Chemical tolerance related to the ABC transporter gene and DNA methylation in cladocera (Daphnia magna).

We performed multigenerational tests to clarify the chemical tolerance mechanisms of a nontarget aquatic organism, Daphnia magna. We continuously exposed D. magna to a carbamate insecticide (pirimicarb) at lethal or sublethal concentrations (0, 3.8, 7.5, and 15 µg/L) for 15 generations (F0-F14). We then determined the 48 h-EC50 values and mRNA expression levels of acetylcholinesterase, glutathione S-transferase, and ATP (Adenosine triphosphate)-binding cassette transporter (ABCt) in neonates (<24 h old) from F0, F4, F9, and F14. To ascertain the effects of DNA methylation on pirimicarb sensitivity, we measured 5-methylcytosine levels (DNA methylation levels) in neonates of parents in the last generation (F14). In addition, we cultured groups exposed to 0 and 7.5 µg/L (the latter of which acquired chemical tolerance to pirimicarb) with or without 5-azacytidine (de-methylating agent) and determined methylation levels and 48 h-EC50 values in neonates (<24 h old) from the treated parents. The EC50 values (30.3-31.6 µg/L) in F14 of the 7.5 and 15 µg/L groups were approximately two times higher than that in the control (16.0 µg/L). A linear mixed model analysis showed that EC50 and ABCt mRNA levels were significantly increased with generational alterations; further analysis showed that the ABCt mRNA level was positively related to the EC50 . Therefore, ABCt may be associated with altered pirimicarb sensitivity. In addition, the EC50 value and DNA methylation levels in pirimicarb-tolerant clones decreased after exposure to 5-azacytidine, suggesting that DNA methylation contributes to chemical tolerance. These findings improved our knowledge regarding the acquisition of chemical tolerance in aquatic organisms.

Insights into the Activation of a Crustacean G Protein-Coupled Receptor: Evaluation of the Red Pigment-Concentrating Hormone Receptor of the Water Flea Daphnia pulex (Dappu-RPCH R).

The validation of a previously developed model of the interaction between the red pigment-concentrating hormone of Daphnia pulex and its cognate receptor (Jackson et al., IJBM 106, 969-978, 2018) was undertaken. Single amino acid replacements, noticeably an Ala scan, of the ligand, Dappu-RPCH, were docked to the receptor, and the binding energies calculated and compared to the one with Dappu-RPCH. As a second step, the same molecules were docked using molecular dynamics (MD) in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane. Changes in binding energy were compared to previous results on in vitro receptor activation (Marco et al., Sci. Rep. 7, 6851, 2017). Residue scanning and MD simulations both gave comparable results for binding energy. For most mutants, there was a good inverse correlation between in vitro activity and binding. There were, however, exceptions; for example: [Ala4]Dappu-RPCH bound as tightly as the cognate ligand but had little activity. This seeming discrepancy was explained when the MD data were analyzed in detail, showing that, although [Ala4]Dappu-RPCH had multiple interactions with the receptor accounting for the high binding energy, the interacting residues of the receptor were quite different to those of Dappu-RPCH. The MD calculations show clearly that the strong binding affinity of the ligand to the receptor is not sufficient for activation. Interaction of the binding of the ligand to two residues of the receptor, Ser 155 and Gln 237, is also essential. A comparison of our computational results with the experimental results of Marco et al. and comparison with the extensive data on GnRH supports the validity of our Dappu-RPCH R model.

The genome of the marine water flea Diaphanosoma celebensis: Identification of phase I, II, and III detoxification genes and potential applications in marine molecular ecotoxicology.

