Combined exposure to hypoxia and nanoplastics leads to negative synergistic oxidative stress-mediated effects in the water flea Daphnia magna.

In this study, we investigated the combined effects of hypoxia and NPs on the water flea Daphnia magna, a keystone species in freshwater environments. To measure and understand the oxidative stress responses, we used acute toxicity tests, fluorescence microscopy, enzymatic assays, Western blot analyses, and Ingenuity Pathway Analysis. Our findings demonstrate that hypoxia and NPs exhibit a negative synergy that increases oxidative stress, as indicated by heightened levels of reactive oxygen species and antioxidant enzyme activity. These effects lead to more severe reproductive and growth impairments in D. magna compared to a single-stressor exposure. In this work, molecular investigations revealed complex pathway activations involving HIF-1α, NF-κB, and mitogen-activated protein kinase, illustrating the intricate molecular dynamics that can occur in combined stress conditions. The results underscore the amplified physiological impacts of combined environmental stressors and highlight the need for integrated strategies in the management of aquatic ecosystems.

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.

Identification of gene isoforms and their switching events between male and female embryos of the parthenogenetic crustacean Daphnia magna.

The cladoceran crustacean Daphnia exhibits phenotypic plasticity, a phenomenon that leads to diverse phenotypes from one genome. Alternative usage of gene isoforms has been considered a key gene regulation mechanism for controlling different phenotypes. However, to understand the phenotypic plasticity of Daphnia, gene isoforms have not been comprehensively analyzed. Here we identified 25,654 transcripts derived from the 9710 genes expressed during environmental sex determination of Daphnia magna using the long-read RNA-Seq with PacBio Iso-Seq. We found that 14,924 transcripts were previously unidentified and 5713 genes produced two or more isoforms. By a combination of Illumina short-read RNA-Seq, we detected 824 genes that implemented switching of the highest expressed isoform between females and males. Among the 824 genes, we found isoform switching of an ortholog of CREB-regulated transcription coactivator, a major regulator of carbohydrate metabolism in animals, and a correlation of this switching event with the sexually dimorphic expression of carbohydrate metabolic genes. These results suggest that a comprehensive catalog of isoforms may lead to understanding the molecular basis for environmental sex determination of Daphnia. We also infer the applicability of the full-length isoform analyses to the elucidation of phenotypic plasticity 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.

Extrapolating Metal (Cu, Ni, Zn) Toxicity from Individuals to Populations Across Daphnia Species Using Mechanistic Models: The Roles of Uncertainty Propagation and Combined Physiological Modes of Action.

Mechanistic effect modeling is a promising tool to improve the ecological realism of environmental risk assessment. An open question for the mechanistic modeling of metal toxicity is whether the same physiological mode of action (PMoA) could be assumed for closely related species. The implications of various modeling choices, such as the use of parameter point estimates and assumption of simplistic toxicodynamic models, are largely unexplored. We conducted life-table experiments with Daphnia longispina, Daphnia magna, and Daphnia pulex exposed to the single metals Cu, Ni, and Zn, and calibrated toxicokinetic-toxicodynamic (TKTD) models based on dynamic energy budget theory. We developed TKTD models with single and combined PMoAs to compare their goodness-of-fit and predicted population-level sensitivity. We identified the PMoA reproduction efficiency as most probable in all species for Ni and Zn, but not for Cu, and found that combined-PMoA models predicted higher population-level sensitivity than single-PMoA models, which was related to the predicted individual-level sensitivity, rather than to mechanistic differences between models. Using point estimates of parameters, instead of sampling from the probability distributions of parameters, could also lead to differences in the predicted population-level sensitivity. According to model predictions, apical chronic endpoints (cumulative reproduction, survival) are conservative for single-metal population effects across metals and species. We conclude that the assumption of an identical PMoA for different species of Daphnia could be justified for Ni and Zn, but not for Cu. Single-PMoA models are more appropriate than combined-PMoA models from a model selection perspective, but propagation of the associated uncertainty should be considered. More accurate predictions of effects at low concentrations may nevertheless motivate the use of combined-PMoA models. Environ Toxicol Chem 2024;43:338-358. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Daphnia magna digestive activity is differentially altered when exposed to equally turbid waters caused by either suspended sediment or suspended microplastics.

