Does salinity mediate the toxicity of perfluorooctanesulfonic acid (PFOS) in an estuarine fish?

Perfluorooctanesulfonic acid (PFOS) is detected in estuarine environments, where salinity levels fluctuate regularly. We investigated the effects of salinity on the toxicity of PFOS in embryos and larvae of Cyprinodon variegatus. We crossed six PFOS treatments (0, 1-10,000 µg/L) with two salinities (10, 30 ppt). Larvae exposed to the highest concentration of PFOS under high salinity accumulated over twice the amount of PFOS compared to larvae maintained under low salinity. Embryonic survival was unaffected by PFOS, salinity, or their interaction. PFOS delayed time to hatch and increased salinity reduced time to hatch regardless of PFOS treatment; however, no salinity by PFOS interactions were observed. Conversely, PFOS and salinity interacted in the larval stage, with decreased survival at 30 ppt salinity. This is one of the first studies evaluating interactive effects of PFOS and high salinity and highlights the importance of assessing PFAS toxicity across life stages.

Rapid vertebrate speciation via isolation, bottlenecks, and drift.

Speciation is often driven by selective processes like those associated with viability, mate choice, or local adaptation, and "speciation genes" have been identified in many eukaryotic lineages. In contrast, neutral processes are rarely considered as the primary drivers of speciation, especially over short evolutionary timeframes. Here, we describe a rapid vertebrate speciation event driven primarily by genetic drift. The White Sands pupfish (Cyprinodon tularosa) is endemic to New Mexico's Tularosa Basin where the species is currently managed as two Evolutionarily significant units (ESUs) and is of international conservation concern (Endangered). Whole-genome resequencing data from each ESU showed remarkably high and uniform levels of differentiation across the entire genome (global FST ≈ 0.40). Despite inhabiting ecologically dissimilar springs and streams, our whole-genome analysis revealed no discrete islands of divergence indicative of strong selection, even when we focused on an array of candidate genes. Demographic modeling of the joint allele frequency spectrum indicates the two ESUs split only ~4 to 5 kya and that both ESUs have undergone major bottlenecks within the last 2.5 millennia. Our results indicate the genome-wide disparities between the two ESUs are not driven by divergent selection but by neutral drift due to small population sizes, geographic isolation, and repeated bottlenecks. While rapid speciation is often driven by natural or sexual selection, here we show that isolation and drift have led to speciation within a few thousand generations. We discuss these evolutionary insights in light of the conservation management challenges they pose.

Senescence-associated ß-galactosidase staining over the lifespan differs in a short- and a long-lived fish species.

During the aging process, cells can enter cellular senescence, a state in which cells leave the cell cycle but remain viable. This mechanism is thought to protect tissues from propagation of damaged cells and the number of senescent cells has been shown to increase with age. The speed of aging determines the lifespan of a species and it varies significantly in different species. To assess the progress of cellular senescence during lifetime, we performed a comparative longitudinal study using histochemical detection of the senescence-associated beta-galactosidase as senescence marker to map the staining patterns in organs of the long-lived zebrafish and the short-lived turquoise killifish using light- and electron microscopy. We compared age stages corresponding to human stages of newborn, childhood, adolescence, adult and old age. We found tissue-specific but conserved signal patterns with respect to organ distribution. However, we found dramatic differences in the onset of tissue staining. The stained zebrafish organs show little to no signal at newborn age followed by a gradual increase in signal intensity, whereas the organs of the short-lived killifish show an early onset of staining already at newborn stage, which remains conspicuous at all age stages. The most prominent signal was found in liver, intestine, kidney and heart, with the latter showing the most prominent interspecies divergence in onset of staining and in staining intensity. In addition, we found staining predominantly in epithelial cells, some of which are post-mitotic, such as the intestinal epithelial lining. We hypothesize that the association of the strong and early-onset signal pattern in the short-lived killifish is consistent with a protective mechanism in a fast growing species. Furthermore, we believe that staining in post-mitotic cells may play a role in maintaining tissue integrity, suggesting different roles for cellular senescence during life.

Age-related changes in the zebrafish and killifish inner ear and lateral line.

