Chronic handling stress in zebrafish Danio rerio husbandry.

The zebrafish (Danio rerio, Hamilton, 1822) is one of the most important fish model species in scientific research, with millions of fish housed in laboratory animal facilities around the world. During husbandry, it is necessary to regularly handle the fish, which could cause short- and long-term stress, possibly affecting both fish welfare and experimental outcomes. In two experiments, the authors studied effects of transferring adult zebrafish, by chasing them with a net and/or exposing them to air (netting) on different endpoints: cortisol levels, reproduction parameters and behavioural parameters. They used realistic chase and air-exposure times to mimic normal zebrafish husbandry and investigated the potential to habituate to handling stressors. Finally, the potential welfare improvements of a nutritional reward after handling were studied. All types of handling induced a stress response, but the authors did not find a correlation with the intensity of the stressor. Realistic (short) handling routines also caused stress, both after the first time and after regular handling over a long period of time. Cortisol levels peaked after 15 min, were still elevated after 30 min and dropped to resting level after 60 min. This should be taken into account by researchers when carrying out measurements or behavioural trials within an hour after handling. There is a minor potential benefit of nutritional rewards that may contribute to a faster recovery of normal behaviour. They did not find evidence of habituation to chasing and netting stress. Taking the stress response after handling into consideration will improve fish welfare and health and minimise husbandry-associated sources of variation.

Environmental risks of a commonly used pyrethroid: Insights from temporary pond species of the Lake Manyara Basin, Tanzania.

Environmental risks posed by widespread pesticide application have attracted global attention. Currently, chemical risk assessments in aquatic environments rely on extrapolation of toxicity data from classic model species. However, similar assessments based on local species could be complementary, particularly for unusual living environments such as temporary ponds. Here, we carried out an environmental risk assessment (ERA) of a pyrethroid model compound, cypermethrin, based on local temporary pond species. First, we measured cypermethrin residue concentrations in rivers, irrigation canals and temporary ponds in the Lake Manyara Basin (LMB). Then, we estimated the environmental risks of cypermethrin by combining these data with acute toxicity data of three resident species across three trophic levels: primary producers (Arthrospira platensis), invertebrate grazers (Streptocephalus lamellifer) and fish (Nothobranchius neumanni). Furthermore, we compared the derived ERA to that obtained using toxicity data from literature of classic model species. Cypermethrin residue concentrations in contaminated systems of the LMB ranged from 0.01 to 57.9 ng/L. For temporary pond species, S. lamellifer was the most sensitive one with a 96 h-LC50 of 0.14 ng/L. Regardless of the assumed exposure concentration (0.01 and 57.9 ng/L), the estimated risks were low for primary producers and high for invertebrate grazers, both for local species as well as for classic model species. The highest detected cypermethrin concentration resulted in a moderate risk estimation for local fish species, while the estimated risk was high when considering classic fish models. Our results confirm that, at least for pyrethroids, ERAs with classic model species are useful to estimate chemical risks in temporary pond ecosystems, and suggest that complementary ERAs based on local species could help to fine-tune environmental regulations to specific local conditions and conservation targets.

Combined effects of global warming and chlorpyrifos exposure on the annual fish Nothobranchius furzeri.

Global warming and environmental pollution threaten aquatic ecosystems. While interactive effects between both stressors can have more than additive consequences, these remain poorly studied for most taxa. Especially chronic exposure trials with vertebrates are scarce due to the high time- and monetary costs of such studies. We use the recently-established fish model Nothobranchius furzeri to assess the separate and combined effects of exposure to the pesticide chlorpyrifos (at 2 µg/L and 4 µg/L) and a 2 °C temperature increase. We performed a full life-cycle assessment to evaluate fitness-related endpoints including survival, total body length, maturation time, fecundity, critical thermal maximum (CTmax) and locomotor activity. Exposure to 4 µg/L chlorpyrifos slowed down male maturation, reduced fecundity and impaired growth of the fish. While the temperature increase did not affect any of the measured endpoints on its own, the combination of exposure to 2 µg/L CPF with an increase of 2 °C reduced growth and severely reduced fecundity, with almost no offspring production. Together, these findings suggest that climate change may exacerbate the impact of environmental pollution, and that interactive effects of chronic exposure to multiple stressors should be considered to predict how populations will be affected by ongoing global change.

