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Climate change alters multiple abiotic environmental factors in aquatic environments but relatively little is known about their interacting impacts, particularly in developing organisms where these exposures have the potential to cause long-lasting effects. To explore these issues, we exposed developing killifish embryos (Fundulus heteroclitus) to 26 °C or 20 °C and 20 ppt or 3 ppt salinity in a fully-factorial design. After hatching, fish were transferred to common conditions of 20 °C and 20 ppt to assess the potential for persistent developmental plasticity. Warm temperature increased hatching success and decreased hatch time, whereas low salinity negatively affected hatching success, but this was only significant in fish developed at 20 °C. Temperature, salinity, or their interaction affected mRNA levels of genes typically associated with thermal and hypoxia tolerance (hif1a, hsp90b, hsp90a, hsc70, and hsp70.2) across multiple developmental timepoints. These differences were persistent into the juvenile stage, where the fish that developed at 26 °C had higher expression of hif1a, hsp90b, hsp90a, and hsp70.2 than fish developed at 20 °C, and this was particularly evident for the group developed at both high temperature and salinity. There were also long-lasting effects of developmental treatments on body size after four months of rearing under common conditions. Fish developed at low salinity or temperature were larger than fish developed at high temperature or salinity, but there was no interaction between the two factors. These data highlight the complex nature of the developmental effects of interacting stressors which has important implications for predicting the resilience of fishes in the context of climate change., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Hypoxia tolerance in aquatic ectotherms involves a suite of behavioral and physiological responses at the organismal, tissue, and cellular levels. The current study evaluated two closely related killifish species (Fundulus heteroclitus, Fundulus majalis) to evaluate responses to acute moderate and acute severe hypoxia. Routine metabolic rate and loss of equilibrium were assessed, followed by analysis in skeletal muscle of markers of oxidative damage to proteins (2,4-DNPH), lipids (4-HNE), and DNA (8-OHdG), hypoxia signaling (HIF1α, HIF2α), cellular energy state (p-AMPK: AMPK), and protein degradation (Ubiquitin, LC3B, Calpain 2, Hsp70). Both species had a similar reduction in metabolic rate at low PO2. However, F. heteroclitus was the more hypoxia-tolerant species based on a lower PO2 at which there was loss of equilibrium, perhaps due in part to a lower oxygen demand at all oxygen tensions. Despite the differences in hypoxia tolerance between the species, skeletal muscle molecular markers were largely insensitive to hypoxia, and there were few differences in responses between the species. Thus, the metabolic depression observed at the whole animal level appears to limit perturbations in skeletal muscle in both species during the hypoxia treatments., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

Thermal performance curves (TPCs) provide a framework for understanding the effects of temperature on ectotherm performance and fitness. TPCs are often used to test hypotheses regarding local adaptation to temperature or to develop predictions for how organisms will respond to climate warming. However, for aquatic organisms such as fishes, most TPCs have been estimated for adult life stages, and little is known about the shape of TPCs or the potential for thermal adaptation at sensitive embryonic life stages. To examine how latitudinal gradients shape TPCs at early life stages in fishes, we used two populations of Fundulus heteroclitus that have been shown to exhibit latitudinal variation along the thermal cline as adults. We exposed embryos from both northern and southern populations and their reciprocal crosses to eight different temperatures (15°C, 18°C, 21°C, 24°C, 27°C, 30°C, 33°C, and 36°C) until hatch and examined the effects of developmental temperature on embryonic and larval traits (shape of TPCs, heart rate, and body size). We found that the pure southern embryos had a right-shifted TPC (higher thermal optimum (T opt ) for developmental rate, survival, and embryonic growth rate) whereas pure northern embryos had a vertically shifted TPC (higher maximum performance (P max ) for developmental rate). Differences across larval traits and cross-type were also found, such that northern crosses hatched faster and hatched at a smaller size compared to the pure southern population. Overall, these observed differences in embryonic and larval traits are consistent with patterns of both local adaptation and countergradient variation., (© 2024 The Author(s). Journal of Experimental Zoology Part A: Ecological and Integrative Physiology published by Wiley Periodicals LLC.)

Annual killifishes have active and voracious rates in acquisition of food resources with display of behaviors that allow them to maintain high metabolic rates to deal with the harsh and unpredictable conditions prevalent in temporary pools where they inhabit. The objective of this research was to describe histologically and ultrastructurally, the digestive system of the annual killifish Millerichthys robustus to identify morphological traits related to its annual life history and digestive physiology. Also, we quantify food items along the intestine as a proxy for rates of digestion. Millerichthys has a short digestive system, associated with a carnivorous diet, with no evidence of a stomach. Instead, the presence of pharyngeal jaws with caniform teeth was documented, related to the breakdown of invertebrate exoskeletons, allowing prey fluids to be tasted by taste corpuscles related to selection of food items, and that digestive enzymes penetrate once in the intestine. The histological morphology of the intestine showed four different regions, associated with its digestive rates: (i) reception of food from the esophagus with intact pray; (ii) digestion with enzymes from the pancreas and liver/gallbladder of simple exoskeleton prey (Entognatha), and beginning of absorption; (iii) absorption of nutrients, and digestion of large-complex exoskeleton prey (Hexanauplia, Brachiopoda, and Ostracoda); and (iv) probable absorption of intact macromolecules. The second region of the intestine presented two anatomical loops and the highest thickness that may be related to reducing the speed of food transit, allowing for more efficient digestion given the large amount of food ingested by this species., (© 2024 American Association for Anatomy.)

Externally laid eggs are often responsive to environmental cues; however, it is unclear how such plasticity evolves. In Trinidad, the killifish ( Anablepsoides hartii ) is found in communities with and without predators. Here, killifish inhabit shallower, ephemeral habitats in sites with predators. Such shifts may increase the exposure of eggs to air and lead to possible desiccation. We compared egg-hatching plasticity between communities by rearing eggs terrestrially on peat moss or in water. The timing of hatching did not differ between communities when eggs were reared in water. Eggs from sites with predators responded to terrestrial incubation by hatching significantly earlier compared with water-reared eggs. These responses were weaker in sites with no predators. Such divergent trends show that the presence of predators is associated with evolutionary shifts in hatching plasticity. Our results provide evidence for local adaptation in embryonic plasticity at the population scale.

The maximum rate at which fish can take up oxygen from their environment to fuel aerobic metabolism is an important feature of their physiology and ecology. Methods to quantify maximum oxygen uptake rate (ṀO 2 ), therefore, should reliably and reproducibly estimate the highest possible ṀO 2 by an individual or species under a given set of conditions (peak ṀO 2 ). This study determined peak ṀO 2 and its repeatability in Gulf killifish, Fundulus grandis, subjected to three methods to elevate metabolism: swimming at increasing water speeds, during recovery after an exhaustive chase, and after ingestion of a large meal. Estimates of peak ṀO 2 during swimming and after an exhaustive chase were repeatable across two trials, whereas peak ṀO 2 after feeding was not. Peak ṀO 2 determined by the three methods was significantly different from one another, being highest during swimming, lowest after an exhaustive chase, and intermediate after feeding. In addition, peak ṀO 2 during recovery from an exhaustive chase depended on the length of time of recovery: in nearly 60% of the trials, values within the first hour of the chase were lower than those measured later. A novel and important finding was that an individual's peak ṀO 2 was not repeatable when compared across methods. Therefore, the peak ṀO 2 estimated for a group of fish, as well as the ranking of individual ṀO 2 within that group, depends on the method used to elevate aerobic metabolism., (© 2024 Fisheries Society of the British Isles.)

