Is This Science? Students' Experiences of Failure Make a Research-Based Course Feel Authentic.

Course-based undergraduate research experiences (CUREs) and inquiry-based curricula both expose students to the scientific process. CUREs additionally engage students in novel and scientifically relevant research, with the intention of providing an "authentic" research experience. However, we have little understanding of which course design elements impact students' beliefs that they are experiencing "authentic" research. We designed a study to explore introductory biology students' perceptions of research authenticity in CURE and inquiry classes. Using the Laboratory Course Assessment Survey, we found that students in CURE sections perceived higher levels of authentic research elements than students in inquiry-based sections. To identify specific factors that impact perceptions of research authenticity, we administered weekly reflection questions to CURE students. Coding of reflection responses revealed that experiences of failure, iteration, using scientific practices, and the relevant discoveries in their projects enhanced students' perceived authenticity of their research experiences. Although failure and iteration can occur in both CUREs and inquiry-based curricula, our findings indicate these experiences-in conjunction with the Relevant Discovery element of a CURE-may be particularly powerful in enhancing student perceptions of research authenticity in a CURE.

Male fetal sex affects uteroplacental angiogenesis in growth restriction mouse model†.

Abnormally increased angiotensin II activity related to maternal angiotensinogen (AGT) genetic variants, or aberrant receptor activation, is associated with small-for-gestational-age babies and abnormal uterine spiral artery remodeling in humans. Our group studies a murine AGT gene titration transgenic (TG; 3-copies of the AGT gene) model, which has a 20% increase in AGT expression mimicking a common human AGT genetic variant (A[-6]G) associated with intrauterine growth restriction (IUGR) and spiral artery pathology. We hypothesized that aberrant maternal AGT expression impacts pregnancy-induced uterine spiral artery angiogenesis in this mouse model leading to IUGR. We controlled for fetal sex and fetal genotype (e.g., only 2-copy wild-type [WT] progeny from WT and TG dams were included). Uteroplacental samples from WT and TG dams from early (days 6.5 and 8.5), mid (d12.5), and late (d16.5) gestation were studied to assess uterine natural killer (uNK) cell phenotypes, decidual metrial triangle angiogenic factors, placental growth and capillary density, placental transcriptomics, and placental nutrient transport. Spiral artery architecture was evaluated at day 16.5 by contrast-perfused three-dimensional microcomputed tomography (3D microCT). Our results suggest that uteroplacental angiogenesis is significantly reduced in TG dams at day 16.5. Males from TG dams are associated with significantly reduced uteroplacental angiogenesis from early to late gestation compared with their female littermates and WT controls. Angiogenesis was not different between fetal sexes from WT dams. We conclude that male fetal sex compounds the pathologic impact of maternal genotype in this mouse model of growth restriction.

GABA metabolism is crucial for long-term survival of anoxia in annual killifish embryos.

In most vertebrates, a lack of oxygen quickly leads to irreparable damages to vital organs, such as the brain and heart. However, there are some vertebrates that have evolved mechanisms to survive periods of no oxygen (anoxia). The annual killifish (Austrofundulus limnaeus) survives in ephemeral ponds in the coastal deserts of Venezuela and their embryos have the remarkable ability to tolerate anoxia for months. When exposed to anoxia, embryos of A. limnaeus respond by producing significant amounts of γ-aminobutyric acid (GABA). This study aims to understand the role of GABA in supporting the metabolic response to anoxia. To explore this, we investigated four developmentally distinct stages of A. limnaeus embryos that vary in their anoxia tolerance. We measured GABA and lactate concentrations across development in response to anoxia and aerobic recovery. We then inhibited enzymes responsible for the production and degradation of GABA and observed GABA and lactate concentrations, as well as embryo mortality. Here, we show for the first time that GABA metabolism affects anoxia tolerance in A. limnaeus embryos. Inhibition of enzymes responsible for GABA production (glutamate decarboxylase) and degradation (GABA-transaminase and succinic acid semialdehyde dehydrogenase) led to increased mortality, supporting a role for GABA as an intermediate product and not a metabolic end-product. We propose multiple roles for GABA during anoxia and aerobic recovery in A. limnaeus embryos, serving as a neurotransmitter, an energy source, and an anti-oxidant.

