Effects of environmental concentrations of 6PPD and its quinone metabolite on the growth and reproduction of freshwater cladoceran.

The widespread occurrence and accumulation of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and its quinone metabolite, 6PPD quinone (6PPD-Q), have been globally recognized as a critical environmental issue. However, knowledge on the adverse effects of 6PPD and 6PPD-Q on freshwater invertebrates is limited. This study investigated the effects of 6PPD and its oxidative byproduct, 6PPD-Q, on the growth and reproduction of Daphnia pulex. Through 21-day exposure experiments, we measured the uptake of 0.1, 1, and 10 µg/L 6PPD and 6PPD-Q by D. pulex and assessed the effects on growth and fecundity of D. pulex. While 6PPD and 6PPD-Q did not affect the mortality rate of D. pulex, 6PPD-Q exposure inhibited the growth of D. pulex, indicating potential ecological risks. In particular, the reproductive capacity of D. pulex remained unaffected across the tested concentrations of 6PPD and 6PPD-Q, suggesting specific toxicological pathways that warrant further investigation. This study underscored the importance of evaluating the sublethal effects of emerging contaminants such as 6PPD and 6PPD-Q on aquatic invertebrates, and highlighted the need for comprehensive risk assessments to better understand their environmental impacts.

Combined effects of heat waves and pesticide pollution on zooplankton communities: Does the timing of stressor matter?

Most studies assessing the combined effects of chemical and non-chemical stressors on aquatic ecosystems have been based on synchronous stressor applications. However, asynchronous exposure scenarios may be more common in nature, particularly for pulsed stressors such as heatwaves and pesticide concentration peaks. In this study, we investigated the single and combined effects of the insecticide chlorpyrifos (CPF) and a heatwave (HW) on a zooplankton community representative of a Mediterranean coastal wetland using synchronous (CPF+HW) and asynchronous (HW→CPF and CPF→HW) exposure scenarios. CPF was applied at a concentration of 0.8 µg/L (single pulse), and the HW was simulated by a temperature increase of 8°C above the control temperature (20°C) for 7 days in freshwater microcosms. The interaction between stressors in synchrony resulted in synergistic effects at the population level (Daphnia magna) and additive at the community level. The partial reduction of sensitive species resulted in an abundance increase of competing species that were more tolerant to the evaluated stressors (e.g. Moina sp.). The asynchronous exposure scenarios resulted in a similar abundance decline of sensitive populations as compared to the synchronous one; however, the timing of stressor resulted in different responses in the long term. In the HW→CPF treatment, the D. magna population recovered at least one month faster than in the CPF+HW treatment, probably due to survival selection and cross-tolerance mechanisms. In the CPF→HW treatment, the effects lasted longer than in the CPF+HW, and the population did not recover within the experimental period, most likely due to the energetic costs of detoxification and effects on internal damage recovery. The different timing and magnitude of indirect effects among the tested asynchronous scenarios resulted in more severe effects on the structure of the zooplankton community in the CPF→HW treatment. Our study highlights the relevance of considering the order of stressors to predict the long-term effects of chemicals and heatwaves both at the population and community levels.

Toxicity Evaluation of Water and Pore Water from a Pilot-Scale Pit Lake in the Alberta Oil Sands Region to Daphnia Species.

