Differential DNA methylation and metabolite profiling of Atlantic killifish (Fundulus heteroclitus) from the New Bedford Harbor Superfund site.

Atlantic killifish (Fundulus heteroclitus) is a valuable model in evolutionary toxicology to study how the interactions between genetic and environmental factors serve the adaptive ability of organisms to resist chemical pollution. Killifish populations inhabiting environmental toxicant-contaminated New Bedford Harbor (NBH) show phenotypes tolerant to polychlorinated biphenyls (PCBs) and differences at the transcriptional and genomic levels. However, limited research has explored epigenetic alterations and metabolic effects in NBH killifish. To identify the involvement of epigenetic and metabolic regulation in the adaptive response of killifish, we investigated tissue- and sex-specific differences in global DNA methylation and metabolomic profiles of NBH killifish populations, compared to sensitive populations from a non-polluted site, Scorton Creek (SC). The results revealed that liver-specific global DNA hypomethylation and differential metabolites were evident in fish from NBH compared with those from SC. The sex-specific differences were not greater than the tissue-specific differences. We demonstrated liver-specific enriched metabolic pathways (e.g., amino acid metabolic pathways converged into the urea cycle and glutathione metabolism), suggesting possible crosstalk between differential metabolites and DNA hypomethylation in the livers of NBH killifish. Additional investigation of methylated gene regions is necessary to understand the functional role of DNA hypomethylation in the regulation of enzyme-encoding genes associated with metabolic processes and physiological changes in NBH populations.

Hypoxia-inducible factor 1 alpha protein increases without changes in mRNA during acute hypoxic exposure of the Gulf killifish, Fundulus grandis.

The hypoxia inducible factor 1 (HIF1) is a central regulator of the molecular responses of animals to low oxygen. While the hypoxia-responsiveness of HIF1 is generally attributed to the stabilization of the alpha protein subunit (HIF1α) at low oxygen, several studies on fish report increased tissue levels of HIF1A mRNA during hypoxia, suggesting transcriptional regulation. In the current study, HIF1α protein and HIF1A mRNA were determined in parallel in tissues of Gulf killifish, Fundulus grandis, exposed to short-term hypoxia (24 h at 1 mg O2 l-1). HIF1α protein was higher in brain, ovary, and skeletal muscle from fish exposed to hypoxia compared with normoxic controls by 6 h, and it remained elevated in brain and ovary at 24 h. In contrast, HIF1A mRNA levels were unaffected by hypoxia in any tissue. Moreover, HIF1α protein and HIF1A mRNA levels in the same tissues were not correlated with one another during either normoxia or hypoxia. Hence, an increase in HIF1α protein does not depend upon an increase in HIF1A mRNA during acute exposure to low oxygen in this species. The results support the widely accepted mechanism of post-translational protein stabilization, rather than new transcription, during the initial response of fish to hypoxia.

Fast satellite DNA evolution in Nothobranchius annual killifishes.

Satellite DNA (satDNA) is a rapidly evolving class of tandem repeats, with some monomers being involved in centromere organization and function. To identify repeats associated with (peri)centromeric regions, we investigated satDNA across Southern and Coastal clades of African annual killifishes of the genus Nothobranchius. Molecular cytogenetic and bioinformatic analyses revealed that two previously identified satellites, designated here as NkadSat01-77 and NfurSat01-348, are associated with (peri)centromeres only in one lineage of the Southern clade. NfurSat01-348 was, however, additionally detected outside centromeres in three members of the Coastal clade. We also identified a novel satDNA, NrubSat01-48, associated with (peri)centromeres in N. foerschi, N. guentheri, and N. rubripinnis. Our findings revealed fast turnover of satDNA associated with (peri)centromeres and different trends in their evolution in two clades of the genus Nothobranchius.

A scalable and tunable platform for functional interrogation of peptide hormones in fish.

