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.

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.

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.

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.

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.

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.

Taxonomic classification of the bacterial endosymbiont Wolbachia based on next-generation sequencing: is there molecular evidence for its presence in tardigrades?

We used high-throughput sequencing of 16S rRNA to test whether tardigrade species are infected with Wolbachia parasites. We applied SILVA and Greengenes databases that allowed taxonomic classification of bacterial sequences to OTUs. The results obtained from both databases differed considerably in the number of OTUs, and only the Greengenes database allowed identification of Wolbachia (infection was also supported by comparison of sequences to NCBI database). The putative bacterial endosymbiont Wolbachia was discovered only in adult eutardigrades, while bacteria identified down to the order Rickettsiales were detected in both eutardigrade eggs and adult specimens. Nevertheless, the frequency of Wolbachia in the bacterial communities of the studied eutardigrades was low. Similarly, in our positive control, i.e., a fairy shrimp Streptocephalus cafer, which was found to be infected with Wolbachia in our previous study using Sanger sequencing, only the Rickettsiales were detected. We also carried out phylogenetic reconstruction using Wolbachia sequences from the SILVA and Greengenes databases, Alphaproteobacteria putative endosymbionts and Rickettsiales OTUs obtained in previous studies on the microbial community of tardigrades, and Rickettsiales and Wolbachia OTUs obtained in the current study. Our discovery of Wolbachia in tardigrades can fuel new research to uncover the specifics of this interaction.

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.

Conspecific density and environmental complexity impact behaviour of turquoise killifish (Nothobranchius furzeri).

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.

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.

Detection of bacterial endosymbionts in freshwater crustaceans: the applicability of non-degenerate primers to amplify the bacterial 16S rRNA gene.

Bacterial endosymbionts of aquatic invertebrates remain poorly studied. This is at least partly due to a lack of suitable techniques and primers for their identification. We designed a pair of non-degenerate primers which enabled us to amplify a fragment of ca. 500 bp of the 16S rRNA gene from various known bacterial endosymbiont species. By using this approach, we identified four bacterial endosymbionts, two endoparasites and one uncultured bacterium in seven, taxonomically diverse, freshwater crustacean hosts from temporary waters across a wide geographical area. The overall efficiency of our new WOLBSL and WOLBSR primers for amplification of the bacterial 16S rRNA gene was 100%. However, if different bacterial species from one sample were amplified simultaneously, sequences were illegible, despite a good quality of PCR products. Therefore, we suggest using our primers at the first stage of bacterial endosymbiont identification. Subsequently, genus specific primers are recommended. Overall, in the era of next-generation sequencing our method can be used as a first simple and low-cost approach to identify potential microbial symbionts associated with freshwater crustaceans using simple Sanger sequencing. The potential to detected bacterial symbionts in various invertebrate hosts in such a way will facilitate studies on host-symbiont interactions and coevolution.