Turbidity can be a result of suspended natural particles, such as sediment, or anthropogenic particles such as microplastics. This study assessed whether Daphnia magna, a pelagic filter feeder known to ingest suspended particles, have an altered response to equally turbid environments caused by the presence of either suspended bentonite or suspended polyethylene microplastics. Compared to controls, daphnids exposed to suspended bentonite maintained their feeding efficiency and increased their digestive activity, as measured by mandibular movement, peristalsis, and expulsion, to pass bentonite through the digestive tract. The same effects were not seen in microplastic-exposed individuals, in which feeding efficiency was decreased and only peristaltic movement was increased but without a coordinated increase in expulsion, suggesting that microplastics do not have the same ability as bentonite to pass through the digestive tract. This study highlights the need to discern the identities of particulates contributing to turbid environments as different particles, even of the same size, can have different effects on filter feeders, which inherently ingest suspended particles.

Daphnia's phototaxis as an indicator in ecotoxicological studies: A review.

Animal-based sensors have been increasingly applied to many water monitoring systems and ecological studies. One of the staple organisms used as living sensors for such systems is Daphnia. This organism has been extensively studied and, with time, used in many toxicological and pharmaceutical bioassays, often used for exploring the ecology of freshwater communities. One of its behaviours used for evaluating the state of the aquatic environment is phototaxis. A disruption in the predicted behaviour is interpreted as a sign of stress and forms the basis for further investigation. However, phototaxis is a result of complex processes counteracting and interacting with each other. Predator presence, food quality, body pigmentation and other factors can greatly affect the predicted phototactic response, hampering its reliability as a bioindicator. Therefore, a holistic approach and meticulous documentation of the methods are needed for the correct interpretation of this behavioural indicator. In this review, we present the current methods used for studying phototaxis, the factors affecting it and proposed ways to optimise the reliability of the results.

Thermal dependence of Daphnia life history reveals asymmetries between key vital rates.

Temperature variation affects virtually every aspect of ectotherms' ecological performance, such as their foraging rate, reproduction, and survival. Although these changes influence what happens at higher levels of organizations, such as populations and communities, qualitative changes in dynamics usually require some degree of asymmetry between key vital rates, i.e. that different vital rates, such as growth, development, fecundity and mortality rates, respond differently to temperature. In order to identify possible asymmetries among vital rates and/or life stages, we characterized the thermal response of individuals a clone of Daphnia sinensis, drawn from a high-mountain environment in Taiwan, and examined the temperature dependence of growth, maturation, reproduction, and mortality rates, as well as fitness measures (r and R0) at eight temperatures. Daphnia sinensis was able to maintain reproductive success over a broad range of temperatures, much wider than the one experienced in its environment. However, negative effects of temperature were perceptible at temperatures much lower than the highest one at which they can achieve reproductive success. Adult mortality greatly increased for temperatures above 23 °C, and other vital rates started to decelerate, resulting in a large drop in lifetime reproductive success. This finding implies that D. sinensis may be able to persist over a wide range of temperatures, but also that it may become more sensitive to the detrimental effect of species interactions at increased temperatures. Different vital rates responded relatively similarly at low temperatures, but the degree of asymmetry among these rates was much more pronounced at higher temperatures. In particular, rates associated with adult performance decelerated more strongly than juveniles' rates. These findings indicate that elevated temperatures affect the balance between juvenile and adult performance, which is known to have a crucial role in Daphnia population dynamics. We discuss the implications of these results for the dynamics of structured populations.

Ultra-low Esfenvalerate concentrations increase biomass and may reduce competitiveness of Daphnia magna populations.

Aquatic communities are frequently exposed to pesticides at sublethal concentrations, known to affect fitness parameters such as feeding, reproduction and population growth. Beside adverse effects, beneficial responses to toxicants at low concentrations may also occur. Positive effects, however, are thought to involve trade-offs. To identify such trade-offs, we quantified the population level effects on Daphnia magna during population carrying capacity in laboratory nanocosms after exposure to a single pulse of the pyrethroid insecticide esfenvalerate, including ultra-low concentrations ≤1/30 EC50. Population abundance and biomass were monitored 3 times per week for 3 months using a non-invasive imaging technique. High concentrations ≥1/10 EC50 resulted in reduced fitness endpoints. In contrast, ultra-low concentrations in the range of 0.01 µg/L significantly increased the population abundance of small (+160 %), medium (+130 %) and large organisms (+340 %) and their combined biomass (+200 %) during the 2 months after exposure. During the first five days after exposure to 0.01 µg/L and 0.03 µg/L esfenvalerate, population biomass increased by 0.1 mg/day while staying constant in the controls. While high control mortality makes firm conclusions about population responses of D. magna to esfenvalerate difficult, we hypothesize that population increases at ultra-low concentrations may be due to a hormetic response, where reduced intraspecific competitiveness is the trade-off that enables this response.