Age-related hearing loss (ARHL) is a debilitating disorder for millions worldwide. While there are multiple underlying causes of ARHL, one common factor is loss of sensory hair cells. In mammals, new hair cells are not produced postnatally and do not regenerate after damage, leading to permanent hearing impairment. By contrast, fish produce hair cells throughout life and robustly regenerate these cells after toxic insult. Despite these regenerative abilities, zebrafish show features of ARHL. Here, we show that aged zebrafish of both sexes exhibited significant hair cell loss and decreased cell proliferation in all inner ear epithelia (saccule, lagena, utricle). Ears from aged zebrafish had increased expression of pro-inflammatory genes and significantly more macrophages than ears from young adult animals. Aged zebrafish also had fewer lateral line hair cells and less cell proliferation than young animals, although lateral line hair cells still robustly regenerated following damage. Unlike zebrafish, African turquoise killifish (an emerging aging model) only showed hair cell loss in the saccule of aged males, but both sexes exhibit age-related changes in the lateral line. Our work demonstrates that zebrafish exhibit key features of auditory aging, including hair cell loss and increased inflammation. Further, our finding that aged zebrafish have fewer lateral line hair cells yet retain regenerative capacity, suggests a decoupling of homeostatic hair cell addition from regeneration following acute trauma. Finally, zebrafish and killifish show species-specific strategies for lateral line homeostasis that may inform further comparative research on aging in mechanosensory systems.

Neurotrophins and Trk Neurotrophin Receptors in the Retina of Adult Killifish (Nothobranchius guentheri).

Specific subpopulations of neurons in nerve and sensory systems must be developed and maintained, and this is accomplished in significant part by neurotrophins (NTs) and the signaling receptors on which they act, called tyrosine protein kinase receptors (Trks). The neurotrophins-tyrosine protein kinase receptors (NTs/Trks) system is involved in sensory organ regulation, including the visual system. An NTs/Trks system alteration is associated with neurodegeneration related to aging and diseases, including retinal pathologies. An emergent model in the field of translational medicine, for instance, in aging study, is the annual killifish belonging to the Nothobranchius genus, thanks to its short lifespan. Members of this genus, such as Nothobranchius guentheri, and humans share a similar retinal stratigraphy. Nevertheless, according to the authors' knowledge, the occurrence and distribution of the NTs/Trks system in the retina of N. guentheri has never been investigated before. Therefore, the present study aimed to localize neurotrophin BDNF, NGF, and NT-3 and TrkA, TrkB, and TrkC receptors in the N. guentheri retina using the immunofluorescence method. The present investigation demonstrates, for the first time, the occurrence of the NTs/Trks system in N. guentheri retina and, consequently, the potential key role of these proteins in the biology and survival of the retinal cells.

Ecological character displacement among Nothobranchius annual killifishes in Tanzania.

Divergent ecological character displacement (ECD) is the competition-driven divergence in resource use-related phenotypic traits between coexisting species. It is considered one of the primary drivers of ecological diversification and adaptive radiation. We analyzed phenotypic and ecological variation in 2 African annual killifish species of the genus Nothobranchius: N. eggersi and N. melanospilus in sympatry and N. melanospilus in allopatry. Our aim was to test whether allopatric and sympatric populations of N. melanospilus differ morphologically from each other and from N. eggersi and examine whether these differences are consistent with the predictions of ECD. We find that sympatric N. melanospilus differ from allopatric N. melanospilus and differ from N. eggersi more strongly than the latter. Our data satisfy four criteria for demonstrating ECD: Differences in phenotypes between allopatric and sympatric N. melanospilus are greater than expected by chance; the divergence pattern between allopatric and sympatric N. melanospilus results from an evolutionary shift rather than from ecological sorting; morphological differences observed reflect differences in resource use; and, lastly, sites of allopatry and sympatry do not differ in food resource availability or other ecological conditions. Our results suggest that competition is the main driver of the observed divergence between two N. melanospilus populations.

Modeling familial and sporadic Parkinson's disease in small fishes.

