Histone Changes as a Response to Lactic Acid Selection in Cells of Austrofundulus limnaeus

The killifish Austrofundulus limnaeus is an extremophile native to small temporary ponds of Venezuela. Normal embryonic development of A. limnaeus is segmented into up to three stages of metabolic depression (diapause), and both developing and diapausing embryos can survive long bouts of the complete absence of oxygen (anoxia) by using anaerobic metabolic pathways. While there has been much focus on how mutations in DNA drive evolution, environmental stress can cause heritable changes to histone post-translational modifications (hPTMs) in multiple species. However, this evolutionary role of hPTMs has never been studied in A. limnaeus. Additionally, the possible evolutionary role of extracellular lactate via histone lactylation has never been studied in any species to our knowledge. To study these phenomena at the cellular level, we will force PSU-AL-WS40NE cells to undergo artificial selection. PSU-AL-WS40NE, is a neuroepithelial cell line that was isolated from embryonic A. limnaeus tissue explant. These cells can survive for 49 days without oxygen while accumulating lactate at a lower rate than mammalian cells. In mammalian cells, 25mM extracellular sodium lactate is sufficient to increase histone lactylation. However, our preliminary data has indicated that 30mM-100mM sodium lactate is not a strong selection pressure, while similar concentrations of lactic acid are lethal to the vast majority of cells. This suggests acidity may be an important component for selection and preconditioning. Therefore, repeated lactic acid exposure will be used as an artificial selection pressure to derive cells with a greater tolerance to high concentrations of extracellular lactate, and therefore potentially greater anoxia tolerance. Should a new cell line be developed, changes in hPTMs will be quantified to assess their potential role in driving changes in gene expression

Small Noncoding RNA Expression During Extreme Anoxia Tolerance of Annual Killifish (Austrofundulus limnaeus) Embryos

Small noncoding RNAs (sncRNA) have recently emerged as specific and rapid regulators of gene expression, involved in a myriad of cellular and organismal processes. MicroRNAs, a class of sncRNAs, are differentially expressed in diverse taxa in response to environmental stress, including anoxia. In most vertebrates, a brief period of oxygen deprivation results in severe tissue damage or death. Studies on sncRNA and anoxia have focused on these anoxia-sensitive species. Studying sncRNAs in anoxia-tolerant organisms may provide insight into adaptive mechanisms supporting anoxia tolerance. Embryos of the annual killifish Austrofundulus limnaeus are the most anoxia-tolerant vertebrates known, surviving over 100 days at their peak tolerance at 25°C. Their anoxia tolerance and physiology vary over development, such that both anoxia-tolerant and anoxia-sensitive phenotypes comprise the species. This allows for a robust comparison to identify sncRNAs essential to anoxia-tolerance. For this study, RNA sequencing was used to identify and quantify expression of sncRNAs in four embryonic stages of A. limnaeus in response to an exposure to anoxia and subsequent aerobic recovery. Unique stage-specific patterns of expression were identified that correlate with anoxia tolerance. In addition, embryos of A. limnaeus appear to constitutively express stress-responsive miRNAs. Most differentially expressed sncRNAs were expressed at higher levels during recovery. Many novel groups of sncRNAs with expression profiles suggesting a key role in anoxia tolerance were identified, including sncRNAs derived from mitochondrial tRNAs. This global analysis has revealed groups of candidate sncRNAs that we hypothesize support anoxia tolerance

Embryonic Development of Natural Annual Killifish populations of the genus Austrolebias: Evolutionary parallelism and the role of environment