Pituitary hormones play a central role in shaping vertebrate life history events, including growth, reproduction, metabolism, and aging. The regulation of these traits often requires precise control of hormone levels across diverse timescales. However, fine tuning circulating hormones in-vivo has traditionally been experimentally challenging. Here, using the naturally short-lived turquoise killifish (N. furzeri), we describe a high-throughput platform that combines loss- and gain-of-function of peptide hormones. Mutation of three primary pituitary hormones, growth hormone (gh1), follicle stimulating hormone (fshb), and thyroid stimulating hormone (tshb), alters somatic growth and reproduction. Thus, suggesting that while the killifish undergoes extremely rapid growth and maturity, it still relies on vertebrate-conserved genetic networks. As the next stage, we developed a gain-of-function vector system in which a hormone is tagged using a self-cleavable fluorescent reporter, and ectopically expressed in-vivo through intramuscular electroporation. Following a single electroporation, phenotypes, such as reproduction, are stably rescued for several months. Notably, we demonstrate the versatility of this approach by using multiplexing, dose-dependent, and doxycycline-inducible systems to achieve tunable and reversible expression. In summary, this method is relatively high-throughput, and facilitates large-scale interrogation of life-history strategies in fish. Ultimately, this approach could be adapted for modifying aquaculture species and exploring pro-longevity interventions.

In humans and other vertebrates, a pea-size gland at the base of the brain called the pituitary gland, produces many hormones that regulate how individuals grow, reproduce, and age. Three of the most prominent hormones are known as the growth hormone, the follicle-stimulating hormone, and the thyroid-stimulating hormone. It is important that the body precisely controls the levels of these hormones throughout an individual's life. One way researchers can investigate how hormones and other molecules work is to artificially alter the levels of the molecules in living animals. However, this has proved to be technically challenging and time-consuming for pituitary gland hormones. Moses et al. studied the growth hormone, follicle-stimulating hormone, and thyroid-stimulating hormone in the turquoise killifish, a small fish that grows and matures more rapidly than any other vertebrate research model. The experiments revealed that mutant fish lacking one of the three primary pituitary hormones were smaller, took longer to reach maturity, or were completely sterile. This suggests these three hormones play a similar role in killifish as they do in other vertebrates. The team then developed a new experimental platform to precisely control the levels of the three hormones in killifish. Genes encoding individual hormones were expressed in the muscles of the mutant fish, effectively making the muscles a 'factory' for producing that hormone. Treating mutant fish this way once was enough to restore growth and to fully return reproduction to normal levels for several months. Moses et al. also demonstrated that it is possible to use this platform to express more than one hormone gene at a time and to use drugs to switch hormone production on and off in a reversible manner. For example, this reversible approach made it possible to effectively adjust fertility levels. The new platform developed in this work could be adapted for modifying a variety of traits in animals to explore how they impact health and longevity. In the future, it may also have other applications, such as optimizing how farmed fish grow and reproduce and regulating hormone levels in human patients with hormone imbalances.

Transcriptomes of aging brain, heart, muscle, and spleen from female and male African turquoise killifish.

The African turquoise killifish is an emerging vertebrate model organism with great potential for aging research due to its naturally short lifespan. Thus far, turquoise killifish aging 'omic' studies have examined a single organ, single sex and/or evaluated samples from non-reference strains. Here, we describe a resource dataset of ribosomal RNA-depleted RNA-seq libraries generated from the brain, heart, muscle, and spleen from both sexes, as well as young and old animals, in the reference GRZ turquoise killifish strain. We provide basic quality control steps and demonstrate the utility of our dataset by performing differential gene expression and gene ontology analyses by age and sex. Importantly, we show that age has a greater impact than sex on transcriptional landscapes across probed tissues. Finally, we confirm transcription of transposable elements (TEs), which are highly abundant and increase in expression with age in brain tissue. This dataset will be a useful resource for exploring gene and TE expression as a function of both age and sex in a powerful naturally short-lived vertebrate model.

Widespread sex dimorphism across single-cell transcriptomes of adult African turquoise killifish tissues.