To assemble the genome of the marine water flea Diaphanosoma celebensis, a sentinel model for marine environmental monitoring, we constructed a high-quality genome using PromethION and HiSeq 2500 platforms. The total length of the assembled genome was 100.08 Mb, with N50 = 2.56 Mb (benchmarking universal single-copy orthologs, 96.9%) and consisted of 179 scaffolds. A total of 15,427 genes were annotated, and orthologous gene clusters in D. celebensis were analyzed and compared with those of the cladocerans Daphnia magna and Daphnia pulex. In addition, phase I, II, and III detoxification gene families of cytochrome P450s, glutathione S-transferases, and ATP-binding cassette were fully identified and revealed lineage-specific gene loss and/or expansion, suggesting that the evolution of detoxification gene families likely modulates fitness and susceptibility in response to environmental stressors. The study improves our understanding of the detoxification-related gene system and should contribute to future studies of molecular ecotoxicology in cladoceran species and their responses to emerging pollutants.

Combined effects of heavy metals (Cd, As, and Pb): Comparative study using conceptual models and the antioxidant responses in the brackish water flea.

The combined effect of toxic inducers has emerged as a challenging topic, particularly due to their inconsistent impacts on the environment. Using toxic unit (TU) based on LC50 value, we investigated the 48 h acute toxicities of the following combinations: Cd + As, Cd + Pb, As + Pb, and Cd + As + Pb, and binary and ternary combined effects were interpreted using concentration addition (CA) and independent action (IA) model. The molecular effects of these combinations were further examined on the basis of gene expression (four GST and two SOD isoforms) and antioxidant enzymes activity (SOD and GST). The CA-predicted LC50 was similar to the observed results, indicating that the CA model is more applicable for evaluating the combined effects of the metal mixtures. Synergistic effects (ΣTULC50 < 0.8) were observed for the mixtures As + Pb and Cd + Pb, while additive effects (0.8 < ΣTULC50 < 1.2) were observed for the mixtures Cd + As + Pb and Cd + As. No antagonistic effects were observed in this study. Molecular biomarkers for oxidative stress caused by metals, as well as traditional endpoints such as lethality, have shown a clear response in assessing the toxicity of binary and ternary mixtures. This study opens up a new avenue for the use of biomarkers to assess the combined effects of metals in aquatic environments.

Oxidative stress responses in brackish water flea exposed to microcystin-LR and algal bloom waters from Nakdong River, Republic of Korea.

Microcystis blooms and the impact of their toxins, particularly microcystin (MC), in coastal ecosystems is an emerging threat, but the species-specific effects of MC and the potential for bioconcentration are not fully understood. We exposed the brackish water flea, Diaphanosoma celebensis, to MC-LR, which showed antioxidant responses measured at the molecular to enzyme levels but no acute toxicity. We extended our experimental investigation to measure the released MC and its uptake by D. celebensis exposed to river water. In a short-term exposure (48 h) experiment, D. celebensis exposed to water from an algal bloom (approximately 2 µg L-1 MC) assimilated more than 50 pg MC per individual. The significant increase of MCs suggests the potential for the species to accumulate MCs. The dose-dependent increase in the antioxidant response observed in the mRNA levels also showed that D. celebensis exposed to diluted algal bloom waters were affected by toxins from cyanobacteria.

Influence of Dissolved Organic Carbon on the Acute Toxicity of Copper and Zinc to White Sturgeon (Acipenser transmontanus) and a Cladoceran (Ceriodaphnia dubia).

We conducted acute lethality tests with white sturgeon (Acipenser transmontanus) and Ceriodaphnia dubia exposed to copper and zinc at dissolved organic carbon concentrations ranging from 0.5 to 5.5 mg/L. Dissolved organic carbon had minimal effects on zinc toxicity but did have a protective effect on acute copper toxicity, which was equal to that predicted by the copper biotic ligand model (BLM). The BLM-adjusted copper median effect concentrations for A. transmontanus ranged from 2.4 to 8.2 mg/L. Environ Toxicol Chem 2019;38:2682-2687. Published 2019 Wiley Periodicals, Inc. on behalf of SETAC. This article is a US government work, and as such, is in the public domain in the United States of America.

Relationships between zooplankton biomass and environmental factors of Xiaoxingkai Lake in northeastern China.