The establishment of animal models for Parkinson's disease (PD) has been challenging. Nevertheless, once established, they will serve as valuable tools for elucidating the causes and pathogenesis of PD, as well as for developing new strategies for its treatment. Following the recent discovery of a series of PD causative genes in familial cases, teleost fishes, including zebrafish and medaka, have often been used to establish genetic PD models because of their ease of breeding and gene manipulation, as well as the high conservation of gene orthologs. Some of the fish lines can recapitulate PD phenotypes, which are often more pronounced than those in rodent genetic models. In addition, a new experimental teleost fish, turquoise killifish, can be used as a sporadic PD model, because it spontaneously manifests age-dependent PD phenotypes. Several PD fish models have already made significant contributions to the discovery of novel PD pathological features, such as cytosolic leakage of mitochondrial DNA and pathogenic phosphorylation in α-synuclein. Therefore, utilizing various PD fish models with distinct degenerative phenotypes will be an effective strategy for identifying emerging facets of PD pathogenesis and therapeutic modalities.

Cortisol enhances aerobic metabolism and locomotor performance during the transition to land in an amphibious fish.

Amphibious fishes on land encounter higher oxygen (O2) availability and novel energetic demands, which impacts metabolism. Previous work on the amphibious mangrove killifish (Kryptolebias marmoratus) has shown that cortisol becomes elevated in response to air exposure, suggesting a possible role in regulating metabolism as fish move into terrestrial environments. We tested the hypothesis that cortisol is the mechanism by which oxidative processes are upregulated during the transition to land in amphibious fishes. We used two groups of fish, treated fish (+metyrapone, a cortisol synthesis inhibitor) and control (-metyrapone), to determine the impact of cortisol during air exposure (0 and 1 h, 7 days) on O2 consumption, terrestrial locomotion, the phenotype of red skeletal muscle, and muscle lipid concentration. Metyrapone-treated fish had an attenuated elevation in O2 consumption rate during the water to air transition and an immediate reduction in terrestrial exercise performance relative to control fish. In contrast, we found no short- (0 h) or long-term (7 days) differences between treatments in the oxidative phenotype of red muscles, nor in muscle lipid concentrations. Our results suggest that cortisol stimulates the necessary increase in aerobic metabolism needed to fuel the physiological changes that amphibious fishes undergo during the acclimation to air, although further studies are required to determine specific mechanisms of cortisol regulation.

Preparation of High-Viability Single-Cell Suspensions from African Turquoise Killifish Brain Tissue.

Studying the brain at the single-cell level has become increasingly popular in recent years. This, however, remains challenging, especially in emerging model organisms. To carry out single-cell sequencing, the preparation of a high-viability single-cell suspension is critical. In this protocol, we describe how to prepare a high-viability single-cell suspension starting from brain tissue of the African turquoise killifish (Nothobranchius furzeri). The protocol consists of dissection, enzymatic and mechanical dissociation of the brain tissue, and debris removal. The protocol described here has been successfully used for 10× Genomics single-cell sequencing of the telencephalon of adult killifish, which requires a cell viability of at least 70%. In addition to single-cell sequencing experiments, the single-cell suspension generated can be used for other applications, including cell culture and flow cytometry.

A Robust and Reproducible Method to Study Neurorepair after Stab Injury in the African Turquoise Killifish Telencephalon.

The aging population (people >60 yr old) is steadily increasing worldwide, resulting in an increased prevalence of age-related neurodegenerative diseases. Despite intensive research efforts in the past decades, there are still no therapies available to stop, cure, or prevent these diseases. Induction of successful neuroregeneration (i.e., the production of new neurons that can functionally integrate into the existing neural circuitry) could represent a therapy to replace neurons lost by injury or disease in the aged central nervous system. The African turquoise killifish, with its particularly short life span, has emerged as a useful model to study how aging influences neuroregeneration. Here, we describe a robust and reproducible stab-injury protocol to study regeneration in the telencephalon of the African turquoise killifish. After the injury, newborn cells are traced by conducting a BrdU pulse-chase experiment. To identify newborn neurons, a double immunohistochemical staining for BrdU and HuCD is carried out. Techniques such as bromodeoxyuridine (BrdU) labeling, intracardial perfusion, cryosectioning, and immunofluorescence staining are described as separate sections.

Fluorescent In Situ Hybridization in Embryos of the African Turquoise Killifish Nothobranchius furzeri.