The African turquoise killifish (Nothobranchius furzeri), the shortest-lived vertebrate that can be bred in captivity, is an emerging model organism for aging research. Here, we describe a multitissue, single-cell gene expression atlas of female and male blood, kidney, liver, and spleen. We annotate 22 cell types, define marker genes, and infer differentiation trajectories. We find pervasive sex-dimorphic gene expression across cell types. Sex-dimorphic genes tend to be linked to lipid metabolism, consistent with clear differences in lipid storage in female vs. male turquoise killifish livers. We use machine learning to predict sex using single-cell gene expression and identify potential markers for molecular sex identity. As a proof of principle, we show that our atlas can be used to deconvolute existing bulk RNA sequencing (RNA-seq) data to obtain accurate estimates of cell type proportions. This atlas can be a resource to the community that could be leveraged to develop cell-type-specific expression in transgenic animals.

Bioenergetic Effects of Polycyclic Aromatic Hydrocarbon Resistance Manifest Later in Life in Offspring of Fundulus heteroclitus from the Elizabeth River.

Shifts in key physiological processes can confer resistance to chemical pollutants. However, these adaptations may come with certain trade-offs, such as altered energy metabolic processes, as evident in Atlantic killifish (Fundulus heteroclitus) in Virginia's Elizabeth River (ER) that have evolved resistance to polycyclic aromatic hydrocarbons (PAHs). We seek to understand the bioenergetic costs of PAH resistance among subpopulations of Atlantic killifish with differing contamination levels in order to examine how these changes manifest across multiple life stages and how these costs might be exacerbated by additional stressors. Bioenergetics data revealed differences in metabolic rates between offspring of PAH-resistant fish and reference fish were absent or minimal in both the embryo and larval stages but pronounced at the juvenile life stage, suggesting that bioenergetic changes in pollution-adapted killifish manifest later in life. We also provide evidence that killifish from remediated sites are more sensitive to PAH exposure than killifish from nonremediated sites, suggesting loss of PAH tolerance following relaxed selection. Collectively, our data suggest that the fitness consequences associated with evolved resistance to anthropogenic stressors may manifest differently over time and depend on the magnitude of the selection pressure. This information can be valuable in effective risk and remediation assessments as well as in broadening our understanding of species responses to environmental change.

A review on neurodegeneration in the fast-ageing killifish, the first animal model to study the natural occurrence of neuronal cell loss.

Thanks to medical and technological improvements, our world population has become ever-greying. In consequence, the incidence and prevalence of age-related central nervous system neuropathies, such as Alzheimer's (AD) and Parkinson's disease (PD), are increasing tremendously. Despite many research efforts, the precise aetiology of these age-related neurodegenerative disorders remains elusive, highlighting the urgent need for more effective treatments. Current preclinical research mainly uses animal models that do not fully recapitulate the complex cellular context in which these diseases occur, thereby lacking good construct validity. Indeed, most investigations are performed using relatively young animals, thereby ignoring the ageing environment in which neurodegenerative diseases manifest. This points out a major hiatus in current research: a vertebrate model organism that combines the complex disease context (onset, spreading and further manifestation into functional impairment) with an ageing environment. In recent years, the African turquoise killifish has emerged as a promising novel animal model to study age-related neurodegenerative disorders that combines these essential features. In this review, we bundle all reported findings up till now and provide a detailed overview of the neurodegenerative events within the central nervous system of this teleost fish, with a focus on PD.

The effects of winter cold acclimation on acute and chronic cadmium bioaccumulation and toxicity in the banded killifish (Fundulus diaphanus).

