Shores Florida DEGREES AWARDED: Doctor of Philosophy, Biology, 2020, University of Oregon Bachelor of Science, Biology, 2014, Barry University AREAS OF SPECIAL INTEREST: Population Genetics Developmental Biology GRANTS, AWARDS, AND HONORS: Deans List, University of Oregon, 2014 Research Fellowship National Institute of Aging, University of Oregon, 2015- 2018 Genetics Training Grant Diversity Training Fellow, University of Oregon, 2015- 2017 Diversity Equity and Inclusion Award, University of Oregon, 2018-19 PUBLICATIONS: Maxime Jean, Precious deVerteuil, Joshua Tapia, Nicole Lopez, Brenda Schoffstall. Adult Zebrafish Hearts Efficiently Compensate for Excessive Forced Overload Cardiac Stress with Hyperplastic Cardiomegaly. BioResearch Open Access 2012. Volume 1, No. 2, pg. 88-91. vi S-q Shi, MJ white, HM Borsetti, JS Pendergast, A Hida, CM Ciarleglio, PA de Verteuil, AG Cadar, C Cala, DG McMahon, RC Shelton, SM Williams, and CH Johnson. Molecular analyses of circadian gene variants reveal sex-dependent links between depression and clocks.Translational Psychiatry Open Access 2016. doi:10.1038/tp.2016.9 vii ACKNOWLEDGMENTS I wish to express sincere appreciation to my dissertation advisor Dr. Patrick C. Phillips for all his guidance, support and mentorship. He has been an invaluable resource for successfully navigating a complex academic setting as a first-generation underrepresented minority student. I would also like to thank all of my committee members, my chair Judith Eisen, my core members William Cresko and Carrie McCurdy and my institutional representative Josh Snodgrass for their and valuable insights and contributions to my dissertation work. This work would not be possible without the continued support of the laboratory personnel and resources. I would like to thank all my fellow lab members for their contributions to this work, specifically laboratory technician Christine Ann Sedore for her technical expertise and assistance in compound intervention applications and automated lifespan assays. Dr. Stephan Banse for his collegial support and assistance in manuscript editing. Dr. Gavin Woodruff for his assistance in quantitative developmental techniques. My undergraduate students Juliana Rantisi, Natalia Narh, and Runpeng Nie. Fellow graduate students Katja Kasimatis, Heather Archer, Christine Oconnor and Zachary Stevenson for all their support. Special thanks to my husband Frank Yeboah for all his love and support, and my parents Rory and Jacqueline de Verteuil. The investigation was supported in part by a Research Fellowship Grant through the National Institutes of Aging at the University of Oregon Grants U01 AG045829, U24 AG056052, and R01 AG056436 to Patrick C. Phillips. viii To my Hero Rory Anthony John de Verteuil ix TABLE OF CONTENTS Chapter Page I. INTRODUCTION .......................................................................................................... 1 Dissertation Outline ................................................................................................... 05 II. THE IMPACT OF MATERNAL STARVATION ON PROGENY FITNESS IN CAENORHABDITIS ELEGANS ...................................................................................... 07 Introduction ................................................................................................................ 07 Methods ..................................................................................................................... 10 Nematode Strains and Maintenance ........................................................... 10 Microfluidic Device Fabrication ................................................................. 10 Microfluidic Perfusate ................................................................................ 11 Loading the Microfluidic Device ................................................................ 11 C. elegans Husbandry in the Microfluidic Device ..................................... 12 Progeny Collection ..................................................................................... 12 Progeny Maintenance Post Microfluidic Capture ....................................... 13 Lifespan Measurements .............................................................................. 13 Reproduction Measurements ...................................................................... 13 Embryo Size Measurements ....................................................................... 13 Results ........................................................................................................................ 14 Design of the Embryo Gathering Gadget (EGG)........................................ 14 Starvation in EGG-Chip Alters Egg Laying ............................................... 16 Maternal Starvation in EGG-Chip Does Not Alter Progeny x Chapter Page Lifespan ...................................................................................................... 18 Maternal Starvation in EGG-Chip Does Not Alter Progeny Fecundity .................................................................................................... 19 Embryo Size is Not Altered by Parental Adult Starvation ......................... 20 Discussion .................................................................................................................. 21 Bridge ........................................................................................................................ 23 III. LARVAL EXPOSURE TO LIFESPAN EXTENDING COMPOUND THIOFLAVINT DELAYS DEVELOPMENT AND ALTERS ADULT LIFESPAN IN CAENORHABDITIS ELEGANS ............................................................. 25 Introduction ................................................................................................................ 25 Methods ..................................................................................................................... 27 C. elegans Strains and Culture .................................................................... 27 Thioflavin T (ThT)...................................................................................... 28 Larval Exposure to ThT .............................................................................. 28 Larval Recovery from ThT ......................................................................... 29 Length Measurements ................................................................................. 29 Lifespan Analysis ....................................................................................... 30 Pharyngeal Pump Rate ................................................................................ 30 Fecundity .................................................................................................... 30 RNAi ........................................................................................................... 31 Results ........................................................................................................................ 32 xi Chapter Page Developmental Exposure to [ThT] Induces L3 Like Developmental Delay ................................................................................. 32 Larval Pharyngeal Pump Rate Decreases with Respective [ThT] .............. 34 ThT Delayed Progeny are Sensitive to 1% SDS Exposure......................... 35 Larval Recovery Rate Post [ThT] Exposure ............................................... 38 Larval ThT Exposure Alters Adult Lifespan .............................................. 38 Larval ThT Exposure Reduces Adult Fecundity ........................................ 42 Discussion ........................................................................................................................ 43 Larval ThT Exposure Induces Novel Developmental Delay ...................... 44 ThT Induced Larval Developmental Delay Results in Adult Fitness Tradeoffs ..................................................................................................... 44 Conclusion ....................................................................................................................... 46 IV. CONCLUSION ......................................................................................................... 47 REFERENCES CITED ................................................................................................... 51 xii LIST OF FIGURES Figure Page 1. The Egg Gathering Gadget (EGG) Platform ............................................................. 16 2. Egg Laying Behavior of Adults in EGG .................................................................... 18 3. C. elegans Adult Lifespan Post Being Laid in EGG ................................................. 19 4. Fecundity of Adults Post Being Laid in EGG ........................................................... 20 5. Embryo Circumference .............................................................................................. 21 2.1 Larval Exposure to ThT Delays Development .......................................................... 33 2.2 ThT Exposure Reduces Larval Pharyngeal Pump Rates ........................................... 35 2.3 ThT Exposed Larvae are Sensitive to 1% SDS Exposure ......................................... 37 2.4 Larval ThT Exposure Alters Adult Lifespan ............................................................. 41 2.5 Larval ThT Exposure Reduces Adult Fecundity ....................................................... 42 xiii CHAPTER I INTRODUCTION Scientists have grappled to understand life history theory and aging since the early 20th century. While we have come a long way since the works of theorist August Weismann (Weismann et al. 1904) and naturalist Alfred Russel Wallace (Bulmer 2005), known most for their contributions to the theory of natural selection, in 2020 we are still discovering new paradigms and empirical evidence to better understand the biological foundations and ecological context of senescence (Gladyshev 2016). Historically, senescence has presented itself as an evolutionary paradox because it is assumed that under favorable conditions evolution should prevent aging so as to maximize survival and reproductive success over the lifetime of an individual. This led to the assumption that natural selection favors a “death mechanism” that allows room for younger and more reproductively prolific individuals to enter the population, thereby ensuring the long-term survival of the species (Moorad 2016). While this assumption seems plausible, it has been proven incorrect. J.B.S. Haldane and Peter B Medawar demonstrated that under equivalent reproductive output, longer lived individuals produce more offspring than short lived individuals and that the cost of death exceeds the benefit of the group species. The force or strength of natural selection (a measure of how strongly selection acts on survival and/or reproduction) declines as a function of age is a major theoretical insight that was later mathematically formalized by William D. Hamilton (Akazawa 2016; Flatt & Partridge 2018). In his proof Hamilton describes what is known as the “Selection Shadow” that represents the part of the lifespan post birth and reproductive maturity in 1 which the force of natural selection declines rapidly with age prior to death. The selection shadow prevents natural selection from acting on deleterious mutations that are constrained to late life stages(Kirkwood & Austad 2000). In addition, there is the potential for various mutations to have pleiotropic effects such as being early life beneficial and late life deleterious for the same trait(WELLS 2003). It is likely that such mutations will be passed on to offspring of the individuals bearing it and that selection will thus be inefficient at eliminating such mutations from the population. This concept of the declining force of selection is the fundamental basis for the evolutionary theories of aging we study today. Even in this new era of scientific research with advanced genomics and CRISPR- Cas-9 gene editing technologies(Doudna 2017) there is still some debate about the evolutionary theory of aging and weather numerous small-effect genes, or few large- effect genes are responsible for the biological aging process (Flatt & Partridge 2018). The notion of mutation accumulation was first proposed by Medawar in 1952, suggesting that deleterious late life constrained mutations can rise in population frequency due to weakening selection. Huntington’s Disease is one classical example of said phenomena, in addition to Alzheimer's and Parkinson’s Disease. There is also empirical evidence in support of manipulations that result in biological fitness tradeoffs among various life history traits (Gladyshev 2016). Significant environmental perturbations experienced early in life can shift the allocation of resources between somatic maintenance and reproduction, this can drive adverse fitness consequences to occur later. Nutrient deprivation (caloric restriction) in-utero is the most consistent non-genetic mechanism 2 that can extend lifespan; however, it comes at a cost to growth