Fish predation affects invertebrate community structure of tropical temporary ponds, with downstream effects on phytoplankton that are obscured by pesticide pollution.

Aquatic biota of tropical temporary ponds typically experience a wide range of stressors that can drive the structure and dynamics of natural communities. Particularly in regions with intense agricultural activity, aquatic biota may not only experience predation pressure but also stress from pesticides that inadvertently enter the ponds. We increasingly understand how these different sources of stress affect classic model taxa under controlled laboratory conditions, but how predators and pesticides may jointly affect pond invertebrate communities is still unclear, particularly for tropical systems. Here, we conducted an outdoor mesocosm experiment to study how fish predation combined with exposure to an environmentally relevant concentration of the commonly used insecticide cypermethrin (0.8 ng/L) affects the structure of invertebrate communities, and its potential effects on leaf litter decomposition and invertebrate grazing efficiency as measures of ecosystem functioning. A total of seven invertebrate taxa were recorded in the mesocosm communities. Fish predation effectively lowered the number of invertebrate taxa, with fish mesocosms being dominated by high densities of rotifers, associated with lower phytoplankton levels, but only when communities were not simultaneously exposed to cypermethrin. In contrast, cypermethrin exposure did not affect invertebrate community structure, and neither fish predation nor cypermethrin exposure affected our measures of ecosystem functioning. These findings suggest that predation by killifish can strongly affect invertebrate community structure of tropical temporary ponds, and that downstream effects on phytoplankton biomass can be mediated by exposure to cypermethrin. More broadly, we contend that a deeper understanding of (tropical) temporary pond ecology is necessary to effectively manage these increasingly polluted systems.

Effects of river regulation on aquatic invertebrate community composition: A comparative analysis in two southern African rivers.

While natural floods play a crucial role in shaping the composition of aquatic communities, the most rivers worldwide are regulated or dammed for anthropogenic purposes, resulting in alterations to the biological and chemical composition of these ecosystems. Studies have demonstrated various negative effects of river regulation on aquatic invertebrate communities globally. However, there is a scarcity of research in Africa, despite its vulnerability to anthropogenic impacts. This study aimed to compare aquatic invertebrate communities in the Phongolo River, an impacted regulated river, and the Usuthu River, a less impacted unregulated river, in South Africa. It further aimed to ascertain whether Lake Nyamithi, a naturally saline lake receiving water from both of the aforementioned systems, exhibited a stronger similarity to one of the two rivers in terms of its aquatic invertebrate composition. Aquatic invertebrate and water samples were collected from 2012 to 2018 over several surveys. The Usuthu River demonstrated a diverse and sensitive aquatic invertebrate community, emphasising its high conservation value. The Phongolo River demonstrated effects of anthropogenic impact, with taxa more resilient to changes in water quality and flow compared to the Usuthu River. Mismanagement and excessive water use may lead to the loss of any remaining sensitive aquatic invertebrate communities in this river. The presence of invasive molluscan in the Phongolo River and Lake Nyamithi also poses a threat to the native aquatic invertebrate communities. These invasive species are currently absent from the Usuthu River although other invasive species, such as the Australian redclaw crayfish, are found in both river systems. Lake Nyamithi displayed a unique aquatic invertebrate community, distinct from both rivers and their floodplains. This study provides important baseline information on the Usuthu River's aquatic invertebrates and emphasises the need to maintain adequate water flow in rivers and floodplains to protect biodiversity and sensitive species.

Playing Peekaboo with a Master Manipulator: Metagenetic Detection and Phylogenetic Analysis of Wolbachia Supergroups in Freshwater Invertebrates.