Temperate freshwater fishes can experience large seasonal temperature fluctuations that could affect their exposure and sensitivity to trace metals. Yet, temperature effects are overlooked in ecotoxicology studies, especially for cold temperatures typical of the winter. In the present study, the effects of long-term cold acclimation on Cd bioaccumulation and toxicity were investigated in a freshwater fish, the banded killifish (Fundulus diaphanus). Killifish were acclimated to 14 °C or gradually cooled (2 °C/week) to 4 °C and cold acclimated for 6 weeks. Then, both acclimation groups were exposed to environmentally realistic waterborne Cd concentrations (0, 0.5 or 5 µg Cd L-1) for a further 28 d at their respective acclimation temperatures. Tissue metal bioaccumulation, fish survival, condition, and markers of oxidative and ionoregulation stress, were measured after 0, 2, 5 and 28 days of Cd exposure. Cadmium tissue accumulation increased over the exposure duration and was typically lower in cold-acclimated fish. In agreement with this lower bioaccumulation, fewer Cd toxic effects were observed in cold-acclimated fish. There was little evidence of a difference in intrinsic Cd sensitivity between 4 °C- and 14 °C-acclimated fish, as Cd toxicity appeared to closely follow Cd bioaccumulation. Our study suggests that current environmental water quality guidelines would be protective in the winter for the abundant and ecologically-important banded killifish.

EXPERIMENTAL INFECTIONS WITH EUHAPLORCHIS CALIFORNIENSIS AND A SMALL CYATHOCOTYLID INCREASE CONSPICUOUS BEHAVIORS IN CALIFORNIA KILLIFISH (FUNDULUS PARVIPINNIS).

Some parasites manipulate their host's phenotype to enhance predation rates by the next host in the parasite's life cycle. Our understanding of this parasite-increased trophic transmission is often stymied by study-design challenges. A recurring difficulty has been obtaining uninfected hosts with a coevolutionary history with the parasites, and conducting experimental infections that mimic natural processes. In 1996, Lafferty and Morris provided what has become a classic example of parasite-increased trophic transmission; they reported a positive association between the intensity of a brain-infecting trematode (Euhaplorchis californiensis) in naturally infected California killifish (Fundulus parvipinnis) and the frequency of conspicuous behaviors, which was thought to explain the documented 10-30× increase in predation by the final host birds. Here, we address the primary gap in that study by using experimental infections to assess the causality of E. californiensis infection for increased conspicuous behaviors in F. parvipinnis. We hatched and reared uninfected F. parvipinnis from a population co-occurring with E. californiensis, and infected them 1-2 times/week over half their life span with E. californiensis and a small cyathocotylid trematode (SMCY) that targets the host's muscle tissue. At 3 time points throughout the hosts' lives, we quantified several conspicuous behaviors: contorting, darting, scratching, surfacing, and vertical positioning relative to the water's surface. Euhaplorchis californiensis and SMCY infection caused 1.8- and 2.5-fold overall increases in conspicuous behaviors, respectively. Each parasite was also associated with increases in specific conspicuous behaviors, particularly 1.9- and 1.4-fold more darting. These experimental findings help solidify E. californiensis-F. parvipinnis as a classic example of behavioral manipulation. Yet our findings for E. californiensis infection-induced behavioral change were less consistent and strong than those previously documented. We discuss potential explanations for this discrepancy, particularly the idea that behavioral manipulation may be most apparent when fish are actively attacked by predators. Our findings concerning the other studied trematode species, SMCY, highlight that trophically transmitted parasites infecting various host tissues are known to be associated with conspicuous behaviors, reinforcing calls for research examining how communities of trophically transmitted parasites influence host behavior.

Turquoise killifish: A natural model of age-dependent brain degeneration.