Zooplankton biomass and water environment factors in Xiaoxingkai Lake were investigated, and the correlation between biomass and water environment factors was performed using the Pearson correlation analysis and redundancy analysis (RDA). The results showed that the highest zooplankton biomass was recorded in summer and the lowest in spring. Water depth, transparency (SD), electric conductivity, and total nitrogen were the highest in spring, while temperature and total phosphorus were the highest in summer. The values of pH and turbidity were the highest in autumn, while chloride ion and chlorophyll a were the highest in winter. During the spring period, Protozoa were positively correlated with conductivity, transparency (SD), and total nitrogen. While Rotifera demonstrated a strong correlation with turbidity, pH, temperature, and total phosphorus in summer and autumn seasons, and Cladocera were correlated with water depth. There were negative correlations of chlorophyll a and chlorine on Copepoda during the winter. RDA results displayed that zooplankton had strong relationships with the physicochemical characteristics in Xiaoxingkai Lake.

Acute toxicity of bisphenol A and its structural analogues and transcriptional modulation of the ecdysone-mediated pathway in the brackish water flea Diaphanosoma celebensis.

Bisphenol A (BPA) is a representative endocrine disrupting chemical (EDC) that has estrogenic effects in aquatic animals. In recent years, due to the continuing usage of BPA, its analogues have been developed as alternative substances to replace its use. The molting process is a pivotal point in the development and reproduction of crustaceans. However, studies of the effects of EDCs on molting in crustaceans at the molecular level are scarce. In the present study, we examined the acute toxicity of BPA and its analogues bisphenol F (BPF) and S (BPS) to the brackish water flea Diaphanosoma celebensis. We further identified four ecdysteroid pathway - related genes (cyp314a1, EcRA, EcRB, and USP) in D. celebensis, and investigated the transcriptional modulation of these genes during molting and after exposure to BPA and its analogues for 48 h. Sequencing and phylogenetic analyses revealed that these four genes are highly conserved among arthropods and may be involved in development and reproduction in the adult stage. The mRNA expression patterns of cyp314a1, EcRA and USP were matched with the molting cycle, suggesting that these genes play a role in the molting process in the adult stage in cladocerans. Following relative real-time polymerase chain reaction (RT-PCR) analyses, BPA and its analogues were found to modulate the expression of each of these four genes differently, indicating that these compounds can disrupt the normal endocrine system function of D. celebensis. This study improves our understanding of the molecular mode of action of BPA and its analogues in D. celebensis.

Determination of Bioavailable Aluminum in Natural Waters in the Presence of Suspended Solids.

Analyses of natural waters frequently show elevated levels of total aluminum (Al) attributable to acid extraction of Al from the total suspended solids (TSS) minerals. Hence, there is a need for an analytical method that measures only bioavailable Al. Natural waters high in TSS were collected to study the chronic effects of Al on Ceriodaphnia dubia. In the collected waters TSS ranged from 30 to 411 mg/L; total Al concentrations ranged from 2.0 to 44.8 mg/L. The TSS in natural waters inhibited reproduction of C. dubia up to 40% in comparison to the same filtered waters. This inhibition did not correlate with the concentration of TSS or total Al; it was attributed to nutritional deficiency and was prevented by increasing the food supply. To demonstrate that toxicity can be measured in natural waters, samples with elevated TSS were spiked with soluble Al, and survival and reproduction were measured in chronic studies performed at pH 6.3 and 8.0. To properly characterize the Al concentrations in the toxicity studies, a method was needed that could discriminate bioavailable Al from mineral forms of Al. An extraction method at pH 4 for bioavailable Al was developed and evaluated using C. dubia chronic toxicity studies in the presence of TSS. It is concluded that the proposed method is better able to discriminate chronic toxicity effects attributable to bioavailable Al from mineralized nontoxic forms of Al compared with existing methods using total or total recoverable Al (i.e., extraction at pH ≤ 1.5). We propose that this new method be used when assessing the potential for Al in natural surface waters to cause toxicity. Environ Toxicol Chem 2019;38:1668-1681. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Energy budget in Alona guttata (Chydoridae: Aloninae) and toxicant-induced alterations.

