Crystal Structure of H227A Mutant of Arginine Kinase in Daphnia magna Suggests the Importance of Its Stability.

Arginine kinase (AK) plays a crucial role in the survival of Daphnia magna, a water flea and a common planktonic invertebrate sensitive to water pollution, owing to the production of bioenergy. AK from D. magna (DmAK) has four highly conserved histidine residues, namely, H90, H227, H284, and H315 in the amino acid sequence. In contrast to DmAK WT (wild type), the enzyme activity of the H227A mutant decreases by 18%. To identify the structure-function relationship of this H227A mutant enzyme, the crystal 3D X-ray structure has been determined and an unfolding assay using anilino-1-naphthalenesulfonic acid (ANS) fluorescence has been undertaken. The results revealed that when compared to the DmAK WT, the hydrogen bonding between H227 and A135 was broken in the H227A crystal structure. This suggests that H227 residue, closed to the arginine binding site, plays an important role in maintaining the structural stability and maximizing the enzyme activity through hydrogen bonding with the backbone oxygen of A135.

Evaluation of boscalid toxicity on Daphnia magna by using antioxidant enzyme activities, the expression of genes related to antioxidant and detoxification systems, and life-history parameters.

Boscalid is a succinate dehydrogenase inhibitor fungicide commonly used to control a range of plant pathogens. Although it is one of the most common fungicides in the aquatic environment, the potential adverse effects of boscalid on freshwater invertebrates still remain unclear. This study aimed to evaluate the toxicity of boscalid on Daphnia magna (D. magna) and provide new information to assess the eco-toxicity of the boscalid on aquatic invertebrates. The effects of boscalid on malondialdehyde (MDA) level, activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) and the mRNA level of genes associated with antioxidant system (sod, cat, and gst) and detoxification (cytochrome P450 4 (cyp4) and nuclear respiratory factor 1 (nrf1)) were determined after 48 h treatment. The effect of boscalid on reproduction and development of D. magna was evaluated by a 21-d-chronic toxicity test. Boscalid dose-dependently altered activities of SOD, CAT, and GST and led to lipid peroxidation during acute exposure in D. magna. Exposure to 5 and 10 mg/L boscalid also significantly decreased gene expression of sod, gst, cyp4 and nrf1 but increased cat gene expression. Furthermore, chronic toxicity results showed that exposure to boscalid decreased molting frequency, number of neonates per Daphnia, and the number of broods per female as compared to the control groups. The above results indicated that boscalid had significant negative impacts on D. magna, and information present here helps to evaluate the eco-toxicity caused by boscalid on aquatic invertebrates.

Positive selection of digestive proteases in Daphnia: A mechanism for local adaptation to cyanobacterial protease inhibitors.

Due to the combined effects of global warming and eutrophication, the frequency of deleterious cyanobacterial blooms in freshwater ecosystems has increased. In line with this, local adaptation of the aquatic keystone herbivore Daphnia to cyanobacteria has received major attention. Besides microcystins, the most frequent cyanobacterial secondary metabolites in such blooms are protease inhibitors (PIs). Recently, it has been shown that a protease gene showed copy number variation between four D. magna populations that differed in tolerance to PIs. From that study, we chose two distinct populations of D. magna which had or had not coexisted with cyanobacteria in the past. By calculating FST values, we found that the two populations were genetically more distant in the protease loci than in neutral loci. Population genetic tests applied to the tolerant population revealed that positive selection was most probably acting on the gene loci of the digestive protease CT448 and CT802. We conclude that the selection of digestive proteases and subsequent reduction in copy number is the molecular basis of evolutionary changes leading to local adaptation to PIs.

A comparative study of acetylcholinesterase and general-esterase activity assays using different substrates, in vitro and in vivo exposures and model organisms.

Acetylcholinesterase (AChE) and general-esterase (GE) activities are important to understand detoxification processes of xenobiotics. The assays to quantify them have employed different substrates, inhibitors, types of experiments (in vitro and in vivo) and model organisms. The aim of this work was to give a systematic overview of the effect of the above factors on the outcome of AChE and GE activity measurements. We showed that AChE activity could be measured with the substrate acetylthiocholine iodide (AChI) but not with acetylcholine bromide (AChB) and only in in vitro assays. For GE activity, Michaelis-Menten kinetics differed between the substrates 4-methylumbellifery butyrate (4-MUB) and 1-naphtyl acetate (1-NA) in the measurements of in vitro activity, but their inhibition curves and IC50 values for the general inhibitor tetraisopropyl pyrophosphoramide (iso-OMPA) were similar, confirming that both substrates targeted the same group of enzymes. The GE substrate 4-MUB was applicable both in vitro and in vivo, while 1-NA was only applicable in vitro due to its high acute toxicity. When comparing the zooplankton crustacean Daphnia magna and the sediment dwelling Chironomus riparius, the latter had a four-fold higher maximal AChE activity (Vmax) and a higher susceptibility to the AChE inhibitor BW284c51 (four-fold lower 50% inhibitory concentration, IC50), but a lower maximal GE activity and lower susceptibility to iso-OMPA (higher IC50), indicating significant species differences between in C. riparius and D. magna. We conclude that both choice of substrate and exposure method matters for the outcome of esterase assays and that esterase compositions between species may vary significantly.