Significant amounts of tailings and oil sands process-affected water (OSPW) are generated by bitumen extraction in the Alberta Oil Sands region. These by-products are potentially toxic to aquatic organisms and require remediation. The study site was Lake Miwasin, a pilot-scale pit lake integrated into broader reclamation efforts. It consists of treated tailings overlaid with blended OSPW and freshwater, exhibiting meromictic conditions and harboring aquatic communities. This study assessed the potential toxicity of Lake Miwasin surface water (LMW) and pore water (LMP) using saline-acclimated Cladocera, including lab strains of Daphnia magna and Daphnia pulex and native Daphnia species collected in brackish Humboldt Lake (HL) and Lake Miwasin (LM). The pore water evaluation was used to represent a worst-case water quality scenario during pond stratification. Additionally, the inclusion of native organisms incorporated site-specific adaptations and regional sensitivity into the toxicity evaluation. Our results showed that LMW did not display acute or chronic toxicity to lab species and native Daphnia sp. (HL). Conversely, LMP was acutely toxic to both lab species and native D. pulex (LM). In chronic tests (12 days exposure), LMP negatively affected reproduction in D. pulex (lab), with reductions in the number of offspring. Limited ability to acclimated organisms to the high salinity levels of LMP resulted in a shortened exposure duration for the chronic toxicity test. In addition to salinity being identified as a stressor in LMP, toxicity identification evaluation (TIE) phase I findings demonstrated that the observed toxicity for D. magna (lab) and D. pulex (LM, native) might be attributed to ammonia and metals in LMP. Further investigations are required to confirm the contributions of these stressors to LMP toxicity.

Feeding on stressed algae exerts important effects on life history traits of Daphnia magna in a multi-stressor environment.

Freshwater ecosystems are increasingly exposed to anthropogenic eutrophication, including high nitrogen. In addition, climate change is leading to more intense and frequent heatwaves, which have enormous impacts on all trophic levels of the ecosystem. Any change in the lower trophic levels, e.g., the phytoplankton, also introduces stress to higher trophic levels e.g., the zooplankton crustacean Daphnia. Individual effects of heatwaves, high nitrate, and changing feed quality have been studied in daphnia, but less is known about their interactive effects. This study used a 3 × 3 × 2 factorial design in which daphnia were exposed to combinations of ecologically relevant nitrate concentrations (0, 50, or 200 mg/L) and different heatwave scenarios (no, short-moderate, or long-intense) in which individuals were either fed with microalgae (P. subcapitata and C. reinhardtii) grown at 20 °C and 50 mg/L nitrate (control feed) or the same conditions as daphnia was exposed to (experimental feed). Throughout the experiment, the interactive effects of high nitrate, heatwave, and feed on mortality, maturation, offspring, and body size were evaluated. In general, heatwaves shorten the lifespan of daphnia. Exposing daphnia to a long-intense heatwave combined with high nitrate resulted in poor performance. In the nitrate-limited condition, however, the restricted proliferation of microalgae reduced feed availability, which also had a major impact on daphnia's life history traits. Daphnia cultured in high nitrate and fed control feed performed better than when fed experimental feed, suggesting that in a high nitrate condition, the microalgae grown under the same experimental conditions was either unable to meet energy requirements or introduced extra stress for the daphnia. Most importantly, the effect of nitrate and heatwave as stressors on the availability and quality of the feed had a greater impact on daphnia than its direct impact. Interestingly, a transgenerational adaptation to nitrate was observed which may help to maintain ecological balance in the long run.

Can a shift in dominant species of Microcystis alter growth and reproduction of waterfleas?

The bloom-forming species Microcystis wesenbergii and M. aeruginosa occur in many lakes globally, and may exhibit alternating blooms both spatially and temporally. As environmental changes increase, cyanobacteria bloom in more and more lakes and are often dominated by M. wesenbergii. The adverse impact of M. aeruginosa on co-existing organisms including zooplanktonic species has been well-studied, whereas studies of M. wesenbergii are limited. To compare effects of these two species on zooplankton, we explored effects of exudates from different strains of microcystin-producing M. aeruginosa (Ma905 and Ma526) and non-microcystin-producing M. wesenbergii (Mw908 and Mw929), on reproduction by the model zooplankter Daphnia magna in both chronic and acute exposure experiments. Specifically, we tested physiological, biochemical, molecular and transcriptomic characteristics of D. magna exposed to Microcystis exudates. We observed that body length and egg and offspring number of the daphnid increased in all treatments. Among the four strains tested, Ma526 enhanced the size of the first brood, as well as total egg and offspring number. Microcystis exudates stimulated expression of specific genes that induced ecdysone, juvenile hormone, triacylglycerol and vitellogenin biosynthesis, which, in turn, enhanced egg and offspring production of D. magna. Even though all strains of Microcystis affected growth and reproduction, large numbers of downregulated genes involving many essential pathways indicated that the Ma905 strain might contemporaneously induce damage in D. magna. Our study highlights the necessity of including M. wesenbergii into the ecological risk evaluation of cyanobacteria blooms, and emphasizes that consequences to zooplankton may not be clear-cut when assessments are based upon production of microcystins alone.