Turquoise killifish (Nothobranchius furzeri) are naturally short-lived vertebrates that display a wide range of spontaneous age-related changes, including onset of cancer, reduced mobility, and cognitive decline. Here, we focus on describing the phenotypic spectrum of the aging killifish brain. As turquoise killifish age, their dopaminergic and noradrenergic neurons undergo a significant decline in number. Furthermore, brain aging in turquoise killifish is associated with several glial-specific changes, such as an increase in the astrocyte-covered surface area and an increase in the numbers of microglial cells, i.e. the brain-specific macrophage population. Killifish brains undergo age-dependent reduced proteasome activity and increased protein aggregation, including the aggregation of the Parkinson's disease marker α-synuclein. Parallel to brain degeneration, turquoise killifish develop spontaneous age-related gut dysbiosis, which has been proposed to affect human neurodegenerative disease. Finally, aged turquoise killifish display declined learning capacity. We argue that, taken together, the molecular, cellular and functional changes that spontaneously take place during aging in killifish brains are consistent with a robust degenerative process that shares remarkable similarities with human neurodegenerative diseases. Hence, we propose that turquoise killifish represent a powerful model of spontaneous brain degeneration which can be effectively used to explore the causal mechanisms underlying neurodegenerative diseases.

Sex-biased response of pollution biomarkers in fish: Insights from the killifish Poecilia vivipara.

Copper ions (Cu) are one of the most frequent trace-contaminants found in Brazilian waters and, although considered as an essential element, in high concentrations can accumulate and induce toxicity. Biomarkers are important tools that can be used to assess these impacts, but to be considered trustworthy, they have to be previously tested in target organisms through laboratory studies under controlled conditions. However, many of these experiments are conducted using only males, as it is believed that the hormonal variation of females can bias the results, increasing data variability. Notwithstanding, few studies have actually tested this hypothesis, highlighting the importance of considering and measuring the role of sex in ecotoxicological studies. The aim this study was to evaluate the influence of sex on biomarkers classically used in environmental monitoring programs using the fish Poecilia vivipara as model. For this, females and males were exposed for 96 h to two Cu concentrations (9 and 20 µg/L) and a control group. In liver and gills, Cu accumulation, total antioxidant capacity (TAC) and lipid peroxidation (LPO) were evaluated. In addition, samples of peripheral blood were used for neutrophil to lymphocyte ratio determination, a measure of the onset of secondary stress. Results show that Cu hepatic accumulation did not differ between females and males, but higher levels of this metal were observed in exposed animals compared to control fish. Additionally, interactive effects were observed for hepatic LPO, as males showed elevated oxidative damage in comparison to females. Moreover, Cu exposure elevated hepatic LPO relative to control only in males, but this increase in oxidative damage was not accompanied by changes in liver TAC. On the other hand, differences in branchial Cu accumulation and LPO were not observed. Conversely, control females showed elevated TAC in comparison to control males, but Cu exposure eliminated this difference. Cu exposure also induced an increase in the N:L ratio, indicating the presence of a secondary stress response unrelated to sex. Ultimately, the findings of this study demonstrate that sex can influence the response of biomarkers that are typically used in ecotoxicological investigations in a multifaceted manner. As a result, using animals from a singular sex in such studies may result in consequential outcomes, potentially leading to underestimation or overestimation of results.

Comparative diets and prey resource competition between early-juvenile common snook Centropomus undecimalis and non-native pike killifish Belonesox belizanus.

Pike killifish Belonesox belizanus is an established non-native fish species in Florida, USA, that was first documented in south Florida in 1957 and then in Tampa Bay tributaries in 1994. Decreases in small-bodied fish abundances have been linked to the introduction of B. belizanus in both of these regions. Increases in the range and abundance of B. belizanus in the Tampa Bay area and overlap in habitat usage have led to concerns about potential competition with, and predation on, early-juvenile common snook Centropomus undecimalis [≤100 mm standard length (SL)]. Stomach contents of B. belizanus (N = 422; 14-127 mm SL) and early-juvenile C. undecimalis (N = 1132; 5-119 mm SL) were collected to examine the dietary overlap of these two species and potential differences in the diet of early-juvenile C. undecimalis from locations with and without B. belizanus co-occurring. Prey resources were collected by seine to assess prey resource limitation and prey selectivity. Stomach content analysis indicated that there was low overlap in the diet of early-juvenile C. undecimalis and B. belizanus (C ≤ 0.40). Early-juvenile C. undecimalis had a wider diet breadth, consuming many organisms that are not consumed by B. belizanus and which make up a large portion of the early-juvenile C. undecimalis diet. Analysis of prey resources indicated that some prey groups may have lower abundances in locations where B. belizanus are present, with some of these differences reflected in the diet of early-juvenile C. undecimalis. Despite these differences, there was minimal difference in the diet overlap of early-juvenile C. undecimalis from locations with and without B. belizanus co-occurring. Currently B. belizanus appear to be competing minimally with early-juvenile C. undecimalis for prey resources, with no substantial impacts being detected.