The infamous "master manipulators"-intracellular bacteria of the genus Wolbachia-infect a broad range of phylogenetically diverse invertebrate hosts in terrestrial ecosystems. Wolbachia has an important impact on the ecology and evolution of their host with documented effects including induced parthenogenesis, male killing, feminization, and cytoplasmic incompatibility. Nonetheless, data on Wolbachia infections in non-terrestrial invertebrates are scarce. Sampling bias and methodological limitations are some of the reasons limiting the detection of these bacteria in aquatic organisms. In this study, we present a new metagenetic method for detecting the co-occurrence of different Wolbachia strains in freshwater invertebrates host species, i.e., freshwater Arthropoda (Crustacea), Mollusca (Bivalvia), and water bears (Tardigrada) by applying NGS primers designed by us and a Python script that allows the identification of Wolbachia target sequences from the microbiome communities. We also compare the results obtained using the commonly applied NGS primers and the Sanger sequencing approach. Finally, we describe three supergroups of Wolbachia: (i) a new supergroup V identified in Crustacea and Bivalvia hosts; (ii) supergroup A identified in Crustacea, Bivalvia, and Eutardigrada hosts, and (iii) supergroup E infection in the Crustacea host microbiome community.

Long-term exposure to a pharmaceutical pollutant affects geotaxic behaviour in the adult but not juvenile life stage of killifish.

Ecosystems around the world are increasingly polluted with pharmaceutical compounds that may perturb wildlife behaviour. Because many pharmaceuticals are continuously present in the aquatic environment, animals are often exposed to them across several life stages or even their entire life. Despite a large body of literature showing various impacts of exposure to pharmaceuticals on fish, hardly any long-term studies across different life stages have been conducted which makes it hard to accurately estimate the ecological outcomes of pharmaceutical pollution. Here, we performed a laboratory experiment in which we exposed hatchlings of the fish model Nothobranchius furzeri to an environmentally relevant concentration (0.5 µg/L) of the antidepressant fluoxetine until well into adulthood. We monitored total body length and geotaxic behaviour (i.e. gravity-mediated activity) of each fish as two traits that are ecologically relevant and naturally differ between juvenile and adult killifish. Fish exposed to fluoxetine were smaller compared to control fish, an effect that became more apparent as fish aged. Even though fluoxetine did not affect average swimming depth of either juveniles or adults, nor the time spent at the surface or bottom of the water column, exposed fish changed their position in the water column (depth) more frequently in the adult but not juvenile phase. These results suggest that important morphological and behavioural responses to pharmaceutical exposure-and their potential ecological consequences-may only emerge later in time and/or during specific life stages. Therefore, our results highlight the importance of considering ecologically relevant timescales across developmental stages when studying the ecotoxicology of pharmaceuticals.

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.

Drought altered trophic dynamics of an important natural saline lake: A stable isotope approach.

Climate change and associated droughts threaten the ecology and resilience of natural saline lakes globally. There is a distinct lack of research regarding their ecological response to climatic events in the Global South. This region is predicted to experience climatic events such as El Niño-Southern Oscillation (ENSO) more often and with greater severity with the potential to alter the structure and functioning of aquatic ecosystems significantly. From 2015 to 2016 South Africa experienced one of the most severe country-wide droughts as a result of a strong ENSO event. Our study aimed to investigate the effect of this supra-seasonal drought on the trophic structure of fish communities in a naturally saline shallow lake of a Ramsar wetland using stable isotope techniques. Fishes and potential basal sources were collected from the lake, during predrought conditions in 2010 and after severe drought (recovery phase; 2017). The δ13C and δ15N values of food web elements were determined and analysed using Bayesian mixing models and Bayesian Laymen metrics to establish the proportional contribution of C3 and C4 basal sources to the fish (consumer) diets, and examine the fish community in terms of isotopic niche and trophic structure, respectively. Fish consumers relied predominantly on C3 basal sources in the predrought and shifted to greater reliance on C4 basal sources, decreased isotopic niche space use and a reduction in trophic length in the recovery phase. Drought altered the type and abundance of the basal sources available by limiting sources to those that are more drought-tolerant, reducing the trophic pathways of the food web with no significant alterations in the fish community. These results demonstrate the resilience and biological plasticity of Lake Nyamithi and its aquatic fauna, highlighting the importance of freshwater inflow to saline lakes with alterations thereof posing a significant threat to their continued functioning.