Metabolomics analysis of annual killifish (Austrofundulus limnaeus) embryos during aerial dehydration stress.

The annual killifish, Austrofundulus limnaeus, survives in ephemeral ponds in the coastal deserts of Venezuela. Persistence through the dry season is dependent on drought-resistant eggs embedded in the pond sediments during the rainy season. The ability of these embryos to enter drastic metabolic dormancy (diapause) during normal development enables A. limnaeus to survive conditions lethal to most other aquatic vertebrates; critical to the survival of the species is the ability of embryos to survive months and perhaps years without access to liquid water. Little is known about the molecular mechanisms that aid in survival of the dry season. This study aims to gain insight into the mechanisms facilitating survival of dehydration stress due to aerial exposure by examining metabolite profiles of dormant and developing embryos. There is strong evidence for unique metabolic profiles based on developmental stage and length of aerial exposure. Actively developing embryos exhibit more robust changes; however, dormant embryos respond in an active manner and significantly alter their metabolic profile. A number of metabolites accumulate in aerial-exposed embryos that may play an important role in survival, including the identification of known antioxidants and neuroprotectants. In addition, a number of unique metabolites not yet discussed in the dehydration literature are identified, such as lanthionine and 2-hydroxyglutarate. Despite high oxygen availability, embryos accumulate the anaerobic end product lactate. This paper offers an overview of the metabolic changes occurring that may support embryonic survival during dehydration stress due to aerial incubation, which can be functionally tested using genetic and pharmacological approaches.

No water, no problem: stage-specific metabolic responses to dehydration stress in annual killifish embryos.

Annual killifish survive in temporary ponds by producing drought-tolerant embryos that can enter metabolic dormancy (diapause). Survival of dehydration stress is achieved through severe reduction of evaporative water loss. We assessed dehydration stress tolerance in diapausing and developing Austrofundulus limnaeus embryos. We measured oxygen consumption rates under aquatic and aerial conditions to test the hypothesis that there is a trade-off between water retention and oxygen permeability. Diapausing embryos survive dehydrating conditions for over 1.5 years, and post-diapause stages can survive for over 100 days. Diapausing embryos respond to dehydration stress by increasing oxygen consumption rates while post-diapause embryos exhibit the same or reduced rates compared with aquatic embryos. Thus, water retention does not always limit oxygen diffusion. Aerial incubation coupled with hypoxia causes some embryos to arrest development. The observed stage-specific responses are consistent with an intrinsic bet-hedging strategy in embryos that would increase developmental variation in a potentially adaptive manner.

MitosRNAs and extreme anoxia tolerance in embryos of the annual killifish Austrofundulus limnaeus.

Embryos of the annual killifish Austrofundulus limnaeus are the most anoxia-tolerant vertebrate. Annual killifish inhabit ephemeral ponds, producing drought and anoxia-tolerant embryos, which allows the species to persist generation after generation. Anoxia tolerance and physiology vary by developmental stage, creating a unique opportunity for comparative study within the species. A recent study of small ncRNA expression in A. limnaeus embryos in response to anoxia and aerobic recovery revealed small ncRNAs with expression patterns that suggest a role in supporting anoxia tolerance. MitosRNAs, small ncRNAs derived from the mitochondrial genome, emerged as an interesting group of these sequences. MitosRNAs derived from mitochondrial tRNAs were differentially expressed in developing embryos and isolated cells exhibiting extreme anoxia tolerance. In this study we focus on expression of mitosRNAs derived from tRNA-cysteine, and their subcellular and organismal localization in order to consider possible function. These tRNA-cys mitosRNAs appear enriched in the mitochondria, particularly near the nucleus, and also appear to be present in the cytoplasm. We provide evidence that mitosRNAs are generated in the mitochondria in response to anoxia, though the precise mechanism of biosynthesis remains unclear. MitosRNAs derived from tRNA-cys localize to numerous tissues, and increase in the anterior brain during anoxia. We hypothesize that these RNAs may play a role in regulating gene expression that supports extreme anoxia tolerance.