Pesticide sensitivity of Nothobranchius neumanni, a temporary pond predator with a non-generic life-history.

Pesticides are crucial to improve agricultural productivity, but often adversely affect surrounding aquatic systems and their fauna. To determine the environmental risk of pesticides, routine ecotoxicological tests are performed on several organisms, including standard fish models. However, these typically do not include fish species from variable habitats and with non-generic life-histories. In particular, inhabitants from temporary ponds such as annual killifish are conventionally understood to be resilient to natural stressors which could translate to higher pesticide resistance or, alternatively, trade-off with their resistance to pesticides and render them more sensitive than classic fish models. Using standard exposure tests, we assessed short-term toxicity effects of two commonly used pesticides, Roundup and cypermethrin, on the annual killifish Nothobranchius neumanni, and compared its sensitivity with that of classic fish models. For Roundup, we found a 72 h-LC50 of 1.79 ± 0.11 mg/L, which is lower than the values reported for zebrafish, medaka, fathead minnow and rainbow trout, suggesting that N. neumanni is more sensitive to the compound. The opposite was true for cypermethrin, with a 72 h-LC50 of 0.27 ± 0.03 mg/L. However, these LC50-values do not deviate strongly from those reported for other fish species, supporting earlier findings in the congeneric N. furzeri that the sensitivity of annual killifish to pollutants is similar to that of classic fish models despite their assumed robustness to environmental stress.

Generation-specific and interactive effects of pesticide and antidepressant exposure in a fish model call for multi-stressor and multigenerational testing.

Ecological risks of a pollutant are typically assessed via short-term exposure of model organisms to that single compound. Such tests are informative, but cannot ascertain effects of long-term and multigenerational mixed-stressor exposure with which organisms are often confronted in their natural environment. Therefore, full life-cycle and multigenerational tests are needed. Yet, these are hampered due to long lifespans and generation times of many standard laboratory species, in particular for vertebrates such as fish. With a typical lifespan of 6 months and a generation time of about 3 months, the turquoise killifish (Nothobranchius furzeri) may be an ideal model for multigenerational testing. In this study, we assessed the impact of full life-cycle exposure to the emerging pollutant fluoxetine (0, 0.5 µg/L) in combination with chronic exposure during adulthood to the pesticide 3,4-dichloroaniline (0, 50, 100 µg/L) over two successive generations of N. furzeri. Overall, both life-history and behaviour were affected by exposure to fluoxetine and 3,4-DCA. Inhibitory effects of single chemical exposure on growth and fecundity were generation-dependent, while enhanced swimming acceleration and feeding in response to fluoxetine were dependent on the presence of 3,4-DCA. Together, these findings show the relevance of a multi-stressor approach across successive generations. Although full life-cycle and multigenerational tests are typically assumed to be impractical and costly for fish, we deliver an effective demonstration that such studies are possible within a timespan of less than 6 months with the killifish N. furzeri as a model organism.

Towards improved fish tests in ecotoxicology - Efficient chronic and multi-generational testing with the killifish Nothobranchius furzeri.

As many freshwaters are chemically polluted, one of the challenges for policy makers is to determine the potential impact of these pollutants on ecosystems and to define safe concentrations. Common practice is the use of ecotoxicological assays to assess the response of model organisms from different trophic levels such as algae, invertebrates and fish during exposure to dilutions of a specific compound. Ideally, ecotoxicological assessments of (pseudo-)persistent chemicals should be performed across the life-cycle or even multiple generations for an accurate risk assessment. Multigenerational tests with fish are, however, impractical and costly given the long lifespan and generation time of classic model species. Here, we suggest a framework for more relevant, time- and cost-efficient fish-based testing in ecotoxicology and align it with accredited test guidelines. Next, we introduce an upcoming fish model, the turquoise killifish Nothobranchius furzeri, and show how it facilitates such research agendas due to a short lifespan and generation time. Through a review of fish-based exposure studies with a set of reference toxicants, we position N. furzeri as a sensitive species, suitable for screening effects of different pollutant types. Ultimately, we perform a cost-benefit analysis and propose a plan of action for the introduction of N. furzeri into accredited test guidelines.