Although chydorids are the most diverse cladocerans in the world, there is still little information available related to their biology and even less with respect to their susceptibility to toxicants. Therefore, this work aimed to implement protocols with Alona guttata for acute, chronic, and sublethal toxicity tests, using the environmental concern toxicants deltamethrin (DM) and lead (Pb2+), which are commonly used due to agriculture and vector control or for the automotive industry, respectively. Once the results of LC50 (0.1160 ± 0.0107 µg/L for DM and 1.5797 ± 0.1605 mg/L for Pb2+) were obtained, sublethal concentrations (0.01 to 0.2 LC50) were used for the evaluation of biomarkers and chronic toxicity. Concentrations as low as 0.01 LC50 reduced Alona's survival and fecundity, negatively affecting demographic parameters, and decreased the energy reserves. A significant correlation was found between the natural rate of population increase and the caloric content, which demonstrates the suitability of these biomarkers as endpoints of early warning that allow inferring alterations at higher biological levels. Subsequently, this work could constitute the first report on the evaluation of the energy budget in a non-daphnid species, its alterations due to exposure to toxic substances and the correlation with demographic responses.

Eco-Evolutionary Dynamics of Ecological Stoichiometry in Plankton Communities.

Nitrogen (N) and phosphorus (P) limit primary production in many aquatic ecosystems, with major implications for ecological interactions in plankton communities. Yet it remains unclear how evolution may affect the N∶P stoichiometry of phytoplankton-zooplankton interactions. Here, we address this issue by analyzing an eco-evolutionary model of phytoplankton-zooplankton interactions with explicit nitrogen and phosphorus dynamics. In our model, investment of phytoplankton in nitrogen versus phosphorus uptake is an evolving trait, and zooplankton display selectivity for phytoplankton with N∶P ratios matching their nutritional requirements. We use this model to explore implications of the contrasting N∶P requirements of copepods versus cladocerans. The model predicts that selective zooplankton strongly affect the N∶P ratio of phytoplankton, resulting in deviations from their optimum N∶P ratio. Specifically, selective grazing by nitrogen-demanding copepods favors dominance of phytoplankton with low N∶P ratios, whereas phosphorus-demanding cladocerans favor dominance of phytoplankton with high N∶P ratios. Interestingly, selective grazing by nutritionally balanced zooplankton leads to the occurrence of alternative stable states, where phytoplankton may evolve either low, optimum, or high N∶P ratios, depending on the initial conditions. These results offer a new perspective on commonly observed differences in N∶P stoichiometry between plankton of freshwater and those of marine ecosystems and indicate that selective grazing by zooplankton can have a major impact on the stoichiometric composition of phytoplankton.

The Effects of Sediment Classification Pattern on a Water Column Organism, Ceriodaphnia dubia.

The sediment compartment stands out because it functions as both a temporary sink of pollutants and a potential source of these elements that may become available to the water column.This study aimed to correlate the concentrations of total metals in the crude sediment and in the interstitial water with the ecotoxicity in the water column using an a modified sediment ecotoxicity test with Ceriodaphnia dubia. The results indicate that the sediment may contribute to the toxicity in the water column and that such toxicity is possibly not related to the metals present. Based on the chemical analysis of the metals, the Canadian Sediment Quality Guidelines (SQGs) would frame the sediment as non-toxic to benthic organisms, but the SQGs have no reference standards for possible effects on nektonic organisms. Due to the complexity of this compartment, it is fundamental to evaluate the interactions of the different pollutants in the system and possible effects on the nektonic organisms.

The acute toxicity of major ion salts to Ceriodaphnia dubia. III. Mathematical models for mixture toxicity.