Antioxidant capacity and anoxia tolerance in Austrofundulus limnaeus embryos.

Embryos of Austrofundulus limnaeus can tolerate extreme environmental stresses by entering into a state of metabolic and developmental arrest known as diapause. Oxidative stress is ubiquitous in aerobic organisms and the unique biology and ecology of A. limnaeus likely results in frequent and repeated exposures to oxidative stress during development. The antioxidant capacity of A. limnaeus was explored during development by measuring antioxidant capacity due to small molecules and several enzymatic antioxidant systems. Diapause II embryos can survive for several days in 1% hydrogen peroxide without indications of negative effects. Surprisingly, both small and large molecule antioxidant systems have the highest capacity during early development, which may be due to maternal provisioning. Antioxidant capacity is largely invested in small molecules during early development and in enzymatic systems during late development. The switch in antioxidant mechanisms and decline in small molecule antioxidants during development correlates with the loss of extreme anoxia tolerance.

Establishment and characterization of an anoxia-tolerant cell line, PSU-AL-WS40NE, derived from an embryo of the annual killifish Austrofundulus limnaeus.

Most animal cells rely on aerobic metabolism for survival and are damaged or die within minutes without oxygen. Embryos of the annual killifish Austrofundulus limnaeus, however, survive months without oxygen. Determining how their cells survive without oxygen has the potential to revolutionize our understanding of the cellular mechanisms supporting vertebrate anoxia tolerance and the evolution of such tolerance. Therefore, we aimed to establish and characterize an anoxia-tolerant cell line from A. limnaeus for investigating mechanisms of vertebrate anoxia tolerance. The PSU-AL-WS40NE cell line of neuroepithelial identity was established from embryonic tissue of A. limnaeus using a tissue explant. The cells can survive for at least 49 d without oxygen or replenishment of growth medium, compared to only 3 d of anoxic survival for two mammalian cell lines. PSU-AL-WS40NE cells accumulate lactate during anoxia, indicating use of common metabolic pathways for anaerobic metabolism. Additionally, they express many of the same small noncoding RNAs that are stress-responsive in whole embryos of A. limnaeus and mammalian cells, as well as anoxia-responsive small noncoding RNAs derived from the mitochondrial genome (mitosRNAs). The establishment of the cell line provides a unique tool for investigating cellular mechanisms of vertebrate anoxia tolerance, and has the potential to transform our understanding of the role of oxidative metabolism in cell biology.

Temperature-dependent vitamin D signaling regulates developmental trajectory associated with diapause in an annual killifish.

The mechanisms that integrate environmental signals into developmental programs remain largely uncharacterized. Nuclear receptors (NRs) are ligand-regulated transcription factors that orchestrate the expression of complex phenotypes. The vitamin D receptor (VDR) is an NR activated by 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], a hormone derived from 7-dehydrocholesterol (7-DHC). VDR signaling is best known for regulating calcium homeostasis in mammals, but recent evidence suggests a diversity of uncharacterized roles. In response to incubation temperature, embryos of the annual killifish Austrofundulus limnaeus can develop along two alternative trajectories: active development and diapause. These trajectories diverge early in development, from a biochemical, morphological, and physiological perspective. We manipulated incubation temperature to induce the two trajectories and profiled changes in gene expression using RNA sequencing and weighted gene coexpression network analysis. We report that transcripts involved in 1,25(OH)2D3 synthesis and signaling are expressed in a trajectory-specific manner. Furthermore, exposure of embryos to vitamin D3 analogs and Δ4-dafachronic acid directs continuous development under diapause-inducing conditions. Conversely, blocking synthesis of 1,25(OH)2D3 induces diapause in A. limnaeus and a diapause-like state in zebrafish, suggesting vitamin D signaling is critical for normal vertebrate development. These data support vitamin D signaling as a molecular pathway that can regulate developmental trajectory and metabolic dormancy in a vertebrate. Interestingly, the VDR is homologous to the daf-12 and ecdysone NRs that regulate dormancy in Caenorhabditis elegans and Drosophila We suggest that 7-DHC-derived hormones and their associated NRs represent a conserved pathway for the integration of environmental information into developmental programs associated with life history transitions in animals.