Kryptolebias marmoratus (Kmar), a teleost fish of the order Cyprinodontiformes, has a suite of unique phenotypes and behaviors not observed in other fishes. Many of these phenotypes are discrete and highly plastic-varying over time within an individual, and in some cases reversible. Kmar and its interfertile sister species, K. hermaphroditus, are the only known self-fertile vertebrates. This unusual sexual mode has the potential to provide unique insights into the regulation of vertebrate sexual development, and also lends itself to genetics. Kmar is easily adapted to the lab and requires little maintenance. However, its internal fertilization and small clutch size limits its experimental use. To support Kmar as a genetic model, we compared alternative husbandry techniques to maximize recovery of early cleavage-stage embryos. We find that frequent egg collection enhances yield, and that protease treatment promotes the greatest hatching success. We completed a forward mutagenesis screen and recovered several mutant lines that serve as important tools for genetics in this model. Several will serve as useful viable recessive markers for marking crosses. Importantly, the mutant kissylips lays embryos at twice the rate of wild-type. Combining frequent egg collection with the kissylips mutant background allows for a substantial enhancement of early embryo yield. These improvements were sufficient to allow experimental analysis of early development and the successful mono- and bi-allelic targeted knockout of an endogenous tyrosinase gene with CRISPR/Cas9 nucleases. Collectively, these tools will facilitate modern developmental genetics in this fascinating fish, leading to future insights into the regulation of plasticity., (© 2023 The Authors. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution published by Wiley Periodicals LLC.)

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.

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., Competing Interests: Declaration of Competing Interest Both authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© 2023 Oak Ridge National Laboratory and The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.)

Blood withdrawal is a common procedure performed on laboratory animals to monitor key processes and indicators of fish health and physiology, such as hematopoiesis, hemostasis, and lipid and glucose metabolism. Moreover, the ability to extract blood with minimal invasiveness and without sacrificing animals enables repeated sampling, allowing both longitudinal studies of individual animals, as well as reducing the number of experimental animals needed in a study. The African turquoise killifish is an emerging animal model that is progressively being adopted worldwide for aging studies because of its naturally short life span. However, because of the small body size of this species, nonlethal blood collection is a challenging procedure. Here we present a detailed protocol enabling repeated blood sampling from the same individual fish. This method, if correctly executed, is minimally invasive and does not cause any lasting damage. The protocol has been tested on animals spanning from 6 to 24 wk of age and the amount of blood that could be extracted varied from 0.5 to 8 µL, greatly depending on specimen age, sex, and size. This volume is sufficient to perform analyses such as blood glucose measurement, blood cell counts, or histological stains on blood smears., (© 2023 Cold Spring Harbor Laboratory Press.)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

B cells undergo a process of somatic recombination leading to the synthesis of a staggering diversity of transmembrane and secreted antibodies. This process dates back to the evolution of jawed vertebrates and is pivotal to the origin of lymphocyte-based adaptive immune responses. Here, we optimized a sequencing-based protocol to characterize B-cell-specific IgH transcripts from bulk RNA in the African turquoise killifish, an emerging model organism characterized by naturally short life span and by a vast range of age-dependent dysfunctions., (© 2023 Cold Spring Harbor Laboratory Press.)

Species distribution models predict a poleward migration for marine ectotherms with ocean warming. However, a key limitation in current species distribution models (SDM) is that they do not account for population-specific heterogeneity in physiological responses to temperature change resulting from local adaptations and acclimatization. To address this gap, we developed a novel, Physiology Integrated BioClimate Model (PIBCM) that combines habitat-specific metabolic thermal physiological tolerance of a species into a bioclimate envelope model. Using a downscaling approach, we also established a fine-resolution coastal sea-surface temperature data set for 2050-2080, that showed a high degree of location-specific variability in future thermal regimes. Combining predicted temperature data with the PIBCM model, we estimated habitat distribution for a highly eurythermal intertidal minnow, the Atlantic killifish (Fundulus heteroclitus), a species that likely presents a best-case-scenario for coastal vertebrates. We show that the killifish northern boundary shifts southwards, while distinct habitat fragmentation occurs in the southern sub-population (due to migration of adjacent fish populations to the nearest metabolically optimal thermal habitat). When compared to current SDMs (e.g., AquaMaps), our results emphasize the need for thermal physiology integrated range shift models and indicate that habitat fragmentation for coastal fishes may reshape nursery habitats for many commercially and ecologically important species., (© 2022. The Author(s).)