Based on previous research on the acute toxicity of major ions (Na+ , K+ , Ca2+ , Mg2+ , Cl- , SO42- , and HCO3- /CO32- ) to Ceriodaphnia dubia, a mathematical model was developed for predicting the median lethal concentration (LC50) for any ion mixture, excepting those dominated by K-specific toxicity. One component of the model describes a mechanism of general ion toxicity to which all ions contribute and predicts LC50s as a function of osmolarity and Ca activity. The other component describes Mg/Ca-specific toxicity to apply when such toxicity exceeds the general ion toxicity and predicts LC50s as a function of Mg and Ca activities. This model not only tracks well the observed LC50s from past research used for model development but also successfully predicts LC50s from new toxicity tests on synthetic mixtures of ions emulating chemistries of various ion-enriched effluents and receiving waters. It also performs better than a previously published model for major ion toxicity. Because of the complexities of estimating chemical activities and osmolarity, a simplified model based directly on ion concentrations was also developed and found to provide useful predictions. Environ Toxicol Chem 2018;37:247-259. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Single and combined effects of microcystin- and saxitoxin-producing cyanobacteria on the fitness and antioxidant defenses of cladocerans.

Cyanobacteria produce different toxic compounds that affect animal life, among them hepatotoxins and neurotoxins. Because cyanobacteria are able to produce a variety of toxic compounds at the same time, organisms may be, generally, subjected to their combined action. In the present study, we demonstrate the single and combined effects on cladocerans of cyanobacteria that produce microcystins (hepatotoxins) and saxitoxins (neurotoxins). Animals were exposed (either singly or combined) to 2 strains of cyanobacteria isolated from the same environment (Funil Reservoir, Rio de Janeiro, Brazil). The effects on clearance rate, mobility, survivorship, fecundity, population increase rate (r), and the antioxidant enzymes glutathione-S-transferase (GST) and catalase (CAT) were measured. Cladoceran species showed a variety of responses to cyanobacterial exposures, going from no effect to impairment of swimming movement, lower survivorship, fecundity, and general fitness (r). Animals ingested cyanobacteria in all treatments, although at lower rates than good food (green algae). Antioxidant defense responses were in accordance with fitness responses, suggesting that oxidative stress may be related to such effects. The present study emphasizes the need for testing combined actions of different classes of toxins, because this is often, and most likely, the scenario in a more eutrophic world with global climatic changes. Environ Toxicol Chem 2017;36:2689-2697. © 2017 SETAC.

Development and validation of a metal mixture bioavailability model (MMBM) to predict chronic toxicity of Ni-Zn-Pb mixtures to Ceriodaphnia dubia.

Recently, several bioavailability-based models have been shown to predict acute metal mixture toxicity with reasonable accuracy. However, the application of such models to chronic mixture toxicity is less well established. Therefore, we developed in the present study a chronic metal mixture bioavailability model (MMBM) by combining the existing chronic daphnid bioavailability models for Ni, Zn, and Pb with the independent action (IA) model, assuming strict non-interaction between the metals for binding at the metal-specific biotic ligand sites. To evaluate the predictive capacity of the MMBM, chronic (7d) reproductive toxicity of Ni-Zn-Pb mixtures to Ceriodaphnia dubia was investigated in four different natural waters (pH range: 7-8; Ca range: 1-2 mM; Dissolved Organic Carbon range: 5-12 mg/L). In each water, mixture toxicity was investigated at equitoxic metal concentration ratios as well as at environmental (i.e. realistic) metal concentration ratios. Statistical analysis of mixture effects revealed that observed interactive effects depended on the metal concentration ratio investigated when evaluated relative to the concentration addition (CA) model, but not when evaluated relative to the IA model. This indicates that interactive effects observed in an equitoxic experimental design cannot always be simply extrapolated to environmentally realistic exposure situations. Generally, the IA model predicted Ni-Zn-Pb mixture toxicity more accurately than the CA model. Overall, the MMBM predicted Ni-Zn-Pb mixture toxicity (expressed as % reproductive inhibition relative to a control) in 85% of the treatments with less than 20% error. Moreover, the MMBM predicted chronic toxicity of the ternary Ni-Zn-Pb mixture at least equally accurately as the toxicity of the individual metal treatments (RMSEMix = 16; RMSEZn only = 18; RMSENi only = 17; RMSEPb only = 23). Based on the present study, we believe MMBMs can be a promising tool to account for the effects of water chemistry on metal mixture toxicity during chronic exposure and could be used in metal risk assessment frameworks.