Exposure to 3,4-methylenedioxymethamphetamine (MDMA) induced biochemical but not behavioral effects in Daphnia magna.

Among amphetamine like stimulants (ATS), the 3,4-methylenedioxymethamphetamine (MDMA) is often detected in sewage and surface waters, representing a potential threat for organisms because of its peculiar mechanism of action (i.e., stimulatory and hallucinogenic). The present study aimed at investigating biochemical (i.e., oxidative stress and energetic biomarkers) and behavioral (i.e., swimming activity) effects induced by a 21-days exposure to two concentrations (50 ng/L and 500 ng/L) of MDMA towards Daphnia magna. The amount of reactive oxygen species (ROS), the activity of antioxidant (SOD, CAT, GPx) and detoxifying (GST) enzymes and lipid peroxidation were measured as oxidative stress-related endpoints. Total energy content was estimated from the measurement of protein, carbohydrate and lipid content to assess energy reserves. The modulation of swimming activity was assessed as behavioral endpoint. Slight effects of MDMA exposure on oxidative stress responses and energy reserves were observed, while no alterations of the swimming behavior was noted.

Diet effects on longevity, heat tolerance, lipid peroxidation and mitochondrial membrane potential in Daphnia.

The dietary supply of polyunsaturated fatty acids (PUFA) crucially affects animals' performance at different temperatures. However, the underlying physiological mechanisms are still insufficiently understood. Here, we analyzed lifespan and heat tolerance of four genotypes of Daphnia magna reared on either the green alga Scenedesmus obliquus that lacks long-chain (> C18) PUFA, or the heterokont alga Nannochloropsis limnetica that contains C20 PUFA, both either at saturating and near-starvation levels. A significant genotype-by-diet interaction in lifespan was observed at saturating diets. The C20 PUFA-rich diet eliminated differences in lifespan among genotypes on the PUFA-deficient diet. Corrected for body length, acute heat tolerance was higher at low than at high food concentration, at least in the older of the two age groups analyzed. Genotypes differed significantly in heat tolerance, but there were no genotype-by-diet interactions. As predicted, the C20 PUFA-rich diet resulted in higher lipid peroxidation (LPO) and a lower mitochondrial membrane potential (ΔΨm). LPO levels averaged across clones and rearing conditions were inversely related to acute heat tolerance. Yet, heat tolerance was higher on the PUFA-rich diet than on the PUFA-deficient diet, particularly in older Daphnia, indicating that the C20 PUFA-rich diet allowed Daphnia to compensate for higher LPO. In contrast, Daphnia with intermediate levels of ΔΨm showed the lowest heat tolerance. Neither LPO nor ΔΨm explained the diet effects on lifespan. We hypothesize that antioxidants present in the PUFA-rich diet may have enabled higher heat tolerance of Daphnia despite higher LPO, which may also explain the lifespan expansion of otherwise short-lived genotypes.

Effects of triadimefon fungicide on Daphnia magna: Multigenerational effect and population-level ecological risk.

Triadimefon is ubiquitous in various environmental media. Although toxicity of triadimefon to individual of aquatic organisms has been confirmed, its effect on organisms at population level remain poorly understood. In this study the long-term effect of triadimefon on individual and population of Daphnia magna were studied using multi-generational experiments and matrix model. Development and reproduction of three generations of F1 and F2 were significantly inhibited with the triadimefon concentration of 0.1 mg/L (p < 0.01). Toxicity of triadimefon to the offspring was stronger than to the parent (p < 0.05). When triadimefon concentration was higher than 0.1 mg/L, both population number and intrinsic rate of increase showed a decreasing trend with the increasing exposure concentration. Age structure of the population also tended to decline. Toxicity threshold derived on population-level was between mortality-based LC50 and reproduction-based NOEC of Daphnia magna, and also between acute toxicity and chronic toxicity derived from species sensitivity distribution (SSD). The risk of population level derived from risk quotient was low for most areas, and the results derived from probability risk showed that the expected loss of intrinsic rate of increase of population was 0.0039 without considering other factors. Compared to the individual-level, the ecological risks at the population level were closer to the actual situation of the ecosystem response to the chemical pollution.