Multilevel ecotoxicity assessment of environmentally relevant bisphenol F concentrations in Daphnia magna.

With the pressure to ban or limit the use of Bisphenol A (BPA), substitutes such as bisphenol F (BPF) are applied to various commodities and generally detected in aquatic systems worldwide. To understand the potential ecological risk of BPF, the acute toxicity as well as behavioural, physiological and biochemical parameters of the water flea Daphnia magna were assessed. Following BPF exposure at concentrations ranging from 0.1 µg L-1 to 100 µg L-1, phenotypic traits including growth development, fecundity and swimming activity were significantly inhibited in response to exposure to sublethal concentrations (1-100 µg L-1) of BPF, which had a positive relationship with the activity of antioxidant enzymes. Moreover, the acetylcholinesterase (AChE) activity, which was strictly associated with the behavioural changes, was clearly inhibited, which was also obviously related to the heart rate and thoracic limb activity. Compared to the toxicity of BPA, BPF induces similar toxic effects, and the health concerns regarding the use of these alternatives should be highlighted.

Molecular characterisation of cytochrome P450 enzymes in waterflea (Daphnia pulex) and their expression regulation by polystyrene nanoplastics.

Cytochrome P450 (CYP) enzymes are one of the largest protein families, and they metabolise a wide range of lipophilic organic endogenous and exogenous compounds. Many cytochrome P450 genes have been cloned and characterised, and they are frequently used as biomarkers in environmental toxicology studies because of their sensitivity and inducibility. In the present study, the full-length cDNAs of DpCYP370B and DpCYP4 were cloned from Daphnia pulex for the first time. The sequence of DpCYP370B consisted of an ORF of 1515 bp that encoded a 504 amino acid polypeptide, while the sequence of DpCYP4 comprised an ORF of 1527 bp that encoded a 508 amino acid polypeptide. Homologous alignments revealed the presence of a conserved cysteine haeme-iron ligand signature, FxxGxxxCxG, located in the C-terminal portion. Both the proteins contained a sequence for a transmembrane region that was deduced to be located in the endoplasmic reticulum. Subsequently, the expression levels of DpCYP370B and DpCYP4, as well as those of CYP4AN1, CYP4C33, and CYP4C34, were investigated using quantitative real-time PCR after exposure to five polystyrene nanoplastic concentrations: 0 (control), 0.1, 0.5, 1, and 2 mg/L for 21 days. Except for DpCYP4, the highest mRNA expression was observed at 0.5 mg/L nanoplastics; next, the expression of three of the enzymes (DpCYP370B, CYP4AN1, CYP4C34,) decreased to that of the control level at 1 and 2 mg/L doses of nanoplastics. The expression of DpCYP4 did not significantly change compared with that of the control group. These results indicated that CYP genes might play an important role in protecting D. pulex against nanoplastic pollutants.

Simvastatin affect the expression of detoxification-related genes and enzymes in Daphnia magna and alter its life history parameters.

Simvastatin (SV), as an hypocholesterolaemic drug, has been detected in various aquatic environment. However, limited information is available on the effects of SV on freshwater invertebrates. In the present study, we investigated the toxic effects of SV on Daphnia. magna (D. magna) through measuring the physiological changes (e.g., survival, growth rate, and reproduction) in a 21-d chronic toxicity test We also determined the expression of seven detoxification and reproduction-related genes (i.e. HR96, P-gp, CYP360A8, GST, CYP314, EcR and Vtg) and several enzymes (i.e. APND, ERND, GST and CAT) in a acute test (24 h). Results showed that high concentration (e.g. 50 µg L-1) of SV for short time exposure (e.g. 24 h) significantly induced the expression of HR96 and P-gp (e.g. up to 2.5 folds)and enzymes (e.g. increasing 4.0 folds for ERND and GST activity) in D. magna.. The long-term chronic exposure (21 days) may cause the changes of life history parameters such as decreasing total egg production number per individual and intrinsic growth rates etc. SV may act as a potential endocrine disruptor to D. magna and the reproduction parameters were more sensitive endpoints than the survival and growth for evaluating SV exposure.

Microbial levan and pullulan as potential protective agents for reducing adverse effects of copper on Daphnia magna and Vibrio fischeri.