In fresh water, environmental Ca ameliorates Zn toxicity because Ca 2+ and Zn 2+ compete for uptake at the gills. Zn toxicity is also lower in sea water, but it is unclear whether this is due to increased Ca 2+ concentration, and/or to the other ions present at higher salinity. Using the euryhaline killifish, we evaluated the relative roles of Ca 2+ (as CaNO 3 ) versus the other ions contributing to salinity in protecting against physiological symptoms of Zn 2+ toxicity. Killifish were exposed to a sublethal level of Zn (500 μg/L, as ZnSO 4 ) for 96 h in either fresh water (0 % salinity) at low (1 mmol/L) and High Ca (10 mmol/L) or 35 ppt sea water (100 % salinity) at low (1 mmol/L) and High Ca (10 mmol/L). At 0 % salinity, High Ca partly or completely protected against the following effects of Zn seen at Low Ca: elevated plasma Zn, hypocalcaemia, inhibited unidirectional Ca 2+ influx, inhibited branchial Na + /K + ATPase and Ca 2+ ATPase activities, and oxidative stress in gills, liver, intestine, and muscle. At 100 % salinity, in the presence of 1 mmol/L (Low Ca), Zn caused no disturbances in most of these same parameters, showing that the "non-Ca" components of sea water alone provided complete protection. However, for a few endpoints (inhibited intestinal Ca 2+ ATPase activity, oxidative stress in gill and liver), High Ca (10 mmol/L) was needed to provide full protection against Zn in 100 % salinity. There was no instance where the combination of 100 % salinity and High Ca failed to provide complete protection against Zn-induced disturbances in sea water., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Atlantic killifish, Fundulus heteroclitus, are intertidal marsh fish found along the east coast of North America. Associated with the thermal gradient along this coast, northern and southern killifish populations are known to differ in morphology, behavior, and physiology, including in their cortisol stress response. Our goal was to explore population differences in the stress response and identify underlying molecular mechanisms. We measured responses to both acute and repeated stress in plasma cortisol, stress axis mRNA expression, and body condition in northern and southern killifish. Following an acute stressor, the southern population had higher cortisol levels than the northern population but there was no difference between populations following repeated stress. In the brain, both corticotropin releasing factor and its binding protein had higher expression in the southern than the northern population, but the northern population showed more changes in mRNA levels following a stressor. In the head kidney, Melanocortin 2 Receptor and steroidogenic acute regulatory protein mRNA levels were higher in the southern population suggesting a larger capacity for cortisol synthesis than in the northern fish. Lastly, the glucocorticoid receptor GR1 mRNA levels were greater in the liver of southern fish, suggesting a greater capacity to respond to cortisol, and GR2 had differential expression in the head kidney, suggesting an interpopulation difference in stress axis negative feedback loops. Southern, but not northern, fish were able to maintain body condition following stress, suggesting that these differences in the stress response may be important for adaptation across latitudes., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The absence of a controlled diet is unfortunate in a promising model organism for ageing, the turquoise killifish (Nothobranchius furzeri Jubb, 1971). Currently captive N. furzeri are fed bloodworms but it is not known whether this is an optimal diet. Replacing bloodworms with a practical dry feed would reduce diet variability. In the present study, we estimated the nutritional value of the diet ingested by wild fish and determined the fish-body amino acid profile as a proxy for their nutritional requirements. We compared the performance of fish fed four commercial feeds containing 46%-64% protein to that achieved with bloodworms and that of wild fish. Wild fish target a high-protein (60%) diet and this is supported by their superior performance on high-protein diets in captivity. In contrast, feeds for omnivores led to slower growth, lower fecundity and unnatural liver size. In comparison to wild fish, a bloodworm diet led to lower body condition, overfeeding and male liver enlargement. Out of the four dry feeds tested, the fish fed Aller matched wild fish in body condition and liver size, and was comparable to bloodworms in terms of growth and fecundity. A starter feed for carnivorous species appears to be a practical replacement for bloodworms for N. furzeri. The use of dry feeds improved performance in comparison to bloodworms and thus may contribute to reducing response variability and improving research reproducibility in N. furzeri research., (© 2022 Fisheries Society of the British Isles.)

Phoxinus lagowskii is a popular fish in Chinese cuisine. Though it is found mainly in China's high-latitude regions, where diel-cycling hypoxia (DCH) is known to have unique impacts on aquatic organisms, there is little known about its response to hypoxia. Currently, nothing is known about the changes in blood parameters, gill and liver morphology, glucose and lipid metabolism, or expression of genes involved in clock and glucose metabolism in response to sustained hypoxia (SH) and diel-cycling hypoxia (DCH). To elucidate the influence of sustained and diel-cycling hypoxia on fish hypoxia tolerance, resting oxygen consumption (MO 2 ) analysis was performed after ten days of hypoxia. This analysis revealed that hypoxia tolerance profoundly improved after ten days of either sustained or diel-cycling hypoxia acclimation, with DCH groups showing greater improvements than SH groups. Additionally, an increase in RBCs was found in P. lagowskii, suggesting an increase in the O 2 -carrying capacity of the blood to tolerate hypoxia. Hemoglobin (Hb) concentrations in P. lagowskii were increased at four days of diel-cycling hypoxia, confirming that physiological and metabolic adaptation to hypoxia is based on the duration of O 2 exposure. Increased Hb and hematocrit (Hct) were found in DCH-exposed fish, both of which have been directly linked to high-latitude hypoxia tolerance. In the gills, lamella surface area increased in SH-exposed fish more than DCH-exposed fish, and these increases were accompanied by a decrease in the volume of interlamellar cell mass (ILCM). Histology changes in the liver showed a higher frequency of cytoplasmic vacuolization in DCH-exposed fish. PK increases in SH-exposed fish suggest that fish can use more energy sources in persistent hypoxia. Meanwhile, DCH-exposed fish use TG as an energy source. In SH-exposed fish, self-regulation of Cry1a was observed, whereas Cry1b gene was up-regulated significantly. In DCH-exposed fish, three of eight clock genes studied had increased expression, including Per1a, Clocka, and Cry1b, suggesting that SH and DCH result in different hypoxic responses. This study presents a novel approach to the study of fish responses to hypoxia in high latitude and shows that sustained hypoxia and diel-cycling hypoxia induce large differences in fish physiology., (Copyright © 2021. Published by Elsevier Inc.)

Short-lived turquoise killifish ( Nothobranchius furzeri ) have become a popular model organism for neuroscience. In the present paper we study for the first time their behavior in the novel tank diving test and the levels of mRNA of various 5-HT-related genes in brains of 2-, 4- and 6-month-old males and females of N. furzeri . The marked effect of age on body mass, locomotor activity and the mRNA level of Tph1b , Tph2 , Slc6a4b , Mao , Htr1aa , Htr2a , Htr3a , Htr3b , Htr4 , Htr6 genes in the brains of N. furzeri males was shown. Locomotor activity and expression of the Mao gene increased, while expression of Tph1b , Tph2 , Slc6a4b , Htr1aa , Htr2a , Htr3a , Htr3b , Htr4 , Htr6 genes decreased in 6-month-old killifish. Significant effects of sex on body mass as well as on mRNA level of Tph1a , Tph1b , Tph2 , Slc6a4b , Htr1aa , 5-HT2a , Htr3a , Htr3b , Htr4 , and Htr6 genes were revealed: in general both the body mass and the expression of these genes were higher in males. N. furzeri is a suitable model with which to study the fundamental problems of age-related alterations in various mRNA levels related with the brains 5-HT system.

Over the last decade, the African turquoise killifish, Nothobranchius furzeri, has emerged as an important model system for the study of vertebrate biology and ageing. Propagation of laboratory inbred strains of Nothobranchius furzeri, such as GRZ, however, can pose challenges due to the short window of fertility, the efforts and space requirements involved in continuous strain maintenance, and the risks of further inbreeding. The current method for long term strain preservation relies on arrest of embryos in diapause. To create an alternative for long term maintenance, we developed a robust protocol to cryopreserve and revive sperm for in vitro fertilization (IVF). We tested a variety of extender and activator buffers for sperm IVF, as well as cryoprotectants to achieve practical long-term storage and fertilization conditions tailored to this species. Our protocol enabled sperm to be preserved in a cryogenic condition for months and to be revived with an average of 40% viability upon thawing. Thawed sperm were able to fertilize nearly the same number of eggs as natural fertilization, with an average of ~ 25% and peaks of ~ 55% fertilization. This technical advance will greatly facilitate the use of N. furzeri as a model organism., (© 2021. The Author(s).)