Microplastics enhance the toxicity and phototoxicity of UV filter avobenzone on Daphnia magna.

Microplastics (MPs) and ultraviolet (UV) filters cause pollution in aquatic ecosystems. Moreover, regulations on the use and discharge of UV filters in personal care products are lacking. Therefore, the combined toxicity of MPs (virgin polystyrene (PS) spheres; size: 200 nm) and avobenzone (AVO; a UV filter) on Daphnia magna were assessed. The exposure groups were AVO, AVO + UV irradiation for 6 h [AVO (UV)], AVO with MPs (Mix), and AVO with MPs + UV irradiation for 6 h [Mix (UV)]. The daphnids were exposed to these treatments for 48 h and observed for an additional 6 h. Energy reserves of all treated groups increased compared to that of the control group. Growth in the Mix group was inhibited despite a high food uptake, and food uptake and growth inhibition were validated in the Mix (UV) group. Additionally, the food uptake of the AVO (UV) and Mix (UV) groups decreased during the recovery period, possibly owing to a decrease in the normal feeding ability resulting from an increase in abnormality. These results indicate that the combined toxicity of MPs+AVO can be exacerbated under natural conditions; the complex toxicity should be considered when assessing aquatic environment pollution.

Novel peptide identified from viable-cell based phage display technique regulates growth cycle of Daphnia magna.

Phage display is a widely used technique for selecting specific binding peptides, but presenting antigens in their natural form can be challenging, as protein coating may induce structural changes. In this study, we employed a whole cell-based phage display technique without a coating step to select peptides that bind specifically to Daphnia magna eggs. Boiled eggs were used as a control to ensure that antigens were presented in their natural forms. We identified a peptide, DEP1 (LYALPLSHLKSHGGG), with the highest binding affinity to D. magna eggs. DEP1 did not affect zebrafish eggs, but it inhibited normal hatching and reproductive ability in D. magna eggs, and hindered growth in neonates before their first ecdysis. Morphological analysis revealed that DEP1 caused intestinal damage and tissue abnormalities. Our findings demonstrate that the whole cell-based phage display technique is successful in presenting antigens in their natural form, and that the DEP1 peptide can be applied to regulate the growth cycle of D. magna. These results have implications for the use of phage display in environmental research and the potential use of DEP1 for hazardous organisms in aquatic systems.

Chronic toxicity of broflanilide in Daphnia magna: changes in molting, behavior, and gene expression.

Broflanilide is a novel pesticide used in agriculture that binds to unique receptors on pests; however, the widespread use of broflanilide has led to toxicity in Daphnia magna. At present, little information on the potential threats broflanilide imposes on D. magna is available. Therefore, the present study examined the chronic toxicity of broflanilide in D. magna by comparing changes in molting, neurotransmitter function, and behavior. The results showed that broflanilide caused chronic toxicity in D. magna at a concentration of 8.45 µg/L, and growth, development, reproduction, and the development of offspring were affected. In addition, broflanilide affected the molting of D. magna by significantly inhibiting the expression of chitinase, ecdysteroid, and related genes. Broflanilide also affected the expression of γ-glutamic acid, glutamine, gamma-aminobutyric acid, 5-hydroxytryptamine, 5-hydroxytryptophan, dopa, and dopamine. Furthermore, the swimming distance and speed of D. magna were reduced. Taken together, the results demonstrate the chronic toxicity and exposure risk of broflanilide in D. magna.

Accumulation Kinetics and Gut Microenvironment Responses to Environmentally Relevant Doses of Micro/Nanoplastics by Zooplankton Daphnia Magna.