Rearing and Experimental Uses of Daphnia: Controlling Animal Nutrition and Assessing Lifespan and Life-History Traits.

Caloric restriction has been found to extend the lifespan of many organisms including mammals and other vertebrates. With lifespans exceeding months to years, age-related experiments involving fish and mammals can be overtly costly, both in terms of time and funding. The freshwater crustacean, Daphnia, has a relatively short lifespan (∼50 to 100 days), which makes it a cost-effective alternative animal model for longevity and aging studies. Besides age-specific mortality, there are a suite of physiological responses connected to "healthspan" that can be tracked as these animals age including growth, reproduction, and metabolic rates. These responses can be complemented by assessment of molecular and cellular processes connected to aging and health. Lifespan and metabolism of this model organism is responsive to long studied modulators of aging, such as rearing temperature and nutritional manipulation, but also pharmacological agents that target aging, e.g., rapamycin, which adds to its usefulness as a model organism. Here we describe how to culture Daphnia for aging experiments including maintaining laboratory populations of Daphnia mothers, growing algal food, and manipulating nutrition of these animals. In addition, we provide methods for tracking common physiological and longevity responses of Daphnia. This protocol provides researchers planning to use this model organism with methods to establish and maintain Daphnia populations and to standardize their experimental approaches. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culturing algae for Daphnia food Basic Protocol 2: General methods for culturing Daphnia Basic Protocol 3: Standardizing and controlling nutrition for experimental Daphnia Basic Protocol 4: Monitoring Daphnia lifespan Basic Protocol 5: Evaluating Daphnia health: Heart rate and respiration, body mass and growth rates, and reproduction.

First insight of the intergenerational effects of tri-n-butyl phosphate and polystyrene microplastics to Daphnia magna.

As an emerging organic pollutant, tributyl phosphate (TnBP) can be easily adsorbed by microplastics, resulting in compound toxic effects. In the present work, the effects of polystyrene microplastics (PS-MPs) and TnBP on the survival, growth, reproduction and oxidative stress of Daphnia magna (D. magna) have been evaluated through multigenerational test. Compared with the alone exposure groups, the somatic growth rate and the expression values of growth related genes rpa1, mre11, rnha, and rfc3_5 in the F1 generation of the combined exposure groups were significantly lower (p < 0.05), indicating synergistic effect of PS-MPs and TnBP on the growth toxicity and transgenerational effects. In addition, compared with the PS-MPs groups, significantly lower average number of offspring and expression values of reproduction related genes ccnb, mcm2, sgrap, and ptch1 were observed in the combined exposure group and TnBP group (p < 0.05), indicating TnBP might be the major factor causing reproductive toxicity to D. magna. Although PS-MPs and TnBP alone or in combination also had toxic impacts on the growth, survival and reproduction of D. magna in generations F0 and F2, the effects were less than F1 generation. Regarding oxidative stress, the activity of SOD, CAT and GSH-Px and MDA content in the generations F0 and F1 of combined exposure groups were higher than the TnBP group but lower than the PS-MPs groups, suggesting that PS-MPs might be the dominant cause of the oxidative damage in D. magna and the presence of TnBP would alleviate oxidative stress by reducing the bioaccumulation of PS-MPs. The present work will provide a theoretical basis for further understanding of the toxic effects and ecological risks of combined TnBP and microplastic pollution on aquatic organisms.