Direct effects of elevated dissolved CO2 can alter the life history of freshwater zooplankton.

Dissolved CO2 levels (pCO2) are increasing in lentic freshwaters across the globe. Recent studies have shown that this will impact the nutritional quality of phytoplankton as primary producers. However, the extent to which freshwater zooplankton may also be directly affected remains unclear. We test this in three model species representative of the main functional groups of primary consumers in freshwaters; the water flea Daphnia magna, the seed shrimp Heterocypris incongruens and the rotifer Brachionus calyciflorus. We experimentally exposed individuals to three pCO2 levels (1,500; 25,500 and 83,000 ppm) to monitor changes in life history in response to current, elevated and extreme future pCO2 conditions in ponds and shallow lakes. All species had reduced survival under the extreme pCO2 treatment, but the water flea was most sensitive. Body size and reproduction were reduced at 25,500 ppm in the water flea and the seed shrimp and population growth was delayed in the rotifer. Overall, our results show that direct effects of pCO2 could impact the population dynamics of freshwater zooplankton. By differentially modulating the life history of functional groups of primary consumers, elevated pCO2 has the potential to change the evolutionary trajectories of populations as well as the ecological functioning of freshwater communities.

Stakeholder Analysis on Ecosystem Services of Lake Manyara Sub-basin (Tanzania): How to Overcome Confounding Factors.

Ecosystem services are a telling concept to discuss the integrated management of natural resources, such as integrated water and soil, with non-academic stakeholders. Stakeholders have different perceptions regarding the management of various ecosystem services, which is challenging when aiming to develop and foster sustainable ecosystem management. We performed a stakeholder analysis as part of a social-ecological study in preparation of a decision support system for integrated water management within the Lake Manyara sub-basin (LMSB), Tanzania. The area includes a National Park and UNESCO Biosphere Reserve. A group discussion listed 26 stakeholders, categorized according to the sector, influence, and interest. The stakeholders were grouped into six functional categories: local Non-Governmental Organisations (NGOs), other civil society groups, Belgian and international NGOs, authorities, academics associated to international donors and the private sector. We empirically identified advantages, shortcomings and associated risks when performing a stakeholder analysis with an interest-influence matrix. Confounding factors may include, e.g., the omission of important stakeholders, a different understanding of 'influence' and 'interest', or the omission of fragile groups. Instead of 'low' or 'high' interest and influence, we propose the terms 'supportive', 'potentially supportive', 'unsupportive', 'not interested', 'low or no influence' and 'antagonistic'. Further, we consider stakeholders who directly extract resources from the social-ecological system (SES) as a separate category, because of their direct dependence and impact on the SES. This improved stakeholder analysis framework for developing decision support systems in water basins can contribute to better analysis, understanding and management of aquatic social-ecological systems in general.

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.

The role of anthropogenic container habitats as mosquito oviposition habitats in rural settlements in northern Tanzania.

In many areas, the main sources of mosquito vectors are not natural habitats but small artificial water bodies that are provided unintentionally by humans. Such container habitats have been linked to outbreaks of dengue fever and other arboviral diseases. However, in many parts of the world the possible risks associated with container habitats have not been assessed. Here, we focused on a human population expansion area in northern Tanzania with a high incidence of dengue and other cases of high fever. We explored the importance of anthropogenic container habitats for mosquito production in the Lake Manyara Basin. We also assessed how biotic and physicochemical habitat characteristics limit mosquito abundance in containers. Results showed that Aedes aegypti (Linnaeus), vector of dengue and other arboviruses, and Culex quinquefasciatus (Say), vector of filarial worms, were the dominant mosquitoes ovipositing in large numbers in different containers. Old tires were the dominant and most productive container habitat for mosquitoes in the region. However, there were strong differences among villages, illustrating that the mosquito burden associated with container habitats varies locally. We concluded that in this region, removal of artificial container habitats could be a simple strategy to reduce the mosquito-mediated disease burden within the local population.