Antidepressant exposure reduces body size, increases fecundity and alters social behavior in the short-lived killifish Nothobranchius furzeri.

Social and mating behavior are fundamental fitness determinants in fish. Although fish are increasingly exposed to pharmaceutical compounds that may alter expression of such behavior, potential effects are understudied. Here, we examine the impact of lifelong exposure to two concentrations (0.7 and 5.3 µg/L) of the antidepressant fluoxetine on fecundity and social behavior (i.e. sociability and male-male aggression) in the turquoise killifish, Nothobranchius furzeri. When exposed to the highest concentration of fluoxetine (5.3 µg/L), fish were smaller at maturation but they more frequently engaged in mating. In addition, in both fluoxetine treatments females roughly doubled their overall fecundity while egg fertilization rates were the same for exposed and unexposed fish. Although aggression of male fish was not impacted by fluoxetine exposure, exposed male fish (5.3 µg/L) spent more time in the proximity of a group of conspecifics, which implies an increased sociability in these individuals. Overall, the results of this study indicate that exposure to fluoxetine may result in disrupted male sociability, increased mating frequency and an increased reproductive output in fish populations.

Population-, sex- and individual level divergence in life-history and activity patterns in an annual killifish.

Variation in life-history strategies along a slow-fast continuum is largely governed by life-history trade-offs. The pace-of-life syndrome hypothesis (POLS) expands on this idea and suggests coevolution of these traits with personality and physiology at different levels of biological organization. However, it remains unclear to what extent covariation at different levels aligns and if also behavioral patterns such as diurnal activity changes should be incorporated. Here, we investigate variation in life-history traits as well as behavioral variation at the individual, sex and population level in the Turquoise killifish Nothobranchius furzeri. We performed a common garden laboratory experiment with four populations that differ in pond permanence and scored life-history and behavioral (co-) variation at the individual and population level for both males and females. In addition, we focused on diurnal activity change as a behavioral trait that remains understudied in ecology. Our results demonstrate sex-specific variation in adult body size and diurnal activity change among populations that originate from ponds with differences in permanence. However, there was no pond permanence-dependent divergence in maturation time, juvenile growth rate, fecundity and average activity level. With regard to behavior, individuals differed consistently in locomotor activity and diurnal activity change while, in contrast with POLS predictions, we found no indications for life-history and behavioral covariation at any level. Overall, this study illustrates that diurnal activity change differs consistently between individuals, sexes and populations although this variation does not appear to match POLS predictions.

Interactive effects of 3,4-DCA and temperature on the annual killifish Nothobranchius furzeri.

Although aquatic organisms are increasingly exposed to pollutants and abnormally high temperatures as a consequence of climate change, interactive effects between those stressors remain poorly assessed. Especially in ectotherms, such as fish, increases in ambient temperature are expected to affect fitness-related traits and physiology. We used the turquoise killifish Nothobranchius furzeri to study the effects of a range of 3,4-dichloroaniline concentrations (0, 50, 100 µg/L) in combination with two temperature conditions (control and control +4 °C) during four months of exposure. As part of an integrated multi-level approach, we quantified effects on classic life history traits (size, maturation time, body mass, fecundity), critical thermal maximum and physiology (energy reserves and stress-associated enzymatic activity). While no interactive effects of 3,4-DCA exposure and increased temperature emerged, our results do show a negative effect of 3,4-DCA on thermal tolerance. This finding is of particular relevance in light of increasing temperatures under climate change. Due to increases in pest species and faster degradation of 3,4-DCA under higher temperatures, increased use of the pesticide is expected under climate change which, in turn, could result in a decreased tolerance of aquatic organisms to high temperatures.

Improving the reliability and ecological validity of pharmaceutical risk assessment: Turquoise killifish (Nothobranchius furzeri) as a model in behavioral ecotoxicology.