Anticipating change: The impact of simulated seasonal heterogeneity on heat tolerances along a latitudinal cline.

An understanding of thermal limits and variation across geographic regions is central to predicting how any population may respond to global change. Latitudinal clines, in particular, have been used to demonstrate that populations can be locally adapted to their own thermal environment and, as a result, not all populations will be equally impacted by an increase in temperature. But how robust are these signals of thermal adaptation to the other ecological challenges that animals commonly face in the wild? Seasonal changes in population density, food availability, or photoperiod are common ecological challenges that could disrupt patterns of thermal tolerance along a cline if each population differentially used these signals to anticipate future temperatures and adjust their thermal tolerances accordingly. In this study, we aimed to test the robustness of a cline in thermal tolerance to simulated signals of seasonal heterogeneity. Experimental animals were derived from clones of the Australian water flea, Daphnia carinata, sampled from nine distinct populations along a latitudinal transect in Eastern Australia. We then factorially combined summer (18 h light, 6 h dark) and winter (6 h light, 18 h dark) photoperiods with high (5 million algal cells individual-1 day-1) and low (1 million algal cells individual-1 day-1) food availabilities, before performing static heat shock assays to measure thermal tolerance. We found that the thermal tolerances of the clonal populations were sensitive to both measures of seasonal change. In general, higher food availability led to an increase in thermal tolerances, with the magnitude of the increase varying by clone. In contrast, a switch in photoperiod led to rank-order changes in thermal tolerances, with heat resistance increasing for some clones, and decreasing for others. Heat resistance, however, still declined with increasing latitude, irrespective of the manipulation of seasonal signals, with clones from northern populations always showing greater thermal resistance, most likely driven by adaptation to winter thermal conditions. While photoperiod and food availability can clearly shape thermal tolerances for specific populations, they are unlikely to overwhelm overarching signals of thermal adaptation, and thus, observed clines in heat resistance will likely have remained robust to these forms of seasonal heterogeneity.

Acclimation to warmer temperatures can protect host populations from both further heat stress and the potential invasion of pathogens.

Thermal acclimation can provide an essential buffer against heat stress for host populations, while acting simultaneously on various life-history traits that determine population growth. In turn, the ability of a pathogen to invade a host population is intimately linked to these changes via the supply of new susceptible hosts, as well as the impact of warming on its immediate infection dynamics. Acclimation therefore has consequences for hosts and pathogens that extend beyond simply coping with heat stress-governing both population growth trajectories and, as a result, an inherent propensity for a disease outbreak to occur. The impact of thermal acclimation on heat tolerances, however, is rarely considered simultaneously with metrics of both host and pathogen population growth, and ultimately fitness. Using the host Daphnia magna and its bacterial pathogen, we investigated how thermal acclimation impacts host and pathogen performance at both the individual and population scales. We first tested the effect of maternal and direct thermal acclimation on the life-history traits of infected and uninfected individuals, such as heat tolerance, fecundity, and lifespan, as well as pathogen infection success and spore production. We then predicted the effects of each acclimation treatment on rates of host and pathogen population increase by deriving a host's intrinsic growth rate (rm) and a pathogen's basic reproductive number (R0). We found that direct acclimation to warming enhanced a host's heat tolerance and rate of population growth, despite a decline in life-history traits such as lifetime fecundity and lifespan. In contrast, pathogen performance was consistently worse under warming, with within-host pathogen success, and ultimately the potential for disease spread, severely hampered at higher temperatures. Our results suggest that hosts could benefit more from warming than their pathogens, but only by linking multiple individual traits to population processes can the full impact of higher temperatures on host and pathogen population dynamics be realised.

Declining aqueous calcium and fish predation risk interactively modify the phenotypic plasticity in Daphnia pulex.