The Fundulus genus of killifish includes species that inhabit marshes along the U.S. Atlantic coast and the Gulf of Mexico, but differ in their ability to adjust rapidly to fluctuations in salinity. Previous work suggests that euryhaline killifish stimulate polyamine biosynthesis and accumulate putrescine in the gills during acute hypoosmotic challenge. Despite evidence that polyamines have an osmoregulatory role in euryhaline killifish species, their function in marine species is unknown. Furthermore, the consequences of hypoosmotic-induced changes in polyamine synthesis on downstream pathways, such as ƴ-aminobutyric acid (Gaba) production, have yet to be explored. Here, we examined the effects of acute hypoosmotic exposure on polyamine, glutamate, and Gaba levels in the gills of a marine (F. majalis) and two euryhaline killifish species (F. heteroclitus and F. grandis). Fish acclimated to 32 ppt or 12 ppt water were transferred to fresh water, and concentrations of glutamate (Glu), Gaba, and the polyamines putrescine (Put), spermidine (Spd), and spermine (Spm) were measured in the gills using high-performance liquid chromatography. F. heteroclitus and F. grandis exhibited an increase in gill Put concentration, but showed no change in Glu or Gaba levels following freshwater transfer. F. heteroclitus also accumulated Spd in the gills, whereas F. grandis showed transient increases in Spd and Spm levels. In contrast, gill Put, Spm, Glu, and Gaba levels decreased in F. majalis following freshwater transfer. Together, these findings suggest that increasing polyamine levels and maintaining Glu and Gaba levels in the gills may enable euryhaline teleosts to acclimate to shifts in environmental salinity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Androdioecy, the coexistence of hermaphrodites and males, is very rare in vertebrates but occurs in mangrove killifish living in ephemeral or unstable habitats. Hermaphrodites reproduce both by outcrossing with males and by selfing. Outbreeding is advantageous because of inbreeding depression, but it requires encounters with males. The advantages of a propensity for outcrossing among hermaphrodites and the production of males affect each other very strongly. To study the evolutionary coupling of these two aspects, we here analyze a simple evolutionary game for a population composed of three phenotypes: outcrossing-oriented hermaphrodites, selfing-oriented hermaphrodites, and males. Outcrossing-oriented hermaphrodites first attempt to search for males and perform outcrossing if they encounter males. If they fail to encounter males, they reproduce via selfing. Selfing-oriented hermaphrodites simply reproduce by selfing. The replicator dynamics may show bistability, in which both the androdioecious population (with outcrossing-oriented hermaphrodites and males) and the pure hermaphroditic population are locally stable. The model shows the fraction of males is either zero or relatively high (more than 25%), which is not consistent with the observed low fraction of males (less than 5%). To explain this discrepancy, we studied several models including immigration and enforced copulation. We concluded that the observed pattern can be most likely explained by a population dominated by selfing-oriented hermaphrodites receiving immigration of males., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Historic industrial pollution of the Elizabeth River, Virginia resulted in polycyclic aromatic hydrocarbon (PAH) contamination in sediments. Atlantic killifish (Fundulus heteroclitus) inhabiting the Atlantic Wood (AW) industrial site adapted to complex PAH mixture at this Superfund site. Their embryos have proved highly resistant to cardiac abnormalities indicative of PAH toxicity. In this study, embryos spawned from adults collected at AW and King's Creek (KC), a reference site, were exposed at 24 h post fertilization (hpf) to Elizabeth River Sediment Extract (ERSE), a complex PAH mixture, in a range of concentrations (0, 5.04, 50.45, 100.90, 151.35, or 252.25 µg/L total PAHs). Embryos were processed for histology at 144 hpf to enable evaluations of hearts at tissue and cellular levels. Morphometry and severity scoring were used to evaluate the extent of alterations. Unexposed embryos were similar in both populations. ERSE exposure resulted in multiple changes to hearts of KC embryos but not AW. Alterations were particularly evident in KC embryos exposed to concentrations above 1% ERSE (50.45 µg/L), which had thinner ventricular walls and larger pericardial edema. Individuals with moderate pericardial edema maintained arrangement and proximity of heart chambers, but changes were seen in ventricular myocytes. Severe pericardial edema was prevalent in exposed KC embryos and typically resulted in tube heart formation. Ventricles of tube hearts had very thin walls composed of small, basophilic cells and lacked trabeculae. Edematous pericardial fluid contained small amounts of proteinaceous material, as did controls, and was free of cells. This fluid was primarily unstained, suggesting water influx due to increased permeability. The use of histological approaches provided more specific detail for tissue and cellular effects in hearts of embryos exposed to PAHs and enabled understanding of potential links to later life effects of early life exposure., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Egg size has a crucial impact on the reproductive success of a mother and the performance of her offspring. It is therefore reasonable to employ egg size as a proxy for egg content when studying variation in offspring performance. Here, we tested species differences in allometries of several egg content parameters with egg area. We measured individual eggs in five species of annual killifish (Cyprinodontiformes), a group of fish where egg banks permit population survival over dry season. Apart from comparing allometric scaling exponents, amounts and compositions of egg components across the different species, we assessed the explanatory power of egg area for egg wet and dry weight and for hatchling size. We found notable species-specific allometries between egg area and the other egg parameters (egg dry weight and water content, elemental composition and triglyceride content). Across species, egg area predicted egg wet weight with highest power. Within species, coefficients of determination were largest in A. elongatus, a large piscivorous species with large eggs. Our study shows that systematically using egg area as a proxy of egg content between different species can ignore relevant species-specific differences and mask within-species variability in egg content., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Fish models are essential for research in many biological and medical disciplines. With a typical lifespan of only 6 months, the Turquoise killifish (Nothobranchius furzeri) was recently established as a time- and cost-efficient model to facilitate whole-life and multigenerational studies in several research fields, including behavioural ecotoxicology. Essential information on the behavioural norm and on how laboratory conditions affect behaviour, however, is deficient. In the current study, we examined the impact of the social and structural environment on a broad spectrum of behavioural endpoints in N. furzeri. While structural enrichment affected only fish boldness and exploratory behaviour, fish rearing density affected the total body length, locomotor activity, boldness, aggressiveness and feeding behaviour of N. furzeri individuals. Overall, these results contribute to compiling a behavioural baseline for N. furzeri that increases the applicability of this new model species. Furthermore, our findings will fuel the development of improved husbandry protocols to maximize the welfare of N. furzeri in a laboratory setting., (© 2020 Fisheries Society of the British Isles.)

Aquaporins are the predominant water-transporting proteins in vertebrates, but only a handful of studies have investigated aquaporin function in fish, particularly in mediating water permeability during salinity challenges. Even less is known about aquaporin function in hypoxia (low oxygen), which can profoundly affect gill function. Fish deprived of oxygen typically enlarge gill surface area and shrink the water-to-blood diffusion distance, to facilitate oxygen uptake into the bloodstream. However, these alterations to gill morphology can result in unfavorable water and ion fluxes. Thus, there exists an osmorespiratory compromise, whereby fish must try to balance high branchial gas exchange with low ion and water permeability. Furthermore, the gills of seawater and freshwater teleosts have substantially different functions with respect to osmotic and ion fluxes; consequently, hypoxia can have very different effects according to the salinity of the environment. The purpose of this study was to determine what role aquaporins play in water permeability in the hypoxia-tolerant euryhaline common killifish (Fundulus heteroclitus), in two important osmoregulatory organs-the gills and intestine. Using immunofluorescence, we localized aquaporin-3 (AQP3) protein to the basolateral and apical membranes of ionocytes and enterocytes, respectively. Although hypoxia increased branchial AQP3 messenger-RNA expression in seawater and freshwater, protein abundance did not correlate. Indeed, hypoxia did not alter AQP3 protein abundance in seawater and reduced it in the cell membranes of freshwater gills. Together, these observations suggest killifish AQP3 contributes to reduced diffusive water flux during hypoxia and normoxic recovery in freshwater and facilitates intestinal permeability in seawater and freshwater., (© 2020 Wiley Periodicals LLC.)