Connecting Suborganismal Data to Bioenergetic Processes: Killifish Embryos Exposed to a Dioxin-Like Compound.

A core challenge for ecological risk assessment is to integrate molecular responses into a chain of causality to organismal or population-level outcomes. Bioenergetic theory may be a useful approach for integrating suborganismal responses to predict organismal responses that influence population dynamics. We describe a novel application of dynamic energy budget (DEB) theory in the context of a toxicity framework (adverse outcome pathways [AOPs]) to make quantitative predictions of chemical exposures to individuals, starting from suborganismal data. We use early-life stage exposure of Fundulus heteroclitus to dioxin-like chemicals (DLCs) and connect AOP key events to DEB processes through "damage" that is produced at a rate proportional to the internal toxicant concentration. We use transcriptomic data of fish embryos exposed to DLCs to translate molecular indicators of damage into changes in DEB parameters (damage increases somatic maintenance costs) and DEB models to predict sublethal and lethal effects on young fish. By changing a small subset of model parameters, we predict the evolved tolerance to DLCs in some wild F. heteroclitus populations, a data set not used in model parameterization. The differences in model parameters point to reduced sensitivity and altered damage repair dynamics as contributing to this evolved resistance. Our methodology has potential extrapolation to untested chemicals of ecological concern. Environ Toxicol Chem 2023;42:2040-2053. © 2023 Oak Ridge National Laboratory and The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Rapid and precise genome engineering in a naturally short-lived vertebrate.

The African turquoise killifish is a powerful vertebrate system to study complex phenotypes at scale, including aging and age-related disease. Here, we develop a rapid and precise CRISPR/Cas9-mediated knock-in approach in the killifish. We show its efficient application to precisely insert fluorescent reporters of different sizes at various genomic loci in order to drive cell-type- and tissue-specific expression. This knock-in method should allow the establishment of humanized disease models and the development of cell-type-specific molecular probes for studying complex vertebrate biology.

Detailed whole-body nutrient analysis identifies differences in feeding ecology between related fish species: The case of Orestias native Andean killifish in Lake Titicaca.

Body nutrient profiles in ecological studies allow for relating the nutritional status of consumers and their effects on the movement and retention of elements in ecosystems, as well as reflecting feeding conditions and habitat quality. This study compared the detailed whole-body nutrient composition (macronutrients, minerals, fatty acids and amino acids) of two omnivorous natives Orestias killifish from Lake Titicaca (Orestias agassizii and Orestias luteus, Valenciennes), the largest lake in the Andes, as an indirect tool to understand differences in their feeding ecology. Although both species are usually described as omnivorous fish, both have amphipods (Hyalella spp) as their main food source. Our results showed that both killifish had a comparable macronutrient composition, and the mineral concentrations of Mg, P and Ca (reflecting bony structures) differed between them. Many of the saturated fatty acids were significantly lower in O. luteus, and O. agassizii had higher concentrations of cis-vaccenic acid (18:1n11 (cis)), supporting the idea of a higher algal contribution to the diet of this fish. The lower histidine and higher taurine concentrations in O. agassizii compared with O. luteus (independent of body size) may reflect its ubiquitous behaviour and plasticity. This study shows how whole-body nutrient analysis can identify differences in feeding ecology and feeding behaviour between related species.

Fish Models for Exploring Mitochondrial Dysfunction Affecting Neurodegenerative Disorders.