Microbial polysaccharides, due to their unique physiochemical properties, have found application in the food industry, cosmetics, pharmacy and medicine. In the environment, microbes can use polysaccharides to alleviate the adverse effects of heavy metals in their close proximity. This adaptive property shows interesting potential for bioremediation. Herein, the effects of the exopolysaccharides (EPS) levan, produced by the bacterium Bacillus licheniformis NS032 and pullulan, produced by the fungus Aureobasidium pullulans CH-1 in the presence of copper (Cu2+) have been investigated for the first time on antioxidant enzyme activity, respiration and Cu2+ bioaccumulation of Daphnia magna as well as the bioluminescence of Vibrio fischeri. Both EPS decreased toxicity of Cu2+ in the acute test with D. magna. The activity of catalase (CAT) was significantly diminished after acute exposure to Cu2+ in comparison to treatments with Cu2+ and EPS, while in the prolonged acute exposure the CAT activity did not show statistically significant (P ≤ 0.05) differences between treatments with and without the EPS. According to ICP-MS results, during prolonged acute exposure of neonates, the bioaccumulation of Cu2+ in treatments without the EPS was 52.03 µg/g of biomass (wet), while in treatments with EPS, the bioaccumulation was lower by one order of magnitude. The respiration of neonates during acute exposure to Cu2+ with or without the EPS was monitored using the MicroOxymax respirometer, and the results show the EPS can positively effect the respiration. In the case of bacterial bioluminescence, the toxicity of Cu2+ decreased in treatments with EPS (30 min EC10) from 3.54 mg/L to 140.61 mg/L (levan) and 45.00 mg/L (pullulan). This study demonstrates protective effect of EPS against Cu2+ toxicity on D. magna and V. fischeri, and opens the door for further investigation of potential application of levan and pullulan in bioremediation of heavy metals and mitigation of their adverse effects in the aquatic environment.

Effects of ectoine on behavioral, physiological and biochemical parameters of Daphnia magna exposed to dimethyl sulfoxide.

DMSO is a very common solvent for hydrophobic chemicals that may pose a threat to aquatic organisms. Ectoine (ECT) is a protective amino acid produced by various strains of halophilic bacteria with high potential to alleviate detrimental effects induced by environmental stressors. This amino acid is used in many cosmetics and pharmaceuticals may enter aquatic ecosystems interacting with ions and macromolecules. Little is known on the effects of DMSO and its interaction with ECT on behavioral, physiological and biochemical endpoints of aquatic invertebrates. Therefore, the purpose of the present study was to determine protective effects of DMSO alone and in the combination with ECT on hopping frequency, swimming speed, heart rate, thoracic limb activity, catalase activity and NOx level in an animal model, Daphnia magna subjected to 0.1% and 1% DMSO alone and during combinatorial exposure to ECT (0-25 mg/L) and DMSO for 24 h and 48 h. The results showed that swimming speed, heart rate and thoracic limb activity were inhibited by both 0.1% and 1% DMSO alone however alleviating effects were observed in the combination DMSO + ECT. Thoracic limb activity was higher in the animals exposed to both solutions of DMSO alone, however the parameter was more stimulated at DMSO + ECT. The results suggest that DMSO alone may alter Daphnia behavior and physiological parameters, therefore use of the control group of non-treated animals with DMSO alone would be recommended to avoid data misinterpretation.

Two PBDEs exposure inducing feeding depression and disorder of digestive and antioxidative system of Daphnia magna.

2,2',4,4'-tetrabrominated diphenyl ether (BDE-47) and 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) are two typical polybrominated diphenyl ethers (PBDEs), and studies have proven that these PBDs can disrupt the behaviors and physical function of aquatic organisms. However, little is known about the compositional impacts of BDE-47/BDE-99 compound pollution on the feeding behavior of Daphnia magna. In this study, a response surface methodology (RSM) was introduced into the combined toxicity assessment of BDE-47 and BDE-99 on the feeding depression of D. magna. Low concentrations of BDE-47 (9.2 µg/L) and BDE-99 (5.4 µg/L) had no effect on the feeding behavior of D. magna; nevertheless, the feeding depression was strengthened, and a concentration-dependent effect was observed with increasing concentrations of BDE-47 and BDE-99. The results of RSM indicated that the mixture of BDE-47 and BDE-99 can enhance their toxicity on the feeding behavior of D. magna. Moreover, real-time PCR (qPCR) analysis showed that the down-regulation of α-amylase (AMS) appeared in most of the exposed D. magna. However, there were significant different in the gene expression of trypsin, superoxide dismutase (SOD) and catalase (CAT) between the exposure and control groups. The change in the enzyme activity of AMS, trypsin, SOD and CAT implied that BDE-47 and BDE-99 cause damage to the digestive and antioxidative systems of D. magna. Correlation analysis indicated that a significant positive correlation existed between the gene expression and enzyme activity of SOD and CAT. Our results contribute to the understanding of toxicity caused by BDE-47/BDE-99 compound pollution in D. magna and help to improve traditional toxicity assessment methods for aquatic environments.