Pleistocene allopatric differentiation followed by recent range expansion explains the distribution and molecular diversity of two congeneric crustacean species in the Palaearctic.

Pleistocene glaciations had a tremendous impact on the biota across the Palaearctic, resulting in strong phylogeographic signals of range contraction and rapid postglacial recolonization of the deglaciated areas. Here, we explore the diversity patterns and history of two sibling species of passively dispersing taxa typical of temporary ponds, fairy shrimps (Anostraca). We combine mitochondrial (COI) and nuclear (ITS2 and 18S) markers to conduct a range-wide phylogeographic study including 56 populations of Branchinecta ferox and Branchinecta orientalis in the Palaearctic. Specifically, we investigate whether their largely overlapping ranges in Europe resulted from allopatric differentiation in separate glacial refugia followed by a secondary contact and reconstruct their postglacial recolonization from the inhabited refugia. Our results suggest the existence of distinct refugia for the two species, with genetic divergence among intraspecific lineages consistent with late Pleistocene glacial cycles. While B. ferox lineages originated from Mediterranean refugia, the origin of B. orientalis lineages was possibly located on the Pannonian Plain. We showed that most dispersal events predominantly happened within 100 km, coupled with several recent long-distance events (> 1000 km). Hence the regional habitat density of suitable habitats in Central Europe is possibly a key to the co-existence of the two species. Overall, our study illustrates how isolation in combination with stochastic effects linked to glacial periods are important drivers of the allopatric differentiation of Palaearctic taxa.

Keep your natural enemies close - native predators can maintain low mosquito densities in temporary ponds in a malaria expansion area in Northern Tanzania.

Mosquitoes and the diseases they transmit are a global public health threat that affects most human populations. Mosquito abundances are strongly linked to the number of suitable larval habitats available. However, it is still not well understood how different land uses impact larval habitat quality in different parts of the world. We investigated links between land use practices and abundance of mosquito larvae in temporary ponds located in different land uses in a malaria expansion zone in Tanzania. Temporary ponds are often cited as important sources of mosquitoes but are also of substantial conservation concern being home to a unique fauna and flora. Often, they also have high socio-economic importance. Overall, encountered mosquito larval abundances were very low, both for the collected Anopheles gambiae s.l. and Culex quinquefasciatus. Although temporary ponds are important mosquito larval habitats in other parts of Africa, currently they are unlikely to be an important factor affecting the prevalence of mosquito borne diseases in the Manyara study region. Larval abundances in temporary ponds were higher in agricultural areas where more eutrophic conditions were found and minimal in residential areas. Overall, even though temporary ponds are often modified as rice paddies in the rainy season, they were in a good ecological condition and hosted a diverse assemblage of aquatic predators that likely prevented larval mosquitoes from reaching high densities. Maintaining this good condition by preventing pesticide and nutrient input as much as possible, can be important to prevent pond degradation that is likely to make these habitats more suitable for mosquitoes in the future.

An empirical confirmation of diversified bet hedging as a survival strategy in unpredictably varying environments.

Environmental change jeopardizes the survival of species from variable environments by making the occurrence of favorable conditions less predictable. For organisms with long-lived propagules (e.g., spores, eggs, or seeds), the theory of diversified bet hedging (DBH) predicts that delayed hatching over different growing seasons can help populations avoid extinction. Empirical observations in different organisms are consistent with DBH, but integrated tests that simultaneously validate the main theoretical assumptions and predictions are lacking. In this study, we combine field and multi-generational lab experiments to provide a complete test of DBH. Consistent with DBH predictions, resting egg clutches of the fairy shrimp Branchipodopsis wolfi, which inhabits rain-fed temporary rock pool environments with unpredictable inundations, hatched partially over a succession of inundations with identical hatching cues. Bet hedging was more common in populations from more unpredictable habitats where hatching fractions were lower. This differentiation in hatching strategies was preserved after two generations under common garden conditions, which implies intrinsic (epi-)genetic control of hatching. Finally, a demographic model confirmed that lower hatching fractions increase long-term population growth in unpredictable habitats. With this paper we propose a method to calculate probabilities of successful recruitment for organisms that use imperfect cues and show that this drives selection for variation in life history strategies as part of a DBH strategy.