Microbial Food-Web Drivers in Tropical Reservoirs.

Element cycling in aquatic systems is driven chiefly by planktonic processes, and the structure of the planktonic food web determines the efficiency of carbon transfer through trophic levels. However, few studies have comprehensively evaluated all planktonic food-web components in tropical regions. The aim of this study was to unravel the top-down controls (metazooplankton community structure), bottom-up controls (resource availability), and hydrologic (water residence time) and physical (temperature) variables that affect different components of the microbial food web (MFW) carbon stock in tropical reservoirs, through structural equation models (SEM). We conducted a field study in four deep Brazilian reservoirs (Balbina, Tucuruí, Três Marias, and Funil) with different trophic states (oligo-, meso-, and eutrophic). We found evidence of a high contribution of the MFW (up to 50% of total planktonic carbon), especially in the less-eutrophic reservoirs (Balbina and Tucuruí). Bottom-up and top-down effects assessed through SEM indicated negative interactions between soluble reactive phosphorus and phototrophic picoplankton (PPP), dissolved inorganic nitrogen, and heterotrophic nanoflagellates (HNF). Copepods positively affected ciliates, and cladocerans positively affected heterotrophic bacteria (HB) and PPP. Higher copepod/cladoceran ratios and an indirect positive effect of copepods on HB might strengthen HB-HNF coupling. We also found low values for the degree of uncoupling (D) and a low HNF/HB ratio compared with literature data (mostly from temperate regions). This study demonstrates the importance of evaluating the whole size spectrum (including microbial compartments) of the different planktonic compartments, in order to capture the complex carbon dynamics of tropical aquatic ecosystems.

Diffusive Milli-Gels (DMG) for in situ assessment of metal bioavailability: A comparison with labile metal measurement using Chelex columns and acute toxicity to Ceriodaphnia dubia for copper in freshwaters.

Fluctuations in concentrations of bioavailable metals occur in most natural waters. In situ measurements are desirable to predict risks of adverse effects to aquatic organisms. We evaluated Diffusive Milli-Gels (DMG), a new in situ passive sampler, for assessing the bioavailability and toxicity of copper in waters exhibiting a wide range of characteristics. The performance was compared to an established Chelex-column method that measures labile copper concentrations by discrete sampling, and the ability to predict acute toxicity to the cladoceran, Ceriodaphnia dubia. The labile copper concentrations measured by the DMG and Chelex-column methods decreased with increasing dissolved organic carbon (DOC) (1.9-15 mg L-1) and hardness (21-270 mg CaCO3 L-1 hardness), with 20-70% of total dissolved copper being present as labile copper. Toxicity decreased with increasing DOC and hardness. Strong linear relationships existed between the EC50 for C. dubia and DOC, and when the EC50 was related to either the labile copper concentrations measured by DMG (r2 = 0.874) or the Chelex column (0.956) methods. The study demonstrates that the DMG passive sampler is a relevant tool for the in situ assessment of environmental risks posed by metals whose toxicity is strongly influenced by speciation.

Acute toxicity and accumulation of ZnO NPs in Ceriodaphnia dubia: Relative contributions of dissolved ions and particles.