Aqueous calcium (Ca) decline is threatening freshwater ecosystems worldwide. There are great concerns about the possible ecological consequences of Ca limitation combined with biological pressures like predation. Here we investigated the interactions between Ca restriction and fish predation risk on the phenotypic plasticity in the keystone herbivore Daphnia, together with physiological responses underlying the plastic trait changes. Fish predation risk induced D. pulex to mature earlier and produce more but smaller offspring at adequate Ca. Declining Ca inhibited the expression of defensive traits, with the inhibitive degree showing a linear or threshold-limited dynamic. The presence of predation risk mitigated the negative effect of declining Ca on reducing body size but exacerbated the delay in maturity, indicating a life history trade-off for larger body size rather than the current reproduction in multi-stressed Daphnia. Actin 3-mediated cytoskeleton and AMPK ß-mediated energy metabolism were highly correlated with these plastic trait changes. Altered phenotypic plasticity in planktonic animals is expected to trigger many ecological impacts from individual fitness to community structure, thus providing new insights into the mechanisms underlying decreased Ca affecting lake ecosystems.

Effects of food quantity on the ingestion and egestion of MPs with different colors by Daphnia magna.

Aquatic organism uptake and accumulate microplastics (MPs) through various pathways, with ingestion alongside food being one of the primary routes. However, the impact of food concentration on the accumulation of different types of MPs, particularly across various colors, remains largely unexplored. To address this gap, we selected Daphnia magna as a model organism to study the ingestion/egestion kinetics and the preference for different MP colors under varying concentrations of Chlorella vulgaris. Our findings revealed that as the concentration of Chlorella increased, the ingestion of MPs by D. magna initially increased and then showed a decline. During the egestion phase within clean medium without further food supply, an increase in food concentration during the ingestion phase led to a slower rate of MP discharge; while when food was present during the egestion phase, the discharge rate accelerated for all treatments, indicating the importance of food ingestion/digestion process on the MPs bioaccumulation. Furthermore, in the presence of phytoplankton, D. magna demonstrated a preference for ingesting green-colored MPs, especially at low and medium level Chlorella supply, possibly due to the enhanced food searching activities. Beyond gut passage, we also examined the attachment of MPs to the organism's body surface, finding that the number of adhered MPs increased with increasing food concentration, likely due to the intensified filtering current during food ingestion. In summary, this study demonstrated that under aquatic environment with increasing phytoplankton concentrations, the ingestion and egestion rates, color preferences, as well as surface adherence of MPs to filter feeding zooplanktons will be significantly influenced, which may further pose ecological risks. Our results offer novel insights into the unintentional accumulation of MPs by zooplankton, highlighting the complex interactions between food availability and MPs accumulation dynamics.

Theory of Stoichiometric Intraguild Predation: Algae, Ciliate, and Daphnia.

Consumers respond differently to external nutrient changes than producers, resulting in a mismatch in elemental composition between them and potentially having a significant impact on their interactions. To explore the responses of herbivores and omnivores to changes in elemental composition in producers, we develop a novel stoichiometric model with an intraguild predation structure. The model is validated using experimental data, and the results show that our model can well capture the growth dynamics of these three species. Theoretical and numerical analyses reveal that the model exhibits complex dynamics, including chaotic-like oscillations and multiple types of bifurcations, and undergoes long transients and regime shifts. Under moderate light intensity and phosphate concentration, these three species can coexist. However, when the light intensity is high or the phosphate concentration is low, the energy enrichment paradox occurs, leading to the extinction of ciliate and Daphnia. Furthermore, if phosphate is sufficient, the competitive effect of ciliate and Daphnia on algae will be dominant, leading to competitive exclusion. Notably, when the phosphorus-to-carbon ratio of ciliate is in a suitable range, the energy enrichment paradox can be avoided, thus promoting the coexistence of species. These findings contribute to a deeper understanding of species coexistence and biodiversity.

Pollution offsets the rapid evolution of increased heat tolerance in a natural population.