Neurochemical exposure disrupts sex-specific trade-offs between body length and behaviour in a freshwater crustacean.

Ongoing pollution of aquatic ecosystems with neurochemical compounds warrants an improved understanding of how this affects key organisms. Neurochemicals are shown to alter the behaviour of common study species but it remains difficult to translate these results to biologically meaningful predictions across taxa. This is partly because studies on species with non-generic life-history strategies such as many freshwater crustaceans are currently underrepresented. Here, we use a laboratory experiment to assess baseline behavioural variation (spontaneous activity level and geotaxic behaviour) in the freshwater fairy shrimp Branchipodopsis wolfi and how this is affected by chronic exposure to an environmentally-relevant concentration of the anxiolytic pharmaceutical fluoxetine. The more conspicuously coloured and larger females of the species were overall less active and more benthic than males. Moreover, amongst females, vertical activity was negatively associated with size, while an opposite relationship was found for males. These trade-offs are likely part of an antipredator strategy to reduce the probability of being detected by visual hunters, but disappeared after exposure to fluoxetine. This is of particular interest since it is an effective proof of principle that neurochemicals may impact ecologically-relevant trade-offs between conspicuous morphology and antipredator behaviour. In natural ecosystems, such disturbed antipredator behavioural responses could have far-reaching fitness consequences.

Hydrological, Environmental and Taxonomical Heterogeneity during the Transition from Drying to Flowing Conditions in a Mediterranean Intermittent River.

Intermittent rivers and ephemeral streams (IRES) are increasingly studied because of their often-unique aquatic and terrestrial biodiversity, biogeochemical processes and associated ecosystem services. This study is the first to examine the hydrological, physicochemical and taxonomic variability during the dry-wet transition of an intermittent river in the Chilean Mediterranean Zone. Based on 30-years of river monitoring data and the TREHS tool, the hydrology of the river was characterised. Overall, the river shows a significant reduction in streamflow (-0.031 m3/s per year) and a substantial increase of zero flow days (+3.5 days per year). During the transition of hydrological states, variations were observed in the environmental conditions and invertebrate communities. During the drying phase, abundance, richness, and diversity were highest, while species turn-over was highest during base flow conditions. The disconnected pools and the flow resumption phases were characterised by high proportions of lentic taxa and non-insects, such as the endemic species of bivalves, gastropods, and crustaceans, highlighting the relevance of disconnected pools as refuges. Future climatic change scenarios are expected to impact further the hydrology of IRES, which could result in the loss of biodiversity. Biomonitoring and conservation programmes should acknowledge these important ecosystems.

Natural daily patterns in fish behaviour may confound results of ecotoxicological testing.

Low doses of neuroactive chemicals end up in the environment and disrupt behaviour of non-target organisms. Although a whole range of studies have documented pollutant-induced changes in behaviour, natural daily variability in behaviour is rarely taken into account. This is surprising because biological rhythms may affect the outcome of experiments, are adaptive and are expected to be sensitive to neurochemical exposure. Here, we exploit daily behavioural variation in the fish model Nothobranchius furzeri to examine if behavioural effects of chronic exposure (74 days) to an environmentally relevant level (28 ng/L) of the neurochemical fluoxetine depend on the time of day. Fluoxetine exposure induced an increase in anxiety-related behaviour that was slightly more pronounced in the evening compared to the morning. Moreover, open-field locomotor activity was disrupted and daily patterns in activity lifted upon exposure to the compound. These results imply that short-term behavioural variability should be considered both to standardise ecological risk assessment of neuroactive chemicals as well as to better understand the environmental impact of such compounds in aquatic ecosystems.

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.

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.