Microplastics are emerging pollutants that have been widely reported in aquatic ecosystems. Based on the analysis of environmentally relevant concentrations of microplastics in global freshwater systems, herein, we employed aggregated-induced emission (AIE) microplastic fluorogens and imaged and quantified the bioaccumulation of differentially charged micro- (20 µm)/nano- (200 nm) plastics (MNPs) in zooplankton Daphnia magna. We found that all particles of different sizes and charges were readily ingested, especially larger-sized and positively charged MNPs, with over 50% of the ingested particles accumulating in the gut. Bioaccumulation of MNPs reached 50% of steady-state condition within 1 h. The presence of algae inhibited the ingestion and depuration of MNPs. To further demonstrate the effects of such accumulation on gut health, we further applied the AIE probes for visualizing the pH and esterase in the digestive tract, as well as the gut inflammation. An accumulation of MNPs in D. magna significantly and rapidly induced the acidification of gut pH while inducing esterase activity. The NPs apparently induced gut inflammation in contrast to the MPs, demonstrating the size-dependent effects on oxidative stress. Our results highlighted that MNP exposure at environmentally relevant concentrations perturbed the microenvironments of zooplankton guts, which may significantly affect their digestion and assimilation of food materials as well as contaminant uptake.

Toxicity evaluation of the contaminated area of Crotone from biological indicators: a multispecies approach.

Contamination of terrestrial and aquatic ecosystems by toxic industrial waste has become a major issue in many countries. Of particular concern is the reuse of toxic hazardous waste in construction materials. This paper examined for the first time the chemical and radiation ecotoxicity of site-specific Technological Enhanced Naturally Occurring Radioactive Materials (TENORM) residues from phosphate processing industry in soil environmental matrices through bioindicators. The area under investigation was the former industrial district of Crotone (Calabria, Italy), recently included within the Sites of National Interest (SIN), comprising the 42 Italian national priority contaminated sites. Major biological exposure pathways considered were absorption and bioaccumulation. The marine bacterium Vibrio fischeri and the freshwater crustacean Daphnia magna were employed as aquatic bioindicators, while for the soil ecosystem, the seeds of Sorghum saccharatum and Lepidium sativum were used. Selection of test species aimed at assessing the toxicity of wastes in soil as well as in freshwater or marine systems. Results indicated V. fischeri as the most sensitive of all the species tested (5.56 g/L), while D. magna was found to be affected at 94.27 g/L. An overall inhibition was observed in seedling growth as compared to control at the highest concentration of the pollutants (100 g/L), while seed germination was not adversely affected by the pollutant. At this preliminary level, data indicated a potential risk for biodiversity of the area. In fact, the measured toxicity thresholds, even if above 100 mg/L, are comparable to concentrations of the toxicants spread all over the territory of Crotone.

Combined methods elucidate the multi-organ toxicity of cylindrospermopsin (CYN) on Daphnia magna.

Global water bodies are now at risk from inevitable cyanobacterial blooms and their production of multiple cyanotoxins, in particular cylindrospermopsin (CYN). However, research on the CYN toxicity and its molecular mechanisms is still limited, whilst the responses of aquatic species against CYN are uncovered. By integrating behavioral observations, chemical detections and transcriptome analysis, this study demonstrated that CYN exerted multi-organ toxicity to model species, Daphnia magna. The present study confirmed that CYN could cause protein inhibition by undermining total protein contents, and altered the gene expression related to proteolysis. Meantime, CYN induced oxidative stress by increasing reactive oxygen species (ROS) level, decreasing the glutathione (GSH) concentration, and interfered with protoheme formation process molecularly. Neurotoxicity led by CYN was solidly determined by abnormal swimming patterns, reduced acetylcholinesterase (AChE), and downward expression of muscarinic acetylcholine receptor (CHRM). Importantly, for the first time, this research determined CYN directly interfered with energy metabolism in cladocerans. CYN distinctively reduced filtration and ingestion rate by targeting on heart and thoracic limbs, which declined the energy intake, and could be further displayed by the reduction of motional strength and the trypsin concentration. These phenotypic alterations were supported by transcriptomic profile, including the down-regulation of oxidative phosphorylation and ATP synthesis. Moreover, CYN was speculated to trigger the self-defense responses of D. magna, known as "abandon-ship" by moderating lipid metabolism and distribution. This study, overall, comprehensively demonstrated the CYN toxicity and the responses of D. magna against it, which is of great significance to the advancements of CYN toxicity knowledge.