Neurodegenerative disorders are characterized by the progressive loss of neuronal structure or function, resulting in memory loss and movement disorders. Although the detailed pathogenic mechanism has not been elucidated, it is thought to be related to the loss of mitochondrial function in the process of aging. Animal models that mimic the pathology of a disease are essential for understanding human diseases. In recent years, small fish have become ideal vertebrate models for human disease due to their high genetic and histological homology to humans, ease of in vivo imaging, and ease of genetic manipulation. In this review, we first outline the impact of mitochondrial dysfunction on the progression of neurodegenerative diseases. Then, we highlight the advantages of small fish as model organisms, and present examples of previous studies regarding mitochondria-related neuronal disorders. Lastly, we discuss the applicability of the turquoise killifish, a unique model for aging research, as a model for neurodegenerative diseases. Small fish models are expected to advance our understanding of the mitochondrial function in vivo, the pathogenesis of neurodegenerative diseases, and be important tools for developing therapies to treat diseases.

In Vitro Fertilization of the African Turquoise Killifish Nothobranchius furzeri.

The ability to perform in vitro fertilization, together with sperm cryopreservation, greatly facilitates the long-term laboratory maintenance of wild-type and transgenic model organisms and helps prevent genetic drift. It is also useful in cases where reproduction may be compromised. In this protocol, we present a method for in vitro fertilization of the African Turquoise killifish Nothobranchius furzeri that is compatible with the use of fresh or cryopreserved sperm.

Taxonomic Profiling of Fecal and Intestinal Microbiota in the African Turquoise Killifish Nothobranchius furzeri.

Killifish are emerging as a new laboratory system in which to study a range of questions, from the genetic basis of embryo dormancy to life history trait evolution, age-dependent neurodegeneration, and the connection between microbial community structure and biology of aging. Over the past decade, advances in high-throughput sequencing have helped uncover the vast diversity of microbial communities present in environmental samples and on host epithelia. Here, we describe an optimized protocol to study the taxonomic composition of intestinal and fecal microbiota in laboratory-raised as well as natural killifish populations and provide comprehensive step-by-step instructions for tissue sampling, high-throughput genomic DNA extraction, and the generation of 16S V3V4 rRNA and 16S V4 rRNA gene libraries.

Constant temperature and fluctuating temperature have distinct effects on hypoxia tolerance in killifish (Fundulus heteroclitus).

Climate change is leading to rapid change in aquatic environments, increasing the mean and variability of temperatures, and increasing the incidence of hypoxia. We investigated how acclimation to constant temperatures or to diel temperature fluctuations affects hypoxia tolerance in mummichog killifish (Fundulus heteroclitus). Killifish were acclimated to constant cool (15°C), constant warm (25°C) or a diel temperature cycle (15°C at night, 25°C during day) for 6 weeks. We then measured hypoxia tolerance (time to loss of equilibrium in severe hypoxia, tLOE; critical O2 tension, Pcrit), whole-animal metabolism, gill morphology, haematology and tissue metabolites at 15°C and 25°C in a full factorial design. Among constant temperature groups, tLOE was highest and Pcrit was lowest in fish tested at their acclimation temperature. Warm-acclimated fish had lower metabolic rate at 25°C and greater gill surface area (less coverage of lamellae by interlamellar cell mass, ILCM), but cool-acclimated fish had greater brain glycogen stores. Therefore, effects of constant temperature acclimation on hypoxia tolerance were temperature specific and not exhibited broadly across test temperatures, and they were associated with different underlying mechanisms. Hypoxia tolerance was less sensitive to test temperature in fish acclimated to fluctuating temperatures compared with fish acclimated to constant temperature. Acclimation to fluctuating temperatures also increased haemoglobin-O2 affinity of the blood (decreased P50) compared with constant temperature groups. Therefore, acclimation to fluctuating temperatures helps maintain hypoxia tolerance across a broader range of temperatures, and leads to some distinct physiological adjustments that are not exhibited by fish acclimated to constant temperatures.