Repeated, independent emergence of the same trait within different phylogenetic lineages is termed parallel evolution. It typically occurs as a result of similar selective pressures. Annual killifish have adapted to survive in the extreme habitat of temporary pools on three continents and present an especially amenable system for studying fundamental principles of evolutionary parallelism. When the pools dry, annual killifish embryos survive through the dry phase in the bottom substrate in a stage of dormancy—a diapause. The diapause is a complex set of three different developmental stages, none of which is obligate, thus leading to a multitude of potential developmental trajectories. While the intricacy of the killifishes\u27 embryonic development has been thoroughly studied in the laboratory, information on their natural development is virtually absent. We hypothesised that the natural development of annual killifishes is largely synchronised and governed by ambient conditions as shown in the lineage of the African genus Nothobranchius. We sampled wild embryo banks of the South American genus Austrolebias, which evolved its diapause independently of the African lineage. We sampled during two consecutive dry seasons, using both longitudinal and snapshot monitoring, and conducted transplant experiments to determine the extent of the evolutionary parallelism and role of the environment in Austrolebias spp. embryo development. Main habitat phases were characterised by largely synchronised embryo banks. Different inter-seasonal or local environmental conditions were reflected in a different developmental profile of the embryo banks, suggesting a high degree of environmental control. We found striking similarity in the habitat phase–embryo stage associations between the two lineages. The diapause in the two annual killifish lineages represents a unique example of evolutionary parallelism, with the analogy manifested in very close detail. We highlight the similarity of the selective forces in the two genera despite the different geographic origins, climate zones and reversed seasonality. The repeatedly occurring strict association of the same developmental stages with the same habitat conditions suggests a limited array of developmental settings that can be applied to cope with the given environmental challenges

Locating Vitamin D Receptors (VDRs) in Annual Killifish, Austrofundulus Limnaeus

Austrofundulus limnaeus, a species of annual killifish found in unpredictable temporary habitats in South America, has a profound ability to survive long periods without water and oxygen. This tolerance is associated with the ability to enter metabolic dormancy associated with diapause. Entrance into diapause and tolerance of environmental stress is governed by an interplay of genetic and environmental factors. The vitamin D receptor (VDR) is among these factors and plays a crucial role in determining if an embryo will enter diapause or actively develop. My work aims to identify the genomic locations of VDR proteins in the A. limnaeus genome under conditions that induce diapause and those that do not. This study will provide insight into molecular processes that support metabolic dormancy and survival of extreme environmental stresses

Anoxia Tolerance During Vertebrate Development - Insights from Studies on the Annual Killifish Austrofundulus limnaeus

This is chapter 1 from Anoxia -- This book reviews how severe oxygen deprivation affects biological systems - from the molecular to the ecological level. The contributing authors come from diverse regions of the world, which proves the interest in the academic analysis of oxygen deprivation. The diversity in the experimental approach scientists take, in order to understand the influence oxygen deprivation has on living systems, is apparent throughout this book. One of the presented ideas deals with the exploration and examination of the physiological, cellular and genetic characteristics of killifish embryos and nematodes exposed to anoxia. Furthermore, the book includes material on the mechanisms regulating hypoxia and anoxia tolerance and their implications of on human health issues. Finally, new methodologies to examine oxygen deprivation and the impact of human-related activities on oxygen level, within important ecological systems such as Lake Victoria, are presented. There is no doubt that the oxygen molecule is central to every stratum of biological systems

Mitochondrial DNA Sequence and Lack of Response to Anoxia in the Annual Killifish \u3ci\u3eAustrofundulus limnaeus\u3c/i\u3e

The annual killifish Austrofundulus limnaeus inhabits ephemeral ponds in regions of Venezuela, South America. Permanent populations of A. limnaeus are maintained by production of stress-tolerant embryos that are able to persist in the desiccated sediment. Previous work has demonstrated that A. limnaeus have a remarkable ability to tolerate extended periods of anoxia and desiccating conditions. After considering temperature, A. limnaeus embryos have the highest known tolerance to anoxia when compared to any other vertebrate yet studied. Oxygen is completely essential for the process of oxidative phosphorylation by mitochondria, the intracellular organelle responsible for the majority of adenosine triphosphate production. Thus, understanding the unique properties of A. limnaeus mitochondria is of great interest. In this work, we describe the first complete mitochondrial genome (mtgenome) sequence of a single adult A. limnaeus individual and compare both coding and non-coding regions to several other closely related fish mtgenomes. Mitochondrial features were predicted using MitoAnnotator and polyadenylation sites were predicted using RNAseq mapping. To estimate the responsiveness of A. limnaeus mitochondria to anoxia treatment, we measure relative mitochondrial DNA copy number and total citrate synthase activity in both relatively anoxia-tolerant and anoxia-sensitive embryonic stages. Our cross-species comparative approach identifies unique features of ND1, ND5, ND6, and ATPase-6 that may facilitate the unique phenotype of A. limnaeus embryos. Additionally, we do not find evidence for mitochondrial degradation or biogenesis during anoxia/reoxygenation treatment in A. limnaeus embryos, suggesting that anoxia-tolerant mitochondria do not respond to anoxia in a manner similar to anoxia-sensitive mitochondria