Carbonyl reductase sniffer from the model organism daphnia: Cloning, substrate determination and inhibitory sensitivity.

Carbonyl reductases (CRs) represent a fundamental enzymatic defense mechanism against oxidative stress. While commonly two carbonyl reductases (CBR1 and CBR3) are found in mammalian genomes, invertebrate model organisms like Drosophila melanogaster express no CR but a functional homolog to human CBR1, termed sniffer. The importance of sniffer could be demonstrated in D. melanogaster where it protected against age-dependent neurodegeneration. Interestingly, the microcrustacean Daphnia harbors four copies of the CR gene (CR1, CR2, CR3, CR4) in addition to one sniffer gene. Due to this unique equipment Daphnia is an ideal model organism to investigate the function of sniffer. Recombinant sniffer from D. magna und D. pules were produced in E. coli, purified by Ni-affinity chromatography and tested with a variety of aliphatic and aromatic diketones, reactive aldehydes and precursors of advanced glycation end products (AGE). The highest catalytic activities were determined for sniffer from D. pulex with the aromatic dicarbonyls 9,10-phenanthrenequinone (kcat/Km = 2.6 s-1 x µM-1) and isatin (kcat/Km = 1.5 s-1 x µM-1). While sniffer from D. magna displayed preference for the same two substances, the respective catalytic activities were noticeably lower. Kinetic constants with aliphatic diketones were generally lower than those with aromatic dicarbonyls for both sniffer enzymes. The best aliphatic diketone as substrate for sniffer from D. magna and D. pulex was hexane-3,4-dione with kcat/Km = 0.23 s-1 µM-1 and kcat/Km = 0.35 s-1 µM-1, respectively. Poor or no detectable activity of the two sniffer enzymes was seen with the aliphatic diketones 2,5-hexanedione and 3,5-heptanedione, the aldehydes butanal, hexanal, decanal, crotonaldehyde, acrolein, trans-2-hexenal, and the AGE precursors glyoxal, methylglyoxal, furfural and glyceraldehyde, indicating no physiological function in the metabolism of short-chain aldehydes. Substrate inhibition for both sniffer enzymes was observed with the quinone substrates 1,4-naphthoquinone and 2-methyl-1,4-benzoquinone. From a variety of pesticides endosulfan turned out as an effective inhibitor of the sniffer enzymes (Ki = 9.2 µM for sniffer from D. magna, Ki = 12.0 µM for sniffer from D. pulex). In conclusion, the present results on sniffer from the protein superfamily of the short-chain dehydrogenases/reductases (SDR) in Daphnia ssp. complement earlier studies on carbonyl reductases in the same species and indicate that Daphnia is an interesting model to study the overall response to carbonyl stress.

Cloning and expression of the lifespan-associated protein Sir2 from Daphnia pulex.

The activity of NAD+-dependent deacetylase Sir2, originally discovered in yeast, is known to be essential for effective longevity. The relationship between Sir2 and lifespan in Daphnia pulex was investigated by cloning and analysis the full-length 1901 bp cDNA. The Sir2 protein includes several zinc finger active sites and two readily hydrolysable low-complexity SD-rich regions. The three-domain structure of Sir2 has a distinct crevice that plays an important regulatory role in the binding of NAD+. D. pulex Sir2 shares 90% amino acid sequence identity with Sir2 from D. pulicaria, 89% with D. magna Sir2, 40% with Mus musculus Sir2, and 39% with the Homo sapiens protein. Expression of Sir2 mRNA was measured at 1, 10, 15, 20,25, 30 and 35 days by real-time PCR (p < .05), and was lowest at 1 day and highest at 20 days (p < .05), after which expression decreased with age. In situ hybridisation showed that Sir2 mRNA was expressed in the chest, the first and second antennae, and the gonadal gland in D. pulex. A Sir2 gene fragment was amplified by PCR, ligated into the pEASY-Blunt vector, and recombinant Sir2 was expressed in Escherichia coli and purified by Ni affinity chromatography. The recombinant protein was used as antigen to immunise rabbits, and antiserum was successfully purified using the protein A method, yielding a Sir2 polyclonal antibody. Western blotting showed that Sir2 is expressed at a 69 kDa protein in D. pulex, in accordance with the predicted value. Sir2 protein abundance increased gradually with age, but remained unchanged after 25 days.

Molecular characterization of thioredoxin reductase in waterflea Daphnia magna and its expression regulation by polystyrene microplastics.

Global scale concerns regarding rise in microplastics pollution in the environment have recently aroused. Ingestion of microplastics by biota, including freshwater zooplankton has been well studied, however, