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.

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.

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.

Contrasting parasite communities among allopatric colour morphs of the Lake Tanganyika cichlid Tropheus.

BACKGROUND: Adaptation to different ecological environments is thought to drive ecological speciation. This phenomenon culminates in the radiations of cichlid fishes in the African Great Lakes. Multiple characteristic traits of cichlids, targeted by natural or sexual selection, are considered among the driving factors of these radiations. Parasites and pathogens have been suggested to initiate or accelerate speciation by triggering both natural and sexual selection. Three prerequisites for parasite-driven speciation can be inferred from ecological speciation theory. The first prerequisite is that different populations experience divergent infection levels. The second prerequisite is that these infection levels cause divergent selection and facilitate adaptive divergence. The third prerequisite is that parasite-driven adaptive divergence facilitates the evolution of reproductive isolation. Here we investigate the first and the second prerequisite in allopatric chromatically differentiated lineages of the rock-dwelling cichlid Tropheus spp. from southern Lake Tanganyika (Central Africa). Macroparasite communities were screened in eight populations belonging to five different colour morphs. RESULTS: Parasite communities were mainly composed of acanthocephalans, nematodes, monogeneans, copepods, branchiurans, and digeneans. In two consecutive years (2011 and 2012), we observed significant variation across populations for infection with acanthocephalans, nematodes, monogeneans of the genera Gyrodactylus and Cichlidogyrus, and the copepod Ergasilus spp. Overall, parasite community composition differed significantly between populations of different colour morphs. Differences in parasite community composition were stable in time. The genetic structure of Tropheus populations was strong and showed a significant isolation-by-distance pattern, confirming that spatial isolation is limiting host dispersal. Correlations between parasite community composition and Tropheus genetic differentiation were not significant, suggesting that host dispersal does not influence parasite community diversification. CONCLUSIONS: Subject to alternating episodes of isolation and secondary contact because of lake level fluctuations, Tropheus colour morphs are believed to accumulate and maintain genetic differentiation through a combination of vicariance, philopatric behaviour and mate discrimination. Provided that the observed contrasts in parasitism facilitate adaptive divergence among populations in allopatry (which is the current situation), and promote the evolution of reproductive isolation during episodes of sympatry, parasites might facilitate speciation in this genus.