GABA and lactate preconditioning increases cell division in annual killifish cell line during anoxia

Annual killifish (Austrofundulus limnaeus) live in temporary ponds in Venezuela and experience drastic changes in their environment that cause their ponds to dry up. This species survives by producing drought- and anoxia-tolerant embryos that are deposited in the mud. Embryos survive these conditions by entering metabolic dormancy (diapause) until environmental signals break their dormancy and they continue developing. In order to survive anoxia, embryos rely exclusively on anaerobic metabolism, which leads to abundant lactate accumulation. Previous research has shown that the production and degradation of the neurotransmitter γ-aminobutyric acid (GABA) is crucial for long-term anoxia survival. This study explores the role of lactate and GABA metabolism in anoxia tolerance. To test this, we exposed embryo-derived cells (WS40NE) in anoxia to three treatments: anoxia preconditioning, lactate preconditioning, and GABA supplementation. For all treatments, cell survival was monitored, and extracellular lactate levels were measured. Compared to the control, cells exposed to lactate and GABA proliferated at a higher rate, whereas the anoxia preconditioned treatment proliferated at a lower rate. Rate of lactate accumulation was dependent on time spent in anoxia as well as whether cell media was changed. Media changes led to higher rates of lactate production compared to cells in static media. Understanding survival of cells during anoxia may give insight to how human conditions, such as strokes, can be avoided or damage can be reversed

Guide RNA Design and Delivery for CRISPR/Cas9 Editing in Annual Killifish

The CRISPR-Cas9 genome editing tool has shown to be successful in knocking out genes in model organisms such as zebrafish, turquoise killifish, and cichlid fish. CRISPR-Cas9 genome editing has been demonstrated in many species of fish, but this technology has not been verified in the annual killifish, Austrofundulus limnaeus. We hypothesize that targeted editing of the tyrosinase gene in embryos of A. limnaeus would lead to the development of fish without the ability to produce melanin, the black/brown pigment molecule. Early embryos (1-cell stage) were injected with a Cas9 cocktail containing a mix of guide RNA molecules that target the genomic sequence of the tyrosinase gene and either an mRNA coding for the Cas9 protein, or Cas9 protein. Guide RNAs were designed using ChopChop, and two guides were selected for injection based on a high predicted percent efficiency for binding with low probability for off-target effects. Many injected embryos developed without expressing black pigment. We found for the first time in this species that Cas9 can be successfully used to knockout the tyrosinase gene. In the future, we plan to establish a breeding line of non-pigmented killifish to aid in embryological studies of this species

Mechanisms of animal diapause: Recent developments from nematodes, crustaceans, insects, and fish

© 2016 the American Physiological Society. Life cycle delays are beneficial for opportunistic species encountering suboptimal environments. Many animals display a programmed arrest of development (diapause) at some stage(s) of their development, and the diapause state may or may not be associated with some degree of metabolic depression. In this review, we will evaluate current advancements in our understanding of the mechanisms responsible for the remarkable phenotype, as well as environmental cues that signal entry and termination of the state. The developmental stage at which diapause occurs dictates and constrains the mechanisms governing diapause. Considerable progress has been made in clarifying proximal mechanisms of metabolic arrest and the signaling pathways like insulin/Foxo that control gene expression patterns. Overlapping themes are also seen in mechanisms that control cell cycle arrest. Evidence is emerging for epigenetic contributions to diapause regulation via small RNAs in nematodes, crustaceans, insects, and fish. Knockdown of circadian clock genes in selected insect species supports the importance of clock genes in the photoperiodic response that cues diapause. A large suite of chaperone-like proteins, expressed during diapause, protects biological structures during long periods of energy-limited stasis. More information is needed to paint a complete picture of how environmental cues are coupled to the signal transduction that initiates the complex diapause phenotype, as well as molecular explanations for how the state is terminated. Excellent examples of molecular memory in postdauer animals have been documented in Caenorhabditis elegans. It is clear that a single suite of mechanisms does not regulate diapause across all species and developmental stages