Despite the increasing evidence for rapid thermal evolution in natural populations, evolutionary rescue under global warming may be constrained by the presence of other stressors. Highly relevant in our polluted planet, is the largely ignored evolutionary trade-off between heat tolerance and tolerance to pollutants. By using two subpopulations (separated 40 years in time) from a resurrected natural population of the water flea Daphnia magna that experienced a threefold increase in heat wave frequency during this period, we tested whether rapid evolution of heat tolerance resulted in reduced tolerance to the widespread metal zinc and whether this would affect heat tolerance upon exposure to the pollutant. Our results revealed rapid evolution of increased heat tolerance in the recent subpopulation. Notably, the sensitivity to the metal tended to be stronger (reduction in net energy budget) or was only present (reductions in heat tolerance and in sugar content) in the recent subpopulation. As a result, the rapidly evolved higher heat tolerance of the recent subpopulation was fully offset when exposed to zinc. Our results highlight that the many reports of evolutionary rescue to global change stressors may give a too optimistic view as our warming planet is polluted by metals and other pollutants.

Thresholds and interactive effects of BPA-gradient and temperature on life history traits of Daphnia magna.

Bisphenol A (BPA), a synthetic organic compound widely used in the production of plastics, is recognized as an emerging contaminant because of its toxicity and the potential risks associated with bioaccumulation in organisms. Despite potential environmental hazards, there is a lack of studies examining BPA toxicity mechanisms and its potential impact on various trophic levels, with even fewer exploring whether global stressors such as temperature can affect the toxicity of BPA in organisms. Our aim was to assess the combined impact of BPA and varying temperature regimes on life-history traits in Daphnia magna. Our results revealed a significant impact of BPA on the growth, reproduction, and accumulated moulting of D. magna, with adverse effects primarily associated with the assimilation of BPA in algae rather than the BPA present in the medium, pointing to a trophic transfer mechanism. The interactive effect between BPA and temperature demonstrated a slight stimulatory effect of low BPA level on D. magna growth rate under warming constant conditions, but an inhibitory under warming fluctuating temperatures. Additionally, a BPA threshold was identified, below which growth became temperature-dependent. This study emphasizes the crucial role of considering temperature in predicting how toxins may affect Daphnia within aquatic food webs.

Rapid evolution of consumptive and non-consumptive predator effects on prey population densities, bioenergetics and stoichiometry.

Predators can strongly influence prey populations not only through consumptive effects (CE) but also through non-consumptive effects (NCE) imposed by predation risk. Yet, the impact of NCE on bioenergetic and stoichiometric body contents of prey, traits that are shaping life histories, population and food web dynamics, is largely unknown. Moreover, the degree to which NCE can evolve and can drive evolution in prey populations is rarely studied. A 6-week outdoor mesocosm experiment with Caged-Fish (NCE) and Free-Ranging-Fish (CE and NCE) treatments was conducted to quantify and compare the effects of CE and NCE on population densities, bioenergetic and stoichiometric body contents of Daphnia magna, a keystone species in freshwater ecosystems. We tested for evolution of CE and NCE by using experimental populations consisting of D. magna clones from two periods of a resurrected natural pond population: a pre-fish period without fish and a high-fish period with high predation pressure. Both Caged-Fish and Free-Ranging-Fish treatments decreased the body size and population densities, especially in Daphnia from the high-fish period. Only the Free-Ranging-Fish treatment affected bioenergetic variables, while both the Caged-Fish and Free-Ranging-Fish treatments shaped body stoichiometry. The effects of CE and NCE were different between both periods indicating their rapid evolution in the natural resurrected population. Both the Caged-Fish and Free-Ranging-Fish treatments changed the clonal frequencies of the experimental Daphnia populations of the pre-fish as well as the high-fish period, indicating that not only CE but also NCE induced clonal sorting, hence rapid evolution during the mesocosm experiment in both periods. Our results demonstrate that CE as well as NCE have the potential to change not only the body size and population density but also the bioenergetic and stoichiometric characteristics of prey populations. Moreover, we show that these responses not only evolved in the studied resurrected population, but that CE and NCE also caused differential rapid evolution in a time frame of 6 weeks (ca. four to six generations). As NCE can evolve as well as can drive evolution, they may play an important role in shaping eco-evolutionary dynamics in predator-prey interactions.