Small noncoding RNA profiles along alternative developmental trajectories in an annual killifish.

Embryonic development of Austrofundulus limnaeus can occur along two phenotypic trajectories that are physiologically and biochemically distinct. Phenotype appears to be influenced by maternal provisioning based on the observation that young females produce predominately non-diapausing embryos and older females produce mostly diapausing embryos. Embryonic incubation temperature can override this pattern and alter trajectory. We hypothesized that temperature-induced phenotypic plasticity may be regulated by post-transcriptional modification via noncoding RNAs. As a first step to exploring this possibility, RNA-seq was used to generate transcriptomic profiles of small noncoding RNAs in embryos developing along the two alternative trajectories. We find distinct profiles of mature sequences belonging to the miR-10 family expressed in increasing abundance during development and mature sequences of miR-430 that follow the opposite pattern. Furthermore, miR-430 sequences are enriched in escape trajectory embryos. MiR-430 family members are known to target maternally provisioned mRNAs in zebrafish and may operate similarly in A. limnaeus in the context of normal development, and also by targeting trajectory-specific mRNAs. This expression pattern and function for miR-430 presents a potentially novel model for maternal-embryonic conflict in gene regulation that provides the embryo the ability to override maternal programming in the face of altered environmental conditions.

Small Non-coding RNA Expression and Vertebrate Anoxia Tolerance.

Background: Extreme anoxia tolerance requires a metabolic depression whose modulation could involve small non-coding RNAs (small ncRNAs), which are specific, rapid, and reversible regulators of gene expression. A previous study of small ncRNA expression in embryos of the annual killifish Austrofundulus limnaeus, the most anoxia-tolerant vertebrate known, revealed a specific expression pattern of small ncRNAs that could play important roles in anoxia tolerance. Here, we conduct a comparative study on the presence and expression of small ncRNAs in the most anoxia-tolerant representatives of several major vertebrate lineages, to investigate the evolution of and mechanisms supporting extreme anoxia tolerance. The epaulette shark (Hemiscyllium ocellatum), crucian carp (Carassius carassius), western painted turtle (Chrysemys picta bellii), and leopard frog (Rana pipiens) were exposed to anoxia and recovery, and small ncRNAs were sequenced from the brain (one of the most anoxia-sensitive tissues) prior to, during, and following exposure to anoxia. Results: Small ncRNA profiles were broadly conserved among species under normoxic conditions, and these expression patterns were largely conserved during exposure to anoxia. In contrast, differentially expressed genes are mostly unique to each species, suggesting that each species may have evolved distinct small ncRNA expression patterns in response to anoxia. Mitochondria-derived small ncRNAs (mitosRNAs) which have a robust response to anoxia in A. limnaeus embryos, were identified in the other anoxia tolerant vertebrates here but did not display a similarly robust response to anoxia. Conclusion: These findings support an overall stabilization of the small ncRNA transcriptome during exposure to anoxic insults, but also suggest that multiple small ncRNA expression pathways may support anoxia tolerance, as no conserved small ncRNA response was identified among the anoxia-tolerant vertebrates studied. This may reflect divergent strategies to achieve the same endpoint: anoxia tolerance. However, it may also indicate that there are multiple cellular pathways that can trigger the same cellular and physiological survival processes, including hypometabolism.

The genome of Austrofundulus limnaeus offers insights into extreme vertebrate stress tolerance and embryonic development.