rates, obesity, diabetes, cardiovascular disease and schizophrenia. These and other types of biological fitness tradeoffs have been studied by scientists for centuries, in fact some of the first empirical evidence to support biological fitness tradeoffs was gathered during the Dutch Famine of 1944. Multiple longitudinal studies have examined the relationship between nutrient deficiency and progeny fitness (Lumey et al. 2012; Stein et al. 2009). However, even using cohort sizes of 900+ individuals it is hard to uncouple correlation and causation with respect to fitness tradeoffs. Furthermore, the results of most studies do not account for any genetic predisposition to adverse fitness consequences which we now know to be a potential mitigating factor in population fitness trajectories (Kogenaru et al. 2009; Orr 2009). Alternative animal models for understanding the role of genetic variation and how it results in alternative fitness trajectories include but are not limited to Saccharomyces Cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans (C. elegans). In comparison to Saccharomyces Cerevisiae and Drosophila melanogaster, C. elegans has a well annotated stress response network and its insulin signaling pathway is homologous to the FOXO pathway in humans. Thanks to Sydney Brenner establishing C. elegans as a model, the scientific community has been using C. elegans to examine the effects of nutrient deprivation and environmental perturbations for centuries (Lee et al. 2013; Baugh 2013) Experimental findings within the last decade highlight alternative gene expression profiles associated with L1 arrest (Maxwell et al. 2012). Furthermore, empirical evidence has shown that nutrient deprivation can lead to a maximal lifespan extension in C. elegans through the induction of daf-16 (Ching & Hsu 2011). Additional 3 studies have also demonstrated that alternative developmentally programmed arrest states like dauer can also impact life history traits (Ludewig et al. 2013). Apart from nutrient deprivation, compound interventions and genetic mutations can also affect population lifespan trajectories. Within the last few years, studies supported through the National Institute of Aging have uncovered various compounds that under sustained treatment have the potential to reverse the aging process (Phillips et al. 2018). Screening various compounds for their effects on aging has advanced our understanding about genes and fundamental pathways involved in the aging process that are conserved across species. While there are various model systems that are applicable to understanding senescence of aging, few allow for rapid high throughput population- based screening coupled with advanced genomics. C. elegans is a great model system to explore biological fitness tradeoffs, and population wide lifespan extension fitness trajectories (Tissenbaum 2014). In addition to its robust life cycle (embryo – adult within 4 days), C. elegans also has well characterized stress response pathways and an insulin signaling pathway homologous to humans (Kim et al. 2014). In addition to all the genomic resources, C. elegans also has an average lifespan of 25-30 days, making it an ideal candidate for understand the aging process within and across generations. One outstanding question that is now starting to be addressed in the study of senescence is how epigenetic changes via nutrient deprivation or compound intervention can impact fitness trajectories within and across multiple generations of offspring. C. elegans is the best aging model system to screen for multigenerational effects in a rapid, high throughput manner. However, recapitulating some specific senescence paradigms, 4 such as maternal starvation, are more of a challenge than others due to physiological and biological constraints of the organism. Dissertation Outline For my dissertation I strengthen our understanding of how environmental perturbations impact life history trajectories in within two distinct paradigms (nutrient deprivation and compound intervention): through the development of a novel microfluidic device (which allows for high throughput population based starvation and progeny collection simultaneously), and through developmental exposure to a lifespan extension compound Thioflavin T (ThT). In Chapter I, I describe how I used microfluidic technology to engineer a novel maternal starvation chip to measure the impact of maternal starvation on progeny fitness trajectories. In this chapter, I highlight how, unlike mammalian systems, C. elegans under acute adult starvation do not alter their allocation of resources but rather appear to invest equally in embryos laid. This results in no overall changes to progeny lifespan trajectories or fecundity as adults, despite their parent experiencing acute starvation as adults. In Chapter II, I describe how early life exposure to the compound ThT induces a novel, recoverable, population wide, larval stage three developmental delay. Coupled with a high early life mortality and late life extension in adulthood. Furthermore, based on the novelty of my early life treatment paradigm, I am also able to measure the impact of lifespan extension on reproduction, and I find that it significantly reduces fecundity in adults post exposure. Lastly, I describe how early life compound exposure acts through a generalized hormetic stress response system, as opposed to the previously described HSF-1 and SKN-1 pathway in adulthood treatment. 5 Together, both chapters of my dissertation enhance our fundamental understanding of senescence by uncovering novelties that have the potential to constrain C. elegans ability to significantly restructure the allocation of resources post specific developmental timepoints, as well as discovering a novel developmental delay state induced through compound intervention that has the power to bypass previously characterized arrest response states. 