Preliminary hazard assessment of a new nature-inspired antifouling (NIAF) agent.

A recently synthesized aminated 3,4-dioxygenated xanthone (Xantifoul2) was found to have promising antifouling (AF) effects against the settlement of the macrofouler Mytilus galloprovincialis larvae. Preliminary assessment indicated that Xantifoul2 has reduced ecotoxicological impacts: e.g., being non-toxic to the marine crustacea Artemia salina (<10 % mortality at 50 µM) and showing low bioconcentration factor in marine organisms. In order to meet the EU Biocidal Product Regulation, a preliminary hazard assessment of this new nature-inspired antifouling (NIAF) agent was conducted in this work. Xantifoul2 did not affect the swimming ability of the planktonic crustacean Daphnia magna, the growth of the diatom Phaeodactylum tricornutum, and the cellular respiration of luminescent Gram-negative bacteria Vibrio fischeri, supporting the low toxicity towards several non-target marine species. Regarding human cytotoxicity, Xantifoul2 did not affect the cell viability of retinal human cells (hTERT-RPE-1) and lipidomic studies revealed depletion of lipids involved in cell death, membrane modeling, lipid storage, and oxidative stress only at a high concentration (10 µM). Accelerated degradation studies in water were conducted under simulated sunlight to allow the understanding of putative transformation products (TPs) that could be generated in the aquatic ecosystems. Both Xantifoul2 and photolytic-treated Xantifoul2 in the aqueous matrix were therefore evaluated on several nuclear receptors (NRs). The results of this preliminary hazard assessment of Xantifoul2, combined with the high degradation rates in water, provide strong evidence of the safety of this AF agent under the evaluated conditions, and provide the support for future validation studies before this compound can be introduced in the market.

Daphnia uses its circadian clock for short-day recognition in environmental sex determination.

Some organisms have developed a mechanism called environmental sex determination (ESD), which allows environmental cues, rather than sex chromosomes or genes, to determine offspring sex.1,2,3,4 ESD is advantageous to optimize sex ratios according to environmental conditions, enhancing reproductive success.5,6 However, the process by which organisms perceive and translate diverse environmental signals into offspring sex remains unclear. Here, we analyzed the environmental perception mechanism in the crustacean, Daphnia pulex, a seasonal (photoperiodic) ESD arthropod, capable of producing females under long days and males under short days.7,8,9,10 Through breeding experiments, we found that their circadian clock likely contributes to perception of day length. To explore this further, we created a genetically modified daphnid by knocking out the clock gene, period, using genome editing. Knockout disrupted the daphnid's ability to sustain diel vertical migration (DVM) under constant darkness, driven by the circadian clock, and leading them to produce females regardless of day length. Additionally, when exposed to an analog of juvenile hormone (JH), an endocrine factor synthesized in mothers during male production, or subjected to unfavorable conditions of high density and low food availability, these knockout daphnids produced males regardless of day length, like wild-type daphnids. Based on these findings, we propose that recognizing short days via the circadian clock is the initial step in sex determination. This recognition subsequently triggers male production by signaling the endocrine system, specifically via the JH signal. Establishment of a connection between these two processes may be the crucial element in evolution of ESD in Daphnia.

Food quality shapes gradual phenotypic plasticity in ectotherms facing temperature variability.