BACKGROUND: The annual killifish Austrofundulus limnaeus inhabits ephemeral ponds in northern Venezuela, South America, and is an emerging extremophile model for vertebrate diapause, stress tolerance, and evolution. Embryos of A. limnaeus regularly experience extended periods of desiccation and anoxia as a part of their natural history and have unique metabolic and developmental adaptations. Currently, there are limited genomic resources available for gene expression and evolutionary studies that can take advantage of A. limnaeus as a unique model system. RESULTS: We describe the first draft genome sequence of A. limnaeus. The genome was assembled de novo using a merged assembly strategy and was annotated using the NCBI Eukaryotic Annotation Pipeline. We show that the assembled genome has a high degree of completeness in genic regions that is on par with several other teleost genomes. Using RNA-seq and phylogenetic-based approaches, we identify several candidate genes that may be important for embryonic stress tolerance and post-diapause development in A. limnaeus. Several of these genes include heat shock proteins that have unique expression patterns in A. limnaeus embryos and at least one of these may be under positive selection. CONCLUSION: The A. limnaeus genome is the first South American annual killifish genome made publicly available. This genome will be a valuable resource for comparative genomics to determine the genetic and evolutionary mechanisms that support the unique biology of annual killifishes. In a broader context, this genome will be a valuable tool for exploring genome-environment interactions and their impacts on vertebrate physiology and evolution.

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.

Insulin-like growth factor signaling regulates developmental trajectory associated with diapause in embryos of the annual killifish Austrofundulus limnaeus.

Annual killifishes exhibit a number of unique life history characters including the occurrence of embryonic diapause, unique cell movements associated with dispersion and subsequent reaggregation of the embryonic blastomeres, and a short post-embryonic life span. Insulin-like growth factor (IGF) signaling is known to play a role in the regulation of metabolic dormancy in a number of animals but has not been explored in annual killifishes. The abundance of IGF proteins during development and the developmental effects of blocking IGF signaling by pharmacological inhibition of the insulin-like growth factor I receptor (IGF1R) were explored in embryos of the annual killifish Austrofundulus limnaeus Blocking of IGF signaling in embryos that would normally escape entrance into diapause resulted in a phenotype that was remarkably similar to that of embryos entering diapause. IGF-I protein abundance spikes during early development in embryos that will not enter diapause. In contrast, IGF-I levels remain low during early development in embryos that will enter diapause II. IGF-II protein is packaged at higher levels in escape-bound embryos compared with diapause-bound embryos. However, IGF-II levels quickly decrease and remain low during early development and only increase substantially during late development in both developmental trajectories. Developmental patterns of IGF-I and IGF-II protein abundance under conditions that would either induce or bypass entrance into diapause are consistent with a role for IGF signaling in the regulation of developmental trajectory and entrance into diapause in this species. We propose that IGF signaling may be a unifying regulatory pathway that explains the larger suite of characters that are associated with the complex life history of annual killifishes.

Embryonic development of the annual killifish Austrofundulus limnaeus: An emerging model for ecological and evolutionary developmental biology research and instruction.

BACKGROUND: Austrofundulus limnaeus is an annual killifish from the Maracaibo basin of Venezuela. Annual killifishes are unique among vertebrates in their ability to enter into a state of dormancy at up to three distinct developmental stages termed diapause I, II, and III. These embryos are tolerant of a wide variety of environmental stresses and develop relatively slowly compared with nonannual fishes. RESULTS: These traits make them an excellent model for research on interactions between the genome and the environment during development, and an excellent choice for developmental biology laboratories. Furthermore, A. limnaeus is relatively easy to maintain in a laboratory setting and has a high fecundity, making it an excellent candidate as an emerging model for studies of development, and for defining the limits of developmental buffering in vertebrates. CONCLUSIONS: This study reports for the first time on the detailed development of A. limnaeus and provides a photographic and illustrated atlas of embryos on the two developmental trajectories possible in this species. Developmental Dynamics 246:779-801, 2017. © 2017 The Authors Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Transcriptomic analysis of maternally provisioned cues for phenotypic plasticity in the annual killifish, Austrofundulus limnaeus.