6 CHAPTER II THE IMPACT OF MATERNAL STARVATION ON PROGENY FITNESS IN CAENORHABDITIS ELEGANS The microfluidic device described in this chapter was developed with this assistance of lab member Stephen Banse. My undergraduate mentee Juliana Rantisi contributed substantially to this work by participating in the experimental approach. Patrick C. Phillips and I developed the approaches. I wrote the manuscript and I am the primary investigator for this work. INTRODUCTION Encountering environmental perturbations are a universal feature of the life for all organisms. During periods of high nutrient availability (boom) there remains a positive association among life-history traits such as mating efficiency, lifetime reproductive success, and overall lifespan trajectories. During periods of low nutrient availability (bust) this association can turn negative, generating a trade-off of overall energy investment [1]. For example, in humans low nutrient availability in-utero is associated with health-related impacts later in life, including an increased risk of type-two diabetes, obesity, cardiovascular disease and cancer [2]. This mismatch between early-life and late life environments can result in a “thrifty phenotype” [3], leading to long-term fitness impacts in progeny even after later shifts to high nutrient availability and beneficial environmental conditions. The thrifty phenotype hypothesis postulates that this environmental mismatch is a result of epigenetic inheritance of adverse early-life conditions resulting in increased fat storage and nutrient rationing in subsequent generations [4]. Some early evidence regarding the long-term consequences of starvation 7 and nutrient deprivation on progeny fitness was gathered during the Dutch Famine of 1944-45. During WWII, limited food rationing had significant implications for pregnant mothers, resulting in major health implications for children such as stunted growth, increased rates of obesity, diabetes, cardiovascular disease and schizophrenia [5]. Today, we still find that malnutrition and caloric restriction in-utero is linked to adverse health conditions, however, we also find caloric restriction to be the most consistent non-genetic mechanism that can extend lifespan in mammals [6]. Indeed, dietary restriction in the absence of malnutrition can extend the median and maximal lifespan across a wide variety of organisms, including yeast, worms and flies [6]. The Insulin Signaling Pathway (IIS) is conserved across these species, and its activation in known to accelerate aging [7]. For example, the conserved Forkhead box O (FOXO) pathway within the IIS from animals as diverse as humans and Caenorhabditis elegans (C. elegans) roundworms has been shown to mediate these caloric restriction lifespan extension effects [8]. While the mechanisms for the role that this pathway may play in regulating lifespan in humans is still poorly understood, understanding the impact of caloric restriction in the C. elegans is likely to point the way towards understanding the underlying mechanisms of life-span effects in general [9]. Multiple studies have demonstrated that the strength of caloric restriction, starvation, or malnutrition, as well as the temporal pattern of the onset of nutrient limitation, are linked to the severity of downstream consequences [10]. Furthermore, there is evidence that epigenetic changes in gene expression profiles persist for multiple generations following the initial environmental perturbation [11]. These changes are independent of the current environmental conditions experienced by the individual. 8 Here, we sought to examine if the effects observed during nutrient deprivation early in an individual’s life are also observed when that individual’s parent is starved, rather than the individual itself (i.e., intergenerational embryonic effects). Within the C. elegans model system, traditional methods used to evaluate adult starvation are highly problematic because of the high loss of hermaphrodites from desiccation, burrowing, and facultative vivipary. Facultative vivipary is somewhat unique to nematodes, in which unlaid embryos hatch, grow and develop within the gonad of the hermaphrodite, killing the parent/mother [12]. In order to maintain normal reproductive patterns while achieving complete, acute adult starvation, we developed a novel microfluidic device (“chip”), the Embryo Gathering Gadget (EGG), to induce adult starvation in C. elegans while simultaneously collecting progeny in the form of laid eggs. Our new device allows for the separation of brood at specific time intervals, giving us temporal resolution of egg laying behavior under starvation conditions in real time. Overall, we find that eggs produced from starved parents display few ill effects of the treatment, indicating that parents are apparently able to compensate for shifting nutrient availability (likely via changes in total reproductive output) to compensate for this deprivation. Beyond dietary responses, our new microfluidic design provides the basis for addressing questions that depend on the clean separation of embryos from adults under tightly controlled environmental conditions. 9 Methods Nematode strains and maintenance Caenorhabditis elegans strain N2-PD1073 [25] was obtained from the Caenorhabditis Genetics Center CGC, and maintained using standard culture protocols [26]) unless otherwise noted. All worm cultures were maintained at 20°C in a biological incubator on NGM. Live streptomycin resistant E. coli strain OP50-1 was used as the nematode food source. Every 2 days, a subset of animals from the growing population is transferred to a new plate by excising out a portion of the agar and placing it onto a freshly seeded plate. Approximately 75 animals (at various stages) are transferred to start each new population. Once a week, when gravid hermaphrodites are present, animals are aspirated off the plate and dispensed into a 15 ml conical tube. At this time, a 5% solution of 4M NaOH and bleach is added to extract embryos from gravid adults. The surviving embryos are immediately placed onto freshly seeded plates to mature. Progeny synchronization is independent of L1 arrest, as embryos hatch in the presence of food. Microfluidic device fabrication Microfluidic devices were designed and fabricated using standard soft photolithography techniques[27,28]. In brief, the chip design was drafted in Vectorworks 2013 Fundamentals (Nemetschek SE, Munich, DE) and photomask transparencies were printed at 20k resolution (CAD/Art Services Inc, Bandon OR, United States). The corresponding CAD file is available as Supporting Information (S1). To manufacture the chips, SU-8 (MicroChem Corp.) photoresist masters were made and treated with (Tridecafluoro-1,1,2,2-Tetrahydrooctyl) Trichlorosilane (Gelest Inc., Product # SIT8174.0). These masters served as molds into which polydimethylsiloxane (PDMS) 10 (Sylgard 184, Dow Corning) mixed at a PDMS:developer ratio of 1:10 was poured and cured at 60 °C. Cured PDMS chips were cut out and appropriate holes were punched using 1.5 mm biopsy punches. The cut and punched PDMS chips were then exposed to air plasma (PDC-32G Plasma Cleaner, Harrick Plasma Inc.) and bonded to 50x75 mm glass slides. Microfluidic