Precisely where and when a given gene is expressed is crucial for our understanding of developmental and cell biology but determining this is often constrained by detection limits. Here, we describe a technique for visualization of low-copy mRNA in Nothobranchius furzeri embryos using tyramide signal amplification (TSA). In this protocol, an anti-sense digoxigenin-labeled RNA probe is hybridized to mRNA in situ. Anti-digoxigenin antibodies conjugated to horseradish peroxidase (POD) are then bound to the probe and reacted with fluorescently labeled tyramide. Combining this method with a counterstain, such as DAPI, allows for the detection of mRNA at a single-cell resolution.

The African Turquoise Killifish: A Scalable Vertebrate Model for Aging and Other Complex Phenotypes.

The African turquoise killifish Nothobranchius furzeri is currently the shortest-lived vertebrate that can be bred in captivity. Because of its short life span of only 4-6 months, rapid generation time, high fecundity, and low cost of maintenance, the African turquoise killifish has emerged as an attractive model organism that combines the scalability of invertebrate models with the unique features of vertebrate organisms. A growing community of researchers is using the African turquoise killifish for studies in diverse fields, including aging, organ regeneration, development, "suspended animation," evolution, neuroscience, and disease. A wide range of techniques is now available for killifish research, from genetic manipulations and genomic tools to specialized assays for studying life span, organ biology, response to injury, etc. This protocol collection provides detailed descriptions of the methods that are generally applicable to all killifish laboratories and those that are limited to specific disciplines. Here, we give an overview of the features that render the African turquoise killifish a unique fast-track vertebrate model organism.

Chromatin Accessibility Profiling and Data Analysis Using ATAC-seq in Nothobranchius furzeri.

The state of genome-wide chromatin accessibility in cells, tissues, or organisms can be investigated with a technique called assay for transposase-accessible chromatin using sequencing (ATAC-seq). ATAC-seq is a powerful approach for profiling the epigenomic landscape of cells using very low input materials. Analysis of chromatin accessibility data allows for prediction of gene expression and identification of regulatory elements such as potential enhancers and specific transcription-factor binding sites. Here, we describe an optimized ATAC-seq protocol for the preparation of isolated nuclei and subsequent next-generation sequencing from whole embryos and tissues of the African turquoise killifish (Nothobranchius furzeri). Importantly, we provide an overview of a pipeline for processing and analyzing ATAC-seq data from killifish.

Efficient Transgenesis in African Turquoise Killifish Using a Gibson Assembly-Based Tol2 Transposon System.

The short-lived African killifish Nothobranchius furzeri is an attractive genetic model for vertebrate aging and regeneration studies. The utilization of genetically modified animals is a common strategy for unveiling molecular mechanisms responsible for a biological phenomenon. Here, we report a highly efficient protocol for generating transgenic African killifish using the Tol2 transposon system, which creates random insertions in the genome. Transgenic vectors carrying gene-expression cassettes of interest and an eye-specific marker for transgene identification can be quickly assembled through Gibson assembly. The development of this new pipeline will facilitate transgenic reporter assays and gene-expression-related manipulations in African killifish.

Limited impact of weathered residues from the Deepwater Horizon oil spill on the gut-microbiome and foraging behavior of sheepshead minnows (Cyprinodon variegatus).

The Deepwater Horizon disaster of April 2010 was the largest oil spill in U.S. history and exerted catastrophic effects on several ecologically important fish species in the Gulf of Mexico (GoM). Within fish, the microbiome plays a key symbiotic role in maintaining host health and aids in acquiring nutrients, supporting immune function, and modulating behavior. The aim of this study was to examine if exposure to weathered oil might produce significant shifts in fish gut-associated microbial communities as determined from taxa and genes known for hydrocarbon degradation, and whether foraging behavior was affected. The gut microbiome (16S rRNA and shotgun metagenomics) of sheepshead minnow (Cyprinodon variegatus) was characterized after fish were exposed to oil in High Energy Water Accommodated Fractions (HEWAF; tPAH = 81.1 ± 12.4 µg/L) for 7 days. A foraging behavioral assay was used to determine feeding efficiency before and after oil exposure. The fish gut microbiome was not significantly altered in alpha or beta diversity. None of the most abundant taxa produced any significant shifts as a result of oil exposure, with only rare taxa showing significant shifts in abundance between treatments. However, several bioindicator taxa known for hydrocarbon degradation were detected in the oil treatment, primarily Sphingomonas and Acinetobacter. Notably, the genus Stenotrophomonas was detected in high abundance in 16S data, which previously was not described as a core member of fish gut microbiomes. Data also demonstrated that behavior was not significantly affected by oil exposure. Potential low bioavailability of the oil may have been a factor in our observation of minor shifts in taxa and no behavioral effects. This study lays a foundation for understanding the microbiome of captive sheepshead minnows and indicates the need for further research to elucidate the responses of the fish gut-microbiome under oil spill conditions.