BACKGROUND: Genotype and environment can interact during development to produce novel adaptive traits that support life in extreme conditions. The development of the annual killifish Austrofundulus limnaeus is unique among vertebrates because the embryos have distinct cell movements that separate epiboly from axis formation during early development, can enter into a state of metabolic dormancy known as diapause and can survive extreme environmental conditions. The ability to enter into diapause can be maternally programmed, with young females producing embryos that do not enter into diapause. Alternately, embryos can be programmed to "escape" from diapause and develop directly by both maternal factors and embryonic incubation conditions. Thus, maternally packaged gene products are hypothesized to regulate developmental trajectory and perhaps the other unique developmental characters in this species. RESULTS: Using high-throughput RNA sequencing, we generated transcriptomic profiles of mRNAs, long non-coding RNAs and small non-coding RNAs (sncRNAs) in 1-2 cell stage embryos of A. limnaeus. Transcriptomic analyses suggest maternal programming of embryos through alternatively spliced mRNAs and antisense sncRNAs. Comparison of these results to those of comparable studies on zebrafish and other fishes reveals a surprisingly high abundance of transcripts involved in the cellular response to stress and a relatively lower expression of genes required for rapid transition through the cell cycle. CONCLUSIONS: Maternal programming of developmental trajectory is unlikely accomplished by differential expression of diapause-specific genes. Rather, evidence suggests a role for trajectory-specific splice variants of genes expressed in both phenotypes. In addition, based on comparative studies with zebrafish, the A. limnaeus 1-2 cell stage transcriptome is unique in ways that are consistent with their unique life history. These results not only impact our understanding of the genetic mechanisms that regulate entrance into diapause, but also provide insight into the epigenetic regulation of gene expression during development.

Hit pause: Developmental arrest in annual killifishes and their close relatives.

Killifishes survive and persist in extreme environments by exploiting both aquatic and terrestrial habitats for egg deposition, and by adjusting the length of development to match availability of water to support larval growth and maturation. Annual killifishes persist in ephemeral bodies of water through the production of drought-tolerant embryos. Survival of the environmental stresses associated with their highly variable and seasonal habitat is supported by their ability to enter into at least two states of metabolic and developmental dormancy, diapause or quiescence. There are three stages of diapause in annual killifishes, one occurring prior to gastrulation, one about midway through development, and one in late pre-hatching embryos. Quiescence may occur at any developmental stage. In addition, delayed hatching is known to occur in close relatives of the annual killifishes, and may be superficially confused with pre-hatching diapause. These types of developmental delay are induced by different cues and serve different purposes in the life history of the species. Thus, it is likely that the molecular mechanisms that induce dormancy and support survival are unique in each case. It is imperative that we properly define these forms of developmental dormancy in our studies in order to put our results into the proper ecological and evolutionary context. Here the unique characteristics of these distinct categories of developmental delay are reviewed. Developmental Dynamics 246:858-866, 2017. © 2017 The Authors Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Mitochondrial DNA Sequence and Lack of Response to Anoxia in the Annual Killifish Austrofundulus limnaeus.

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.

Hypoxia and Anoxia Tolerance in the Annual Killifish Austrofundulus limnaeus.

Embryos of the annual killifish Austrofundulus limnaeus are routinely exposed to oxygen limitation during development and are extremely tolerant of anoxia. Importantly, tolerance of anoxia is not strictly associated with entrance into metabolic dormancy associated with diapause II, but rather any embryo will respond to anoxia by entering into a state of anoxia-induced quiescence. Hypoxia causes a reduction in the rate of development, reduced heart rates, and reduced capacities for metabolic enzyme activity in both aerobic and anaerobic pathways. Embryos of A. limnaeus begin life as oxyconformers, and transition into oxyregulators near the completion of embryonic development. As this transition occurs, extreme anoxia tolerance is lost. The rate of early development is independent of oxygen partial pressure, despite the fact that the embryos are oxyconformers. This suggests a contribution from anaerobic pathways to support early development. However, the specific pathways supporting this metabolism are unknown. The response of A. limnaeus embryos to hypoxia and anoxia is unique compared to other fishes and most other vertebrates, and thus future studies on this species may lend insight into novel mechanisms that support survival during prolonged oxygen limitation.