perfusate The EGG-platform uses a flow through system to maintain environmental consistency and to provide a constant food level when food is provided. The perfusate was driven at ~2 psi. Under constant pressure of 2 psi, total volume of liquid in the chip is replaced every 30 seconds. A second microfluidic chip with narrow channels was used upstream in serial to provide additional resistance. This enables the system to be run a higher PSI while minimizing the variability in the flow rate as previously described [13]. The perfusate used in this study was either S-basal or S-basal supplemented with E. coli OP50-1. To prepare the E. coli for feeding, 1 Liter of Terrific Broth is autoclaved in a wide-bottom 2-liter flask, pre inoculation of one OP50-1 CFU. OP50-1 is incubated at 37°C while rotating at 180RPM. Post 24H of incubation, OP50-1 is pelleted (6,000 RPM for 5 mins) and re-suspended into a 50 mls total volume S-basal Solution. For each μ-flux run, the final volume of OP50-1 must be at a minimum concentration of 108 CFU per ml to ensure a positive control. Aliquots of OP50-1 in S-basal Complete can last in 4°C for several weeks and maintain a 108 CFU per ml concentration. 11 Loading the microfluidic device To ensure proper animal synchronization without L1 arrest, three days prior to loading in the EGG-Chip, populations are bleached. At 48H post bleaching, animals at larval stages four (L4) are picked to a freshly seeded plate for the duration of development. After 24H, this results in a synchronized population of day 1 adults, ready for microfluidic set-up 700 Day 1 adult hermaphrodites are aspirated off each plate and suspended into a 1.7 ml microcentrifuge tube. A 5 ml syringe pump is used to load animals through the top port, down the worm loading and distribution network, and into the Arena portion of each EGG-chip (Fig 1). C. elegans husbandry in the microfluidic device Adult hermaphrodites remain within the arena portion (Fig 1A, region ii) of the microfluidic chip for the duration of the experiment. Under starvation conditions, the left port and bottom port are closed, forcing liquid to exit the chip through the right port. As adult hermaphrodites in the arena portion of the chip experience starvation, all progeny laid pass through the egg capture chamber and exit the chip through the right port due to the laminar flow of buffer through the chip (Fig 1). Under fed conditions the procedure is the same with one exception: OP50-1 is added to the buffer of fed hermaphrodites at a density of 108 CFUs/ml. A magnetic stir bar and stir plate is used to ensure that OP50-1 is continuously evenly distributed within the buffer. 12 Progeny collection Post the 24H adult starvation, a 5 ml syringe is used to flush buffer through the left port, clearing the egg capture chamber. Post flush, the left and right ports are closed and the bottom port is open. This changes in the direction of laminar flow, allowing fluid to travel in from the top port and out through the bottom port (Fig 1). Once the left and right ports are closed, all laid progeny are collected in the egg capture chamber. The bottom micron filter prevents them from traveling through the waste channels and exiting the chip (Fig 1). Progeny are collected at the 2.5H interval post starvation. A 5 ml syringe is used to collect all eggs from the egg capture chamber into a 1.7 ml microcentrifuge tube. Progeny maintenance post microfluidic capture Following collection in the EGG-chip, embryos were plated onto large, seeded agar plates and left to develop at 20°C (normal developmental conditions). All animals remained under normal developmental conditions until adulthood. Once progeny reached adulthood, a subset of the population was evaluated for lifespan and fecundity. Lifespan Measurements Lifespan was evaluated using Automated Lifespan Machine technology [29]. Plate preparation and worm handling for the Lifespan Machine assays were performed as previously described [30]. Survivorship curves and statistical analyses were all generated using JMP PRO 13 software package [31]. 13 Reproduction measurements Brood size was quantified by isolating 30 adult hermaphrodites at L4 stage to individual plates and transferring daily per treatment. Assays were performed at 20°C and using OP50-1 as a nematode food source using standard agar. The total brood size was measured as hatched larvae for each individual over 4 days or until a 24H period with <5 embryos. Embryos that did not hatch were not included in brood size measures for that individual. Embryo size measurements To determine embryo size, collected embryos were mounted on 2% agar pads and imaged on an inverted microscope (Olympus IX73P1F) at 60X (PLAN APO 60X - NA 1.42). Collected images were analyzed using ImageJ software [32]. Calibration of the pixel/size ratio was performed using images collected with a micrometer and the ImageJ measure function was used to measure the circumference of ten embryos per treatment to generate size measurements. Results Design of the Embryo Gathering Gadget To determine the effects of maternal starvation on offspring life-history traits we developed a microfluidic device (Fig 1 and S1) that enables (1) experimental control of food levels, (2) retention of animals in low-food environments, and (3) collection of embryos with complete temporal flexibility. Our new device, the Embryo Gathering Gadget (EGG) consists of four features. The first is a worm loading and distribution network (Fig 1A, region i) that enables introduction of animals at the start of the experiment and the delivery of perfusate during normal operation. The second is a large 14 arena (Fig 1A, region ii), which houses the adult animals during the experiment. The inside of the arena is patterned with a series of pillars to form an “artificial dirt” environment (see Fig 1B) that elicits normal crawling motion and plate-like behavior [13] (see S2). The pillars are 200 microns in diameter and spaced 300 microns on center, an arrangement that has been published as optimal for normal crawling behavior in C. elegans adults [14]. The animals that reside in the arena experience a constant flow of perfusate that maintains environmental consistency, as well as food levels under feeding conditions, while removing eggs from the arena. Eggs that leave the arena pass through a filter made up of 40-micron wide channels (see Fig 1B) that are sized to allow passage of eggs while retaining adults. Following this, they enter the third chip feature, the embryo capture chamber (Fig 1A, region iii), which sits between the upstream 40-micron filter that separates it from the arena and a downstream 20-micron filter that is sized to retain eggs within the chamber. The chamber also has an independent inlet and outlet that enables extraction of captured eggs from the device at will. The fourth feature of the device is a series of waste removal channels (Fig 1A, region iv) downstream from the 20- micron egg retention filter, which allows for exit of the perfusate during normal operation. 15 Fig 1. The Egg Gathering Gadget (EGG) platform (A) The EGG-chip is a custom microfluidic device consisting of four regions; i - a worm loading and distribution network, ii- a large worm husbandry arena, iii – an egg capture chamber, and iv – a waste removal network. (B) Zoom of the arena and egg capture chamber. The pillars in the arena are 200 microns in diameter (i) and spaced 300 microns apart on center (ii). The egg capture arena sits between an upstream filter made up of 40-micron wide channels (iii) and a downstream 20-micron wide filter (iv). (C) Perfusion of buffer (+/- food) is driven by pressurized air. The egg-flush inlet is connected to a syringe with buffer for removal of eggs. (D) Under normal operation the EGG-platform cycles through four states. Under normal growth the egg flush inlet and the profusion exit are blocked which causes flow from the profusion to travel through the arena which collects the eggs, passes through the 40-micron filter, then exits the egg flush exit into waste. To start a timed egg collection the air pressure that drives the perfusate is turned off and the perfusion exit is blocked. Egg clearance is then accomplished by driving buffer through the egg collection chamber from a syringe attached to the egg flush inlet. After egg clearance the egg flush inlet and exit are blocked, the perfusion exit is opened, and pressure driven flow through the perfusion inlet is resumed to begin egg capture. At the end of the timed egg capture eggs are collected by again turning off the pressure, blocked the perfusion exit, unblocking the egg flush inlet and exit. 16 Starvation in the EGG-Chip alters egg laying To ensure that egg laying behavior was maintained in the EGG-Chip, embryos were collected and quantified from each chip at various timepoints (1H, 3H, 5H, 24H, 31H, and 48H) (Fig 2). The 1H timepoint shows the largest number of embryos laid per treatment group (mean of 684 for fed and 698.55 for starved). This is not surprising as previous literature suggests that the sudden change in environment elicits a “dumping” response in which adults immediately lay all fertilized embryos, presumably to enhance survival under changing conditions [15]. Samples at 3, 5 and 24 hours all trended toward fewer embryos laid (means of 529.5 [fed], 314.55 [starved], 484.5 [fed], 394.05 [starved], 334.05 [fed], 244.05 [starved], respectively) compared to the 1H timepoint. These slight differences are not statistically significant, and the decrease is consistent among starved and fed environments. Furthermore, this confirms that at least within the first 24H starved adult hermaphrodites will continue to lay embryos despite poor environmental conditions. At 31H post starvation, there is a significant decrease in the number of embryos laid from adults in starved versus fed conditions (mean of 293.55 versus 134.55, p=0.0423). Lastly at 48H there is another significant decrease in the number of laid embryos, with starved adults laying no embryos (mean of 0 versus 64.05, p=0.0457). In both starved and fed environmental conditions one major contributing factor for decreased embryo production is the increasing percentage of adults who experience facultative vivipary. 17 1.0 Condition 0.9 Fed Starved 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 1 3 5 24 31 48 Time in Hours Post T= 0 START Fig 2. Egg Laying Behavior of Adults in EGG C. elegans embryos are captured and counted from each EGG chip under fed and starved conditions respectively. For each time interval per treatment condition we collected embryos and calculated the total number of embryos per adult per hour through liquid density calculations. Bars represent the mean per treatment and error bars represent +/- 1 SEM. Fig 2. shows that for the first 24 hours, both starved and fed animals continue to lay progeny in EGG. Only at hours 31 and 48 is there a significant difference between the number of embryos laid under fed versus starved treatment conditions. Maternal starvation in the EGG-chip does not alter progeny lifespan To assesses the effects of the maternal environment on progeny fitness trajectories in the following generation (maternal effects), we isolated embryos laid after 24H of adult starvation and examined their lifespan (Fig 3). To control for environmental changes, we also isolated embryos laid in an EGG-Chip at the same time period from adults who were continuously exposed to food. Our findings show that the maternal environment has no significant effects on progeny lifespan (mean lifespan 19.721[Fed] vs 18 Number of Embryos Laid Per Adult in EGG 19.76 [Starved]). Embryos laid from fed or starved mothers experienced no changes to overall lifespan expectancy. Fig 3. C. elegans Adult Lifespan Post Being Laid in EGG We examined the lifespan of C. elegans adults post being laid in EGG under each treatment condition respectively. We observe no significant difference in adult lifespan (log-rank p value = 0.8964, n=69 [fed], n=96 [starved], combined 2 experimental replicates) trajectories in progeny post maternal environment under fed nor starved conditions. 19 Maternal starvation in the EGG-chip does not alter progeny fecundity To further evaluate the effects of maternal starvation on progeny fitness we examined the fecundity of 30 individuals from each microfluidic treatment group respectively in addition to the adult hermaphrodites who never experienced a microfluidic environment. Among all three treatment groups our results show no significant differences in reproduction (p=0.8614). Fig 4. Fecundity of Adults Post Being Laid in EGG Animals were isolated as L4’s and transferred each day for 4 days or until the total number of embryos laid was 0.001) n=30 individuals per treatment, each dot represents an individual > L3 stage, individual hermaphrodites are isolated at 4th larval stage to capture total reproduction 42 Total fecudity Discussion Here, we sought to examine if the mechanism for ThT lifespan extension found in adults could be activated by exposure earlier in life. In particular, we were interested in if short term early-life treatment would be sufficient for inducing late-life longevity benefits. We made the surprising discovery that early-life exposure leads to a population- wide developmental delay. This is especially surprising since the proposed the mechanism for ThT induced lifespan extension in adults is enhanced protein homeostasis (Alavez et al. 2011), which would seem to be potentially favorable regardless of age. Apart from the adult early life mortality at high concentrations, there was little evidence suggesting that ThT had any toxicity effects in adults. The diverse and complex effects that we observe following larval treatment of ThT (developmental delay, decreased pharyngeal pump rate, bi-phasic N2 adult lifespan and decreased fecundity) suggest the potential for substantially different mechanisms of action for larval and adult response to treatment. Overall, our findings suggest that both ThT induction of larval developmental delay, and recovery post exposure is complex and likely a hormetic stress response, given the observation that daf-16, hsf-1 and skn-1 all prevent the late life extension observed in N2 WT (Jones & Candido 1999). In addition, all strains show decreased fecundity post treatment, suggesting that developmental ThT exposure is acting through multiple stress response pathways and that removing them results in reduced lifespans (Kumsta & Hansen 2017). We were also able to show that fecundity is significantly reduced following larval ThT exposure, highlighting the impact of this stressor to differentially allocate resources between somatic maintenance and reproduction early in development. 43 Larval ThT Exposure Causes Novel Developmental Delay Identification of a compound resulting in a sustained severe developmental delay was a surprise finding. Especially, considering that C. elegans has two programmed developmental arrest states to survive harsh conditions (Baugh & Sternberg 2006)(Angelo & Gilst 2009). The ability of larval ThT exposure to bypass predetermined developmental arrest pathways, both L1 arrest and dauer, highlights the complexity of ThT’s mechanism of action during development. Preliminary data on alternative adult lifespan extension compounds, such as green tea extract, do not result in developmental delay. This is suggestive that ThT is unique in its temporally restrictive response. ThT treatment during embryogenesis, L1, L2, and L3 development lead to a prolonged L3 developmental delay, eventually resulting in death after 3 weeks of exposure (Figure 2.1). It is also associated with a significant reduction in pharyngeal pump rates. ThT treatment during L4 results in facultative vivipary as (opposed to L4 reproductive diapause). Lastly, chronic ThT treatment during adulthood results in a significant lifespan extension (Alavez et al. 2011; Lucanic et al. 2017; Banse, Lucanic, Sedore, Coleman-Hulbert, Todd Plummer, et al. 2019). Remarkably, we show that early developmental delay effects of ThT can be reversed upon removal from the compound with sustained effects to overall adult fitness. ThT Induced Developmental Delay Results in Adult Fitness Tradeoffs In 2011, the Lithgow lab examined that adult exposure to ThT leads to a major adult lifespan extension. Their studies concluded that the lifespan extension of ThT in adults relies on HSF-1 and SKN-1 and that ThT is potentially acting as a stress response mimic to promote protein homeostasis. In 2017, a Caenorhabditis Intervention Testing Program 44 longevity assay including 22 Caenorhabditis strains across three independent laboratories, confirmed the response to adult ThT exposure was both potent and robust across all strains tested (Lucanic et al. 2017). Our results on the examination of ThT adult lifespan post larval developmental delay recapitulate the N2 adult lifespan extension to a lesser degree. Furthermore, our larval treatment paradigm allows us to examine the effects of ThT on reproduction. The fitness tradeoff we observe among increased lifespan and decreased reproduction suggests changes in nutrient allocation resulting in a fitness- tradeoff. We interpret these findings to mean that the enhanced lifespan following larval ThT treatment is achieved by a reallocation of nutrient resources to somatic maintenance at the expense of germline resources. Taken together, our findings support that developmental ThT exposure results in a hormetic stress response in adulthood, conversely to sustained adult ThT exposure resulting in a lifespan extension independently of daf-16 but dependent on hsf-1 and skn-1. Our observations show that the effects of larval exposure to ThT are different than adult ThT exposure in a variety of ways, most distinctly, that it requires multiple stress response pathways (daf-16, skn-1, and hsf-1) to attain late adult lifespan extension. We characterize larval ThT exposure as hormesis due to the biphasic response of larval toxicity coupled with late lifespan extension and reduced fecundity in adults. Additionally, we observe a dose dependent toxicity effect of larval ThT exposure. Such that, under a moderate exposure concentration (100~150µM) for a short (~3 days) exposure period the toxicity effects of ThT are buffered by activation of multiple stress response pathways (hormesis), which remains activated throughout adulthood resulting in the observed adult lifespan extension. Taken together, these data suggest that the 45 consequences of larval ThT exposure on adults is complex, possibly polygenic, and temporally sensitive. Conclusion The developmental delay induced by larval ThT exposure examines an alternative paradigm for how C. elegans can potentially respond to environmental stress. Unlike the traditional L1 arrest or dauer larvae, C. elegans response to larval ThT exposure is bypassing the predetermined developmental stages for surviving adverse environmental conditions. These experiments highlight the need to examine the potential toxicity effects, and tradeoffs that can be associated with exposure to various compounds. It also highlights the various pathways that can be used to metabolize compounds, and that the temporal onset of stressors can result in varied pharmacodynamics and have the potential to lead to adverse consequences. 