Comprehensive Colony Health Management and Emerging Pathogens of the Annual Killifish Species Nothobranchius furzeri.

The Leibniz Institute on Aging has maintained killifish colonies for over 15 y. Our veterinarians, scientists, and animal technicians developed a fish health scoring system and routine colony health surveillance program for our colonies. Over a 4-y period, health data from the African turquoise killifish Nothobranchius furzeri colony were systematically collected and analyzed. The fish health assessment system facilitated categorization of clinical signs and differentiation of fish with mild clinical signs from fish that required euthanasia. This report provides new information on clinical signs and conditions that may occur in young and aged N. furzeri. To be comprehensive, a colony health surveillance program incorporates animal health at both the individual and the population levels. The quarterly routine health monitoring program identified Mycobacterium spp. as the most common agent in our facility and identified the killifish pathogen (Loma acerinae) for the first time. Taken together, these findings demonstrate the importance of a comprehensive colony health management system in a fish research facility. By improving the health and welfare of fish used for research, the scientific community will benefit from less variable and more reliably reproducible research results.

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.

Jaw size variation is associated with a novel craniofacial function for galanin receptor 2 in an adaptive radiation of pupfishes.

Understanding the genetic basis of novel adaptations in new species is a fundamental question in biology. Here we demonstrate a new role for galr2 in vertebrate craniofacial development using an adaptive radiation of trophic specialist pupfishes endemic to San Salvador Island, Bahamas. We confirmed the loss of a putative Sry transcription factor binding site upstream of galr2 in scale-eating pupfish and found significant spatial differences in galr2 expression among pupfish species in Meckel's cartilage using in situ hybridization chain reaction (HCR). We then experimentally demonstrated a novel role for Galr2 in craniofacial development by exposing embryos to Garl2-inhibiting drugs. Galr2-inhibition reduced Meckel's cartilage length and increased chondrocyte density in both trophic specialists but not in the generalist genetic background. We propose a mechanism for jaw elongation in scale-eaters based on the reduced expression of galr2 due to the loss of a putative Sry binding site. Fewer Galr2 receptors in the scale-eater Meckel's cartilage may result in their enlarged jaw lengths as adults by limiting opportunities for a circulating Galr2 agonist to bind to these receptors during development. Our findings illustrate the growing utility of linking candidate adaptive SNPs in non-model systems with highly divergent phenotypes to novel vertebrate gene functions.

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.

Combined toxic effects of cadmium and environmental microplastics in Aphanius fasciatus (Pisces, Cyprinodontidae).

Microplastics (MPs), plastic particles smaller than 5 mm in diameter, have received extensive attention as new environmental pollutants with still unexplored potential ecological risks. The main objective of the present study is to see if the concomitant exposure to MPs and Cd is more toxic than that to MPs or Cd separately in Aphanius fasciatus. Immature female were exposed to Cd and/or MPs for 21 days, and the subsequent effects were monitored by a combination of biochemical, histological and molecular toxicity markers. Exposure to Cd, but not to MPs, increased metallothioneins content and mRNA levels of the metallothioneins gene MTA both in liver and gills. In addition, we observed a significant oxidative stress response at histological, enzymatic (Catalase and Superoxide dismutase), non-enzymatic (proteins sulfhydryl and malondialdehyde) and gene expression levels to both toxicants in both tissues, particularly in gills, but no clear evidence for interaction between the two factors. Our results indicate a major effect of MPs on gills at different organizational levels. Finally, exposure to both MPs and Cd induced spinal deformities, although bone composition was only altered by the latter, whereas MTA mRNA bone levels were only increased realtive to controls in doubly-exposed samples. Interestingly, the simultaneous use of both pollutants produced the same effects as Cd and MPs alone, probably due to reduced bioavailability of this heavy metal.