Although the ecotoxicological effects of various metal oxide nanoparticles on aquatic organisms are being actively studied, the contributions of particles and dissolved ions towards toxicity are still not well understood. The current study aims to assess the contribution of ZnO NP(particle) and ZnO NP(ion) to the overall toxicity and accumulation of ZnO NP(total) in Ceriodaphnia dubia. The aggregation and dissolution kinetics were studied for three different sizes (50nm, 100nm and bulk) of ZnO particles at 0.05, 0.12, 0.25 and 0.5mg/L concentrations in the sterile lake water medium at 6, 12, 24, and 48h intervals. The 48h LC50 of ZnO NP(total) was found to be 0.431, 0.605 and 0.701mg/L for 50, 100nm and bulk particles exposure. However, LC50 of Zn(ion) was found to be 1.048, 1.343 and 2.046mg/L for dissolved ions from different sizes (50nm, 100nm, and bulk) of ZnO particles. At LC50 concentration, the accumulation of 90-95% was noted for the NP(particles) across the sizes employed, while only about 4-5% contribution was from the NP(ion) to the overall accumulation NP(total). The relative contribution of ZnO NP(ion) to overall toxicity and accumulation was found to be lesser than that of ZnO NP(particles) across the sizes used in the study.

NMDA receptor activation upstream of methyl farnesoate signaling for short day-induced male offspring production in the water flea, Daphnia pulex.

BACKGROUND: The cladoceran crustacean Daphnia pulex produces female offspring by parthenogenesis under favorable conditions, but in response to various unfavorable external stimuli, it produces male offspring (environmental sex determination: ESD). We recently established an innovative system for ESD studies using D. pulex WTN6 strain, in which the sex of the offspring can be controlled simply by changes in the photoperiod: the long-day and short-day conditions can induce female and male offspring, respectively. Taking advantage of this system, we demonstrated that de novo methyl farnesoate (MF) synthesis is necessary for male offspring production. These results indicate the key role of innate MF signaling as a conductor between external environmental stimuli and the endogenous male developmental pathway. Despite these findings, the molecular mechanisms underlying up- and downstream signaling of MF have not yet been well elucidated in D. pulex. RESULTS: To elucidate up- and downstream events of MF signaling during sex determination processes, we compared the transcriptomes of daphnids reared under the long-day (female) condition with short-day (male) and MF-treated (male) conditions. We found that genes involved in ionotropic glutamate receptors, known to mediate the vast majority of excitatory neurotransmitting processes in various organisms, were significantly activated in daphnids by the short-day condition but not by MF treatment. Administration of specific agonists and antagonists, especially for the N-methyl-D-aspartic acid (NMDA) receptor, strongly increased or decreased, respectively, the proportion of male-producing mothers. Moreover, we also identified genes responsible for male production (e.g., protein kinase C pathway-related genes). Such genes were generally shared between the short-day reared and MF-treated daphnids. CONCLUSIONS: We identified several candidate genes regulating ESD which strongly suggests that these genes may be essential factors for male offspring production as an upstream regulator of MF signaling in D. pulex. This study provides new insight into the fundamental mechanisms underlying how living organisms alter their phenotypes in response to various external environments.

Microcystin accumulation in cladocerans: first evidence of MC uptake from aqueous extracts of a natural bloom sample.

Bioaccumulation of microcystins (MC) in zooplankton has been shown in several studies, mainly in field samples. A few studies, however, have demonstrated MC bioaccumulation in laboratory experiments. Although ingestion of cell-bound MC is considered the main route of MC accumulation, another important source is the MC from the dissolved fraction (DMC). This study reports the accumulation of DMC from aqueous extracts of natural bloom samples in three cladoceran species: Moina micrura, Daphnia laevis and Daphnia similis. Animals were exposed for 96 h to aqueous extracts of lyophilized matter from two bloom samples from Colombian reservoirs in different concentrations (25-1000 mg DW L(-1)). Analysis by HPLC-MS detected MC-LR in these samples at concentrations of 434-538 µg g(-1). For the analysis of MC in animal tissues the samples were homogenized and sonicated in methanol:water (75%) and analyzed by ELISA. Results showed that the animals uptake of MC increased with increasing exposure concentrations of aqueous extracts, with M. micrura and D. laevis clones presenting the highest MC concentrations in their tissues (up to 1170-1260 µg g(-1)) while D. similis the lowest (184 µg g(-1)). This study shows, for the first time, that MC uptake from dissolved fraction by zooplankton is possible, not only from the ingestion of seston or cell-bound MC as previously demonstrated.