Pharmaceuticals are essential for human well-being, but their increasing and continuous use pollutes the environment. Although behavioral ecotoxicology is increasingly advocated to assess the effects of pharmaceutical pollution on wildlife and ecosystems, a consensus on the actual environmental risks is lacking for most compounds. The main limitation is the lack of standardized reproducible tests that are based on sensitive behavioral endpoints and that accommodate a high ecological relevance. In the present study, we assessed the impact of a 3-wk exposure to the antidepressant fluoxetine on multiple behavioral traits in the promising new model organism Nothobranchius furzeri (turquoise killifish). Overall, our study shows that fluoxetine can impact feeding behavior, habitat choice in a novel environment, and antipredator response of N. furzeri individuals; effects on spontaneous activity and exploration tendency were less pronounced. However, effects became only apparent when individuals were exposed to fluoxetine concentrations that were 10 times higher than typical concentrations in natural aquatic environments. Ecotoxicologists are challenged to maximize both the reliability and ecological validity of risk assessments of pollutants. Our study contributes to the development of a time- and cost-efficient, standardized ecotoxicological test based on sensitive, ecologically relevant behavioral endpoints in N. furzeri. Environ Toxicol Chem 2019;38:262-270. © 2018 SETAC.

Individual behavioral variation reflects personality divergence in the upcoming model organism Nothobranchius furzeri.

In the animal kingdom, behavioral variation among individuals has often been reported. However, stable among-individual differences along a behavioral continuum-reflective of personality variation-have only recently become a key target of research. While a vast body of descriptive literature exists on animal personality, hypothesis-driven quantitative studies are largely deficient. One of the main constraints to advance the field is the lack of suitable model organisms. Here, we explore whether N. furzeri could be a valuable model to bridge descriptive and hypothesis-driven research to further unravel the causes, function and evolution of animal personality. As a first step toward this end, we perform a common garden laboratory experiment to examine if behavioral variation in the turquoise killifish Nothobranchius furzeri reflects personality divergence. Furthermore, we explore if multiple behavioral traits are correlated. We deliver "proof of principle" of personality variation among N. furzeri individuals in multiple behavioral traits. Because of the vast body of available genomic and physiological information, the well-characterized ecological background and an exceptionally short life cycle, N. furzeri is an excellent model organism to further elucidate the causes and implications of behavioral variation in an eco-evolutionary context.

Squeezing out the last egg-annual fish increase reproductive efforts in response to a predation threat.

Both constitutive and inducible antipredator strategies are ubiquitous in nature and serve to maximize fitness under a predation threat. Inducible strategies may be favored over constitutive defenses depending on their relative cost and benefit and temporal variability in predator presence. In African temporary ponds, annual killifish of the genus Nothobranchius are variably exposed to predators, depending on whether larger fish invade their habitat from nearby rivers during floods. Nonetheless, potential plastic responses to predation risk are poorly known. Here, we studied whether Nothobranchius furzeri individuals adjust their life history in response to a predation threat. For this, we monitored key life history traits in response to cues that signal the presence of predatory pumpkinseed sunfish (Lepomis gibbosus). While growth rate, adult body size, age at maturation, and initial fecundity were not affected, peak and total fecundity were higher in the predation risk treatment. This contrasts with known life history strategies of killifish from permanent waters, which tend to reduce reproduction in the presence of predators. Although our results show that N. furzeri individuals are able to detect predators and respond to their presence by modulating their reproductive output, these responses only become evident after a few clutches have been deposited. Overall our findings suggest that, in the presence of a predation risk, it can be beneficial to increase the production of life stages that can persist until the predation risk has faded.

Combined effects of cadmium exposure and temperature on the annual killifish (Nothobranchius furzeri).

Freshwater organisms are increasingly exposed to combinations of stressors. However, because it is time-consuming and costly, research on the interaction of stressors, such as compound toxicity and global warming on vertebrates, is scarce. Studies on multigenerational effects of these combined stressors are almost nonexistent. In the present study, we tested the combined effects of 4 °C warming and cadmium (Cd) exposure on life-history traits, biomarkers, bioaccumulation, and multigenerational tolerance in the turquoise killifish, Nothobranchius furzeri. The extremely short life cycle of this vertebrate model allows for assessment of sublethal and multigenerational effects within 4 mo. The applied Cd concentrations had only limited effects on the measured endpoints, which suggests that N. furzeri is more resistant to Cd than fathead minnow and rainbow trout. In contrast, the temperature increase of 4 °C was stressful: it delayed female maturation and lowered adult mass and fecundity. Finally, indications of synergistic effects were found on peak fecundity and embryonic survival. Overall, these results indicate the importance of studying chronic and multigenerational effects of combined stressors. Environ Toxicol Chem 2018;37:2361-2371. © 2018 SETAC.

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri.

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity testing has been demonstrated. Overall, the sensitivity of the species to toxic compounds is in the range with, or higher than, that of other model species. This work describes protocols for acute, chronic, and multigenerational bioassays of single and combined stressor effects on N. furzeri. Due to its short maturation time and life-cycle, this vertebrate model allows the study of endpoints such as maturation time and fecundity within four months. Transgenerational full life-cycle exposure trials can be performed in as little as 8 months. Since this species produces eggs that are drought-resistant and remain viable for years, the on-site culture of the species is not needed but individuals can be recruited when required. The protocols are designed to measure life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

Acute sensitivity of the killifish Nothobranchius furzeri to a combination of temperature and reference toxicants (cadmium, chlorpyrifos and 3,4-dichloroaniline).

Aquatic organisms of inland waters are often subjected to a combination of stressors. Yet, few experiments assess mixed stress effects beyond a select group of standard model organisms. We studied the joint toxicity of reference toxicants and increased temperature on the turquoise killifish, Nothobranchius furzeri, a promising model for ecotoxicological research due to the species' short life cycle and the production of drought-resistant eggs. The acute sensitivity of the larval stage (2dph) to three compounds (cadmium, 3,4-dichloroaniline and chlorpyrifos) was tested in combination with a temperature increase of 4 °C, mimicking global warming. Dose-response relationships were used to calculate 96h-LC50 of 0.28 mg/L (24 °C) and 0.39 mg/L (28 °C) for cadmium, 96h-LC50 of 9.75 mg/L (24 °C) and 6.61 mg/L (28 °C) for 3,4-dichloroaniline and 96h-LC50 of 15.4 µg/L (24 °C) and 14.2 µg/L (28 °C) for chlorpyrifos. After 24 h of exposure, the toxicity of all tested compounds was exacerbated under increased temperature. Furthermore, the interaction effect of cadmium and temperature could be predicted by the stress addition model (SAM). This suggests the applicability of the model for fish and at the same time indicates that the model could be suitable to predict effects of temperature-toxicant interactions.

Acute and chronic sensitivity to copper of a promising ecotoxicological model species, the annual killifish Nothobranchius furzeri.

Nothobranchius furzeri is a promising model for ecotoxicological research due to the species' short life cycle and the production of drought-resistant eggs. Although the species is an emerging vertebrate fish model for several fundamental as well as applied research domains, its potential for ecotoxicological research has not yet been tested. The aim of this study was to characterise the acute and chronic sensitivity of this species to copper as compared to other model organisms. Effects of both acute and chronic copper exposure were tested on survival, life history and physiological traits. We report a 24h-LC50 of 53.93µg Cu/L, which is situated within the sensitivity range of other model species such as Brook Trout, Fathead Minnow and Rainbow Trout. Moreover, in the full life cycle exposure, we show that an exposure concentration of 10.27µg/L did not cause acute adverse effects (96h), but did cause mortality after prolonged exposure (3-4 weeks). Also chronic, sublethal effects were observed, such as a reduction in growth rate, delayed maturation and postponed reproduction. Based on our results, we define the NOEC at 6.68µg Cu/L, making N. furzeri more sensitive to copper as compared to Brook Trout and Fathead Minnow. We found stimulatory effects on peak fecundity at subinhibitory levels of copper concentrations (hormesis). Finally, we found indications for detoxifying and copper-excreting mechanisms, demonstrating the ability of the fish to cope with this essential metal, even when exposed to stressful amounts. The successful application of current ecotoxicological protocols on N. furzeri and its sensitivity range comparable to that of other model organisms forms the basis to exploit this species in further ecotoxicological practices.