Phenothiazine (PTZ) is a heterocyclic thiazine compound used for industrial and medical purposes. Through environmental surveillance studies, PTZ was found being discharged into a local river in Connecticut. Phenothiazine has been shown to act similarly to endocrine disrupting chemicals. This study sought to identify sex specific hormone receptor changes in Fundulus heteroclitus in response to PTZ exposure. Fundulus heteroclitus, also known as mummichog, are small fish native to the Atlantic coast of the United States and Canada. They reside in brackish waters and can survive harsh toxic environments. This model organism is native to the polluted waters found in Connecticut. In this study, fish were exposed to PTZ concentrations of 0.5 ppm, 1.0 ppm, and 2.0 ppm for 1 week. Following exposure, brain, liver, and gonad tissues were harvested; cDNA was synthesized; and mRNA expression was assessed for 6 different hormone receptors. Compared with vehicle control (ethanol) differences in mRNA expression, levels of hormone receptors were observed in various tissues from male and female fish. Many of the tissues assessed showed changes in expression level, while only female liver and testis showed no change. These results implicate PTZ as a potential endocrine disrupting compound to mummichog at environmentally relevant concentrations.

We used a field experiment to test the effects of population density on the growth rate and survival of Austrolebias bellottii, a Neotropical annual killifish. Effects differed between the sexes: males at high densities achieved a smaller final size and experienced higher mortality while no such effects were observed in females. This sex-specific effect could be an indirect consequence of mate competition., (© 2020 The Fisheries Society of the British Isles.)

Steroids are essential structural components of cell membranes that organize lipid rafts and modulate membrane fluidity. They can also act as signalling molecules that work through nuclear and G protein-coupled receptors to impact health and disease. Notably, changes in steroid levels have been implicated in metabolic, cardiovascular and neurodegenerative diseases, but how alterations in the steroid pool affect ageing is less well understood. One of the major challenges in steroidomic analysis is the ability to simultaneously detect and distinguish various steroids due to low in vivo concentrations and naturally occurring stereoisomers. Here, we established such a method to study the mass spectrometry behaviour of nine sterols/steroids and related molecules (cholesterol precursors: squalene, lanosterol; sterol metabolites; 7 Dehydrocholesterol, 24, 25 and 27 Hydroxycholesterol; and steroids: progesterone, testosterone, and corticosterone) during ageing in the African turquoise killifish, a new model for studying vertebrate longevity. We find that levels of all tested steroids change significantly with age in multiple tissues, suggesting that specific steroids could be used as biomarkers of ageing. These findings pave the way for use of Nothobranchius furzeri as a novel model organism to unravel the role of sterols/steroids in ageing and age-related diseases. Graphical abstract.

Recent studies on the distribution of microplastics in the Charleston Harbor, SC, USA revealed that a large part of the microplastic particles that are found in the intertidal sediments are tire wear particles. These particles originate from the wear of tire treads on roadways, and wash into the estuary during rain events. The abundance of these particles has raised questions about potential toxicity to aquatic organisms that ingest these particles. The synthetic rubber in car tires consists of a large variety of chemicals, which can vary between manufacturers, but usually contains styrene-butadiene rubber, carbon black and zinc. To investigate the potential toxicity of tire wear particles, both mummichogs (Fundulus heteroclitus) and fathead minnow (Pimephales promelas) were exposed to different concentrations of crumb rubber particles (38-355 µm) in a 7-day static renewal exposure. Dissection of the fish revealed that crumb rubber was ingested and accumulated in the intestinal tract. At the highest concentration tested (6 g/L) partial mortality was observed in the fathead minnow, which came close to the assumed LC 50 . To investigate if polynuclear aromatic hydrocarbons (PAHs) were leaching from the particles, bile fluorescence was measured, together with potential induction of cytochrome P450-1A through the ethoxyresorufin-O-deethylase (EROD) assay. Elevated levels of 2-, 4-, and 5-, ring structures representative of PAHs were detected in the bile of exposed animals. Bile fluorescence indicated that 4-ring PAH compounds were the most bioavailable from the crumb rubber particles. Induction of EROD activity was observed in exposed animals at environmentally relevant concentrations of the crumb rubber particles (<1-2 g/L), and this elevated EROD activity indicated that PAH compounds from the crumb rubber particles were being metabolized in both mummichogs and fathead minnow.

Dietary alteration is one of the most universally effective aging interventions, making its standardization a fundamental need for model organisms in aging. In this dietetic study we address the current lack of standardized formulated diet for turquoise killifish Nothobranchius furzeri - a promising model organism. We first demonstrated that N. furzeri can be fully weaned at the onset of puberty onto a commercially available pelleted diet as the sole nutrition when kept in social tanks. We then compared nine somatic and six reproductive parameters between fish fed a typical laboratory diet - frozen chironomid larvae (bloodworms) and fish weaned from bloodworms to BioMar pellets. Both dietary groups had comparable somatic and reproductive performance. There was no difference between diet groups in adult body size, specific growth rate, condition or extent of hepatocellular vacuolation. Fish fed a pelleted diet had higher juvenile body mass and more visceral fat. Pellet-fed males had lower liver mass and possessed a lipid type of hepatocellular vacuolation instead of the prevailing glycogen-like vacuolation in the bloodworm-fed group. No considerable effect was found on reproductive parameters. The negligible differences between dietary groups and good acceptance of pellets indicate their suitability as a useful starting point for the development of standardized diet for Nothobranchius furzeri.