Organisms exhibit reversible physiological adjustments as a response to rapidly changing environments. Yet such plasticity of the phenotype is gradual and may lag behind environmental fluctuations, thereby affecting long-term average performance of the organisms. By supplying energy and essential compounds for optimal tissue building, food determines the range of possible phenotypic changes and potentially the rate at which they occur. Here, we assess how differences in the dietary supply of essential lipids modulate the phenotypic plasticity of an ectotherm facing thermal fluctuations. We use three phytoplankton strains to create a gradient of polyunsaturated fatty acid and sterol supply for Daphnia magna under constant and fluctuating temperatures. We used three different fluctuation periodicities to unravel the temporal dynamics of gradual plasticity and its long-term consequences for D. magna performance measured as juvenile somatic growth rate. In agreement with gradual plasticity theory, we show that in D. magna, fluctuation periodicity determines the differential between observed growth rates and those expected from constant conditions. Most importantly, we show that diet modulates both the size and the direction of the growth rate differential. Overall, we demonstrate that the nutritional context is essential for predicting ectotherm consumers' performance in fluctuating thermal environments.

Impacts of sequential salinity and heat stress are recovery time-specific in freshwater crustacean, Daphnia pulicaria.

Stressors can interact to affect animal fitness, but we have limited knowledge about how temporal variation in stressors may impact their combined effect. This limits our ability to predict the outcomes of pollutants and future dynamic environmental changes. Elevated salinity in freshwater ecosystems has been observed worldwide. Meanwhile, heatwaves have become more frequent and intensified as an outcome of climate change. These two stressors can jointly affect organisms; however, their interaction has rarely been explored in the context of freshwater ecosystems. We conducted lab experiments using Daphnia pulicaria, a key species in lakes, to investigate how elevated salinity and heatwave conditions collectively affect freshwater organisms. We also monitored the impacts of various recovery times between the two stressors. Daphnia physiological conditions (metabolic rate, Na+-K+-ATPase (NKA) activity, and lipid peroxidation level) and life history traits (survival, fecundity, and growth) in response to salt stress as well as mortality in heat treatment were examined. We found that Daphnia responded to elevated salinity by upregulating NKA activity and increasing metabolic rate, causing a high lipid peroxidation level. Survival, fecundity, and growth were all negatively affected by this stressor. These impacts on physiological conditions and life history traits persisted for a few days after the end of the exposure. Heat treatments caused mortality in Daphnia, which increased with rising temperature. Results also showed that individuals that experienced salt exposure were more susceptible to subsequent heat stress, but this effect decreased with increasing recovery time between stressors. Findings from this work suggest that the legacy effects from a previous stressor can reduce individual resistance to a subsequent stressor, adding great difficulties to the prediction of outcomes of multiple stressors. Our work also demonstrates that cross-tolerance/susceptibility and the associated mechanisms remain unclear, necessitating further investigation.

Developmental defects in cognition, metabolic and cardiac function following maternal exposures to low environmental levels of selective serotonin re-uptake inhibitors and tributyltin in Daphnia magna.

Aquatic organisms are exposed to low concentrations of neuro-active chemicals, many of them acting also as neuroendocrine disruptors that can be hazardous during earlier embryonic stages. The present study aims to assess how exposure early in live to environmental low concentrations of two selective serotonin reuptake inhibitors (SSRIs), fluoxetine and sertraline, and tributyltin (TBT) affected cognitive, metabolic and cardiac responses in the model aquatic crustacean Daphnia magna. To that end, newly brooded females were exposed for an entire reproductive cycle (3-4 days) and the response of collected juveniles in the first, second and third consecutive broods, which were exposed, respectively, as embryos, provisioned and un-provisioned egg stages, was monitored. Pre-exposure to the selected SSRIs during embryonic and egg developmental stages altered the swimming behaviour of D. magna juveniles to light in a similar way reported elsewhere by serotonergic compounds while TBT altered cognition disrupting multiple neurological signalling routes. The studied compounds also altered body size, the amount of storage lipids in lipid droplets, heart rate, oxygen consumption rates and the transcription of related serotonergic, dopaminergic and lipid metabolic genes in new-born individuals, mostly pre-exposed during their embryonic and provisioning egg stages. The obtained cognitive, cardiac and metabolic defects in juveniles developed from exposed sensitive pre-natal stages align with the "Developmental Origins of Health and Disease (DoHAD)" paradigm.