46 CHAPTER IV CONCLUSION In an effort to advance the field of senescence and address gaps in our knowledge concerning the biological fitness tradeoffs within the aging process, I address two fundamental questions using C. elegans as a model. First, I examine how nutrient deprivation can impact fitness trajectories within and across generations. Then I examine how compound intervention via Thioflavin T (ThT) can also impact fitness characteristics within a generation and affect various life history traits. In Chapter II I address the role of transgenerational changes under an acute maternal starvation effects paradigm. Through the development and implementation of a novel microfluidic device fabricated to impose maternal starvation and collect laid progeny simultaneously, while mitigating the adverse biological and physiological constraints associated with adult starvation in nematodes. My findings demonstrate that under acute starvation C. elegans decreases overall fecundity while maintaining equal investment per embryo. Progeny laid during maternal starvation display no significant differences in lifespan nor fecundity trajectories when raised under normal environmental conditions. In this study we did not evaluate the potential for increased stress resistance of progeny when exposed to adverse environmental conditions, but rather chose to examine how incongruent environments for adults and progeny shape fitness trajectories under ideal circumstances. We sought to examine if increasing environmental resources could mitigate the consequences of prior adverse conditions. Under a specific set of conditions, we show that C. elegans progeny laid as a result of maternal starvation does not exhibit the same transgenerational consequences as extended L1 arrest nor extended 47 dauer. This highlights that, in C. elegans, transgenerational epigenetic effects are tightly regulated dependent upon the temporal resolution stress exposure. In this system, we see that developmental stravation has a stronger impact on progeny fitness than adult starvation. However, this phenomenon is likely to be inconsistent across biological systems given variation in the dynamics of reproduction and diverse biological tradeoffs between reproduction and lifespan. This finding can be explained by various evolutionary life history strategies that organisms employ to maximize survival and reproductive success in adverse conditions. Senescence is accompanied by a decrease in protein and cellular homoeostasis irrespective of external nutrient availability. Following sexual and reproductive maturity there is a shift in the allocation of resources towards somatic maintenance instead of germline maintenance and gamete formation. This shift in resource allocation that happens naturally during senescence and can be induced via external manipulations that alter nutrient availability prior or during sexual and reproductive maturity. Reproduction is an energetically costly process across all systems; however, it is necessary for species survival. Thus, even when faced with less than ideal conditions, organisms continue to invest in progeny. In Chapter III I address the role of epigenetic changes under compound intervention with the use of the amyloid-binding compound ThT. To examine how a compound that promotes protein homeostasis can impact life history trajectories I developed an early exposure paradigm to treat developing animals. I found that early exposure to ThT results in a population wide developmental arrest that is only recoverable within 6 days of exposure. I also found that the long-term consequences of 48 early life exposure to ThT to be an increased early life mortality, coupled with a late life extension for individuals who are able to overcome the early life mortality. I found a significant reduction to overall fecundity post sexual maturation in hermaphrodites as a result of early life exposure, suggesting a shift in the allocation of resources from reproduction to somatic maintenance. Through the use of insulin signaling mutants and RNAi knockdown I was able to determine that the mechanism for ThT exposure impacting lifespan varies from the previously reported adult exposure treatment such that it causes a widespread hormetic response, as opposed to acting through specific stress response pathways independently of insulin signaling in adults. Using our developmental exposure treatment paradigm, we first show that the previously reported assertion of the impacts of ThT on adult lifespan is not applicable to a developmental exposure treatment paradigm. This further highlights that temporal variation in the treatment exposure is critical to downstream fitness consequences. The incongruent effects of ThT exposure leading to developmental delay in larvae and ThT exposure leading to a two-fold increase in overall lifespan in adults can be explained by a hormesis stress model. That is, exposure to something that is toxic can have beneficial effects later in life. Again, there is a shift in the allocation of resources from germline development to somatic maintenance as a function of age. Similar to the adult starvation model explored in Chapter II, we show that the total progeny output is affected due to adverse environmental conditions. Taken together, both chapters of my dissertation increase our understanding of senescence and highlight the necessity that temporal onset of interventions, both through use of compounds and caloric restriction, is essential in mitigating inheritance within and 49 across generations. We show that the impacts of such perturbations can be sustained within a generation, but also have the potential to impact succeeding generations through high mortality rates. Although, we did not examine the transgenerational responses to species fitness in these studies, that is a reasonable future direction for this research given the information. In addition to protein homeostasis regressing as a function of age, so does the ability of natural selection to purge deleterious mutations from the population. Due to population drift (induced via high mortality rates), pleiotropy, linkage disequilibrium, and genetic hitchhiking there are a multitude of ways for selection to favor alleles that are early life beneficial and late life deleterious. In the case of ThT exposure, we see a late life extension and an early life mortality. While this may not be the case for all lifespan extension compounds, our study highlights that the ability to use a model system to understand the pharmacodynamics of compound interventions both during and after development yields to better insights on senescence as a function of time. Overall, this thesis shows that the when evaluating the fitness consequences of environmental perturbations, it is imperative to consider temporal onset of the stressor. 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