Personality and physiological traits often have close relationships with dominance status, but the significance and/or direction of the relationships vary between studies. This study examines whether two personality traits (aggressiveness and boldness) and three physiological traits (testosterone and cortisol levels and oxygen consumption rates) are associated with contest decisions/performance using a mangrove killifish Kryptolebias marmoratus. The results show that individuals that attacked their own mirror images (an aggressiveness index) at higher rates or had higher levels of testosterone were more likely to attack their opponent and win non-escalated contests, while individuals that had higher levels of cortisol were more likely to lose. After the contests, (1) individuals that had attacked their opponents or won had higher post-contest oxygen consumption rates, and (2) individuals that had attacked their opponents also had higher post-contest levels of cortisol. Although no significant correlations were detected among pre-contest physiological traits, post-contest levels of cortisol were positively correlated with oxygen consumption rates. Overall, personality and physiological traits provide useful predictors for the fish's contest decisions/performance. Contest interactions subsequently modified post-contest physiological traits and potentially also promoted associations between them. Nevertheless, the fish's physiological traits remained rather consistent over the entire study period., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

By investigating evolutionary adaptations that change physiological functions, we can enhance our understanding of how organisms work, the importance of physiological traits, and the genes that influence these traits. This approach of investigating the evolution of physiological adaptation has been used with the teleost fish Fundulus heteroclitus and has produced insights into (i) how protein polymorphisms enhance swimming and development; (ii) the role of equilibrium enzymes in modulating metabolic flux; (iii) how variation in DNA sequences and mRNA expression patterns mitigate changes in temperature, pollution, and salinity; and (iv) the importance of nuclear-mitochondrial genome interactions for energy metabolism. Fundulus heteroclitus provides so many examples of adaptive evolution because their local population sizes are large, they have significant standing genetic variation, and they experience large ranges of environmental conditions that enhance the likelihood that adaptive evolution will occur. Thus, F. heteroclitus research takes advantage of evolutionary changes associated with exposure to diverse environments, both across the North American Atlantic coast and within local habitats, to contrast neutral versus adaptive divergence. Based on evolutionary analyses contrasting neutral and adaptive evolution in F. heteroclitus populations, we conclude that adaptive evolution can occur readily and rapidly, at least in part because it depends on large amounts of standing genetic variation among many genes that can alter physiological traits. These observations of polygenic adaptation enhance our understanding of how evolution and physiological adaptation progresses, thus informing both biological and medical scientists about genotype-phenotype relationships. © 2020 American Physiological Society. Compr Physiol 10:637-671, 2020., (Copyright © 2020 American Physiological Society. All rights reserved.)

Fast-start predator-escape performance of mummichogs Fundulus heteroclitus was tested across field-informed variation in temperature (24, 30 and 36°C) and salinity (2, 12 and 32 ppt). Performance was similar across temperatures and salinities when fish were allowed to acclimate to these conditions. However, when mummichogs experienced acute temperature changes, performance exhibited thermal dependence in two contrasting ways. Fast-start turning rates and linear speeds varied directly with the temperature at which the manoeuvre was executed, but these aspects of performance varied inversely with acclimation temperature, with cool-acclimated fish exhibiting faster starts across test temperatures. Temperature effects were consistent across salinities. These results suggest that while mummichogs increase performance with acute temperature increases, long-term rises in sea temperature may cause these fish to become more susceptible to predation during abrupt cooling events, such as when storm events flood shallow water estuaries with cool rainwater., (© 2020 The Fisheries Society of the British Isles.)

Cystic Fibrosis Transmembrane conductance Regulator (CFTR) anion channels are the regulated exit pathway in Cl - secretion by teleost salt secreting ionocytes of the gill and opercular epithelia of euryhaline teleosts. By confocal light immunocytochemistry using regular and phospho-antibodies directed against conserved sites, we found that killifish CFTR (kfCFTR) and the tyrosine kinase Focal Adhesion Kinase (FAK) phosphorylated at Y407 (FAKpY407) and FAKpY397 are colocalized at the apical membrane and in subjacent membrane vesicles of ionocytes. Hypotonic shock and the α-2 adrenergic agonist clonidine rapidly and reversibly inhibit Cl - secretion by isolated opercular epithelia, simultaneous with dephosphorylation of FAKpY407 and increased FAKpY397, located in the apical membrane of ionocytes in the opercular epithelium. FAKpY407 is re-phosphorylated at the apical membrane of ionocytes and Cl - secretion rapidly restored by hypertonic shock, detectable at 2 min., maximum at 5 min and still elevated at 30 min. In isolated opercular epithelia, the FAK phosphorylation inhibitor Y15 and p38MAP kinase inhibitor SB203580 significantly blunted the recovery of short-circuit current (I sc , equal to Cl - secretion rate) after hypertonic shock. The cSRC inhibitor saracatinib dephosphorylated FAKpY861 seen near tight junctions of pavement cells, and reduced the increase in epithelial resistance normally seen with clonidine inhibition of ion transport, while FAKpY397 was unaffected. The results show rapid osmosensitive responses in teleost fish ionocytes involve phosphorylation of CFTR by FAKpY407, an opposing role for FAKpY397 and a possible role for FAKpY861 in tight junction dynamics., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Hypoxia is a pervasive stressor in aquatic environments, and both phenotypic plasticity and evolutionary adaptation could shape the ability to cope with hypoxia. We investigated evolved variation in hypoxia tolerance and the hypoxia acclimation response across fundulid killifishes that naturally experience different patterns of hypoxia exposure. We compared resting O 2 consumption rate ( Ṁ O 2 ), and various indices of hypoxia tolerance [critical O 2 tension ( P crit ), regulation index (RI), O 2 tension ( P O 2 ) at loss of equilibrium ( P LOE ) and time to LOE ( t LOE ) at 0.6 kPa O 2 ] in Fundulus confluentus , Fundulus diaphanus , Fundulus heteroclitus , Fundulus rathbuni , Lucania goodei and Lucania parva We examined the effects of chronic (28 days) exposure to constant hypoxia (2 kPa) or nocturnal intermittent hypoxia (12 h normoxia:12 h hypoxia) in a subset of species. Some species exhibited a two-breakpoint model in Ṁ O 2  caused by early, modest declines in Ṁ O 2  in moderate hypoxia. We found that hypoxia tolerance varied appreciably across species: F. confluentus was the most tolerant (lowest P LOE and P crit , longest t LOE ), whereas F. rathbuni and F. diaphanus were the least tolerant. However, there was not a consistent pattern of interspecific variation for different indices of hypoxia tolerance, with or without taking phylogenetic relatedness into account, probably because these different indices are underlain by partially distinct mechanisms. Hypoxia acclimation generally improved hypoxia tolerance, but the magnitude of plasticity and responsiveness to different hypoxia patterns varied interspecifically. Our results therefore suggest that hypoxia tolerance is a complex trait that is best appreciated by considering multiple indices of tolerance., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Production of industrial effluents have led to increased copper (Cu) pollution of aquatic ecosystems, impacting the physiology of aquatic vertebrates. Past work has shown that Cu exerts its toxicity by disruption ion regulation and/ or increasing oxidative stress. However, it remains unclear how Cu may influence aerobic metabolism and hypoxia tolerance, two possible targets of its toxicity. To address this issue, we exposed freshwater acclimated killifish (F. heteroclitus) to a 96 h Cu exposure at a target concentration of 100 μg L -1 . We determined resting oxygen consumption (ṀO 2 ), ṀO 2max after exhaustive exercise, and followed ṀO 2 for 3 h in post-exercise recovery in water with either no Cu or 100 μg L -1 Cu. We assessed hypoxia tolerance by determining the critical oxygen tension (P crit ). It was found that killifish exposed to combined 96 h Cu exposure and Cu present during metabolic measurements, showed a significant decrease in ṀO 2max and in aerobic scope (ṀO 2max - ṀO 2rest ), compared to control fish. However, changes in blood and muscle lactate and muscle glycogen were not consistent with an upregulation of anaerobic metabolism as compensation for reduced aerobic performance in Cu exposed fish. Hypoxia tolerance was not influenced by the 96 h Cu exposure or by presence or absence of Cu during the P crit test. This study suggests that Cu differentially influences responses to changes in oxygen demand and oxygen availability., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

The external environment influences brain cell proliferation, and this might contribute to brain plasticity underlying adaptive behavioural changes. Additionally, internal genetic factors influence the brain cell proliferation rate. However, to date, researchers have not examined the importance of environmental versus genetic factors in causing natural variation in brain cell proliferation. Here, we examine brain cell proliferation and brain growth trajectories in free-living populations of Trinidadian killifish, Rivulus hartii , exposed to contrasting predation environments. Compared to populations without predators, populations in high predation (HP) environments exhibited higher rates of brain cell proliferation and a steeper brain growth trajectory (relative to body size). To test whether these differences in the wild persist in a common garden environment, we reared first-generation fish originating from both predation environments in uniform laboratory conditions. Just as in the wild, brain cell proliferation and brain growth in the common garden were greater in HP populations than in no predation populations. The differences in cell proliferation observed across the brain in both the field and common garden studies indicate that the differences are probably genetically based and are mediated by evolutionary shifts in overall brain growth and life-history traits.

Numerous anthropogenic sources, such as pulp mill and sewage treatment effluents, contain androgenic endocrine disrupting compounds that alter the reproductive status of aquatic organisms. The current study injected adult male mummichog (Fundulus heteroclitus) with 0 (control), 1 pg/g, 1 ng/g or 1 μg/g body weight of the model androgen 5α-dihydrotestosterone (DHT) with the intent to induce a period of plasma sex hormone depression, a previously-observed effect of DHT in fish. A suite of gonadal steroidogenic genes were assessed during sex hormone depression and recovery. Fish were sampled 6, 12, 16, 18, 24, 30 and 36 h post-injection, and sections of testis tissue were either snap frozen immediately or incubated for 24 h at 18 °C to determine in vitro gonadal hormone production and then frozen. Plasma testosterone (T) and 11-ketotestosterone (11KT) were depressed beginning 24 h post-injection. At 36 h post-injection plasma T remained depressed while plasma 11KT had recovered. In snap frozen tissue there was a correlation between plasma sex hormone depression and downregulation of key steroidogenic genes including steroidogenic acute regulatory protein (star), cytochrome P450 17a1 (cyp17a1), 3β-hydroxysteroid dehydrogenase (3βhsd), 11β-hydroxysteroid dehydrogenase (11βhsd) and 17β-hydroxysteroid dehydrogenase (17βhsd). Similar to previous studies, 3βhsd was the first and most responsive gene during DHT exposure. Gene responses from in vitro tissue were more variable and included the upregulation of 3βhsd, 11βhsd and star during the period of hormone depression. The differential expression of steroidogenic genes from the in vitro testes compared to the snap frozen tissues may be due to the lack of regulators from the hypothalamo-pituitary-gonadal axis present in whole-animal systems. Due to these findings it is recommended to use snap frozen tissue, not post-incubation tissue from in vitro analysis, for gonadal steroidogenic gene expression to more accurately reflect in vivo responses., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Common killifish Fundulus heteroclitus were acclimated to ecologically relevant temperatures (5, 15 and 33°C) and their maximum heart rate (f Hmax ) was measured at each acclimation temperature during an acute warming protocol. Acclimation to 33°C increased peak f Hmax by up to 32% and allowed the heart to beat rhythmically at a temperature 10°C higher when compared with acclimation to 5°C. Independent of acclimation temperature, peak f Hmax occurred about 3°C cooler than the temperature that first produced cardiac arrhythmias. Thus, when compared with previously published values for the critical thermal maximum of F. heteroclitus, the temperature for peak f Hmax was cooler and the temperature that first produced cardiac arrhythmias was similar to these critical thermal maxima. The considerable thermal plasticity of f Hmax demonstrated in the present study is entirely consistent with eurythermal ecology of killifish, as shown previously for another eurythermal fish Gillichthys mirabilis., (© 2019 The Fisheries Society of the British Isles.)

Species with fast life-histories typically prioritize current over future reproductive events, compared to species with slow life-histories. These species therefore require greater energetic input into reproduction, and also likely have less time to realize their reproductive potential. Hence, behaviors that increase access to both resources and mating opportunities, at a cost of increased mortality risk, could coevolve with the pace of life-history. However, whether this prediction holds across species, remains untested under standardized conditions. Here, we test how risky behaviors, which facilitate access to resources and mating opportunities (i.e., activity, boldness, and aggression), along with metabolic rate, coevolve with the pace of life-history across 20 species of killifish that present remarkable divergences in the pace of life-history. We found a positive association between the pace of life-history and aggression, but interestingly not with other behavioral traits or metabolic rate. Aggression is linked to interference competition, and in killifishes is often employed to secure mates, while activity and boldness are more relevant for exploiting energetic resources. Our results suggest that the trade-off between current and future reproduction plays a more prominent role in shaping mating behavior, while behaviors related to energy acquisition may be influenced by ecological factors., (© 2019 The Author(s). Evolution © 2019 The Society for the Study of Evolution.)

Sites along the Elizabeth River are contaminated with polycyclic aromatic hydrocarbons (PAHs) from historical creosote production and other industrial processes. Previous studies have demonstrated that Atlantic killifish collected from sites throughout the Elizabeth River display resistance to the teratogenic effects of PAH-exposure in a manner commensurate with sediment PAH concentrations. The current study characterized various chemical pollutants in sediment and investigated the effects of aqueous sediment extracts from sites along the Elizabeth River to the cardiac development of Atlantic killifish embryos from fish collected from an uncontaminated reference site. Embryonic cardiac deformities were more prevalent after exposure to extracts from sites with high PAH loads. However, activation of cytochrome P4501A, a gene up-regulated by PAH-induction of the aryl hydrocarbon receptor and measured using an in ovo EROD assay, did not consistently increase with PAH concentrations. This work further characterizes sediments in the Elizabeth River, as well as provides insight into the evolutionary pressures at each ER site.

The characteristics of the fish gill that maximize gas exchange are the same that promote diffusion of ions and water to and from the environment; therefore, physiological trade-offs are likely to occur. Here, we investigated how salinity acclimation affects whole-animal respiratory gas exchange during hypoxia using Fundulus heteroclitus , a fish that inhabits salt marshes where salinity and oxygen levels vary greatly. Salinity had marked effects on hypoxia tolerance, with fish acclimated to 11 and 35 ppt showing much longer time to loss of equilibrium (LOE) in hypoxia than 0 ppt-acclimated fish. Fish acclimated to 11 ppt (isosmotic salinity) exhibited the greatest capacity to regulate oxygen consumption rate ( Ṁ O 2 ) under hypoxia, as measured through the regulation index (RI) and P crit At 35 ppt, fish had a higher routine metabolic rate (RMR) but a lower RI than fish at 11 ppt, but there were no differences in gill morphology, ventilation or blood O 2 transport properties between these groups. In contrast, 0 ppt-acclimated fish had the highest ventilation and lowest O 2 extraction efficiency in normoxia and hypoxia, indicating a higher ventilatory workload in order to maintain similar levels of Ṁ O 2 These differences were related to alterations in gill morphology, where 0 ppt-acclimated fish had the smallest lamellar surface area with the greatest epithelial cell coverage (i.e. thicker lamellae, longer diffusion distance) and a larger interlamellar cell mass, contrasting with 11 ppt-acclimated fish, which had overall the highest respiratory surface area. The alteration of an array of physiological parameters provides evidence for a compromise between salinity and hypoxia tolerance in killifish acclimated to freshwater., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The effects of an ecologically relevant range of salinities (2, 12, 22, 32) on thermal preferences and growth of adult mummichogs Fundulus heteroclitus were determined for fish from a southern Chesapeake Bay population. Salinity did not affect the mean temperature selected by F. heteroclitus in a thermal gradient, which was identified as 26.6°C based on observations of 240 individuals. Salinity and temperature had significant and interacting effects on growth rates of F. heteroclitus measured over 12 weeks. Growth rates were highest overall and remained high over a broader range of temperatures at moderate salinities (12 and 22), while high growth rates were shifted toward lower temperatures for fish grown at a salinity of 2 and higher temperatures at a salinity of 32. Significant reductions in growth relative to the optimal conditions (28.6°C, salinity of 22) were observed at the coolest (19.6°C) and warmest (33.6°C) temperature tested at all salinities, as well as temperatures ≥ 26.6°C at a salinity of 2, ≥ 28.6°C at a salinity of 12 and ≤ 26.6°C at a salinity of 32. Growth rates provide a long-term, organismal measure of performance and results of this study indicate that performance may be reduced under conditions that the highly euryhaline F. heteroclitus can otherwise easily tolerate. The combination of reduced salinity and increased temperature that is predicted for temperate estuaries as a result of climate change may have negative effects on growth of this ecologically important species., (© 2019 The Fisheries Society of the British Isles.)

The mitonuclear species concept hypothesizes that incompatibilities between interacting gene products of the nuclear and mitochondrial genomes are a major factor establishing and maintaining species boundaries. However, most of the data available to test this concept come from studies of genetic variation in mitochondrial DNA, and clines in the mitochondrial genome across contact zones can be produced by a variety of forces. Here, we show that using a combination of population genomic analyses of the nuclear and mitochondrial genomes and studies of mitochondrial function can provide insight into the relative roles of neutral processes, adaptive evolution, and mitonuclear incompatibility in establishing and maintaining mitochondrial clines, using Atlantic killifish (Fundulus heteroclitus) as a case study. There is strong evidence for a role of secondary contact following the last glaciation in shaping a steep mitochondrial cline across a contact zone between northern and southern subspecies of killifish, but there is also evidence for a role of adaptive evolution in driving differentiation between the subspecies in a variety of traits from the level of the whole organism to the level of mitochondrial function. In addition, studies are beginning to address the potential for mitonuclear incompatibilities in admixed populations. However, population genomic studies have failed to detect evidence for a strong and pervasive influence of mitonuclear incompatibilities, and we suggest that polygenic selection may be responsible for the complex patterns observed. This case study demonstrates that multiple forces can act together in shaping mitochondrial clines, and illustrates the challenge of disentangling their relative roles., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

The critical oxygen tension ( P crit ) for fishes is the oxygen level below which the rate of oxygen consumption ( Ṁ O 2 ) becomes dependent upon ambient oxygen partial pressure ( P O 2 ). We compare multiple curve-fitting approaches to estimate P crit of the Gulf killifish, Fundulus grandis , during closed and intermittent-flow respirometry. Fitting two line segments of Ṁ O 2  versus P O 2  produced high and variable estimates of P crit , as did nonlinear regression using a hyperbolic (Michaelis-Menten) function. Using nonlinear regression fit to an exponential (modified Weibull) function, or linear regression of Ṁ O 2 versus P O 2  at low P O 2 , and determining P crit as the P O 2  when Ṁ O 2 equals standard metabolic rate (SMR) yielded values that were consistent across fish and among experimental trials. The magnitude of the difference in P crit determined by alternative calculation methods exceeded the differences determined in closed and intermittent-flow respirometry, highlighting the need to standardize analytical as well as experimental approaches in determining P crit ., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Freshwater- and seawater-acclimated Fundulus heteroclitus were exposed to acute hypoxia (10% air saturation, 3 h), followed by normoxic recovery (3 h). In both salinities, ventilation increased and heart rate fell in the classic manner, while Ṁ O 2  initially declined by ∼50%, with partial restoration by 3 h of hypoxia, and no O 2 debt repayment during recovery. Gill paracellular permeability (measured with [ 14 C] PEG-4000) was 1.4-fold higher in seawater, and declined by 50% during hypoxia with post-exposure overshoot to 188%. A similar pattern with smaller changes occurred in freshwater. Drinking rate (also measured with [ 14 C] PEG-4000) was 8-fold higher in seawater fish, but declined by ∼90% during hypoxia in both groups, with post-exposure overshoots to ∼270%. Gill diffusive water flux (measured with 3 H 2 O) was 1.9-fold higher in freshwater fish, and exhibited a ∼35% decrease during hypoxia, which persisted throughout recovery, but was unchanged during hypoxia in seawater fish. Nevertheless, freshwater killifish gained mass while seawater fish lost mass during hypoxia, and these changes were not corrected during normoxic recovery. We conclude that this hypoxia-tolerant teleost beneficially reduces gill water permeability in a salinity-dependent fashion during hypoxia, despite attempting to simultaneously improve Ṁ O 2 , but nevertheless incurs a net water balance penalty in both freshwater and seawater., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Identifying the cues followed by cells is key to understand processes as embryonic development, tissue homeostasis, or several pathological conditions. Based on a durotaxis model, it is shown that cells moving on predeformed thin elastic membrane follow the direction of increasing strain of the substrate. This mechanism, straintaxis, does not distinguish the origin of the strain, but the active stresses produce large strains on cells or tissues being used as substrates. Hence, straintaxis is the natural realization of duratoaxis in vivo. Considering a circular geometry for the substrate cells, it is shown that if the annular component of the active stress component increases with the radial distance, cells migrate toward the substrate cell borders. With appropriate estimation for the different parameters, the migration speeds are similar to those obtained in recent experiments (Reig et al 2017 Nat. Commun. 8 15431). In these, during the annual killifish epiboly, deep cells that move in contact with the epithelial enveloping cell layer (EVL), migrate toward the EVL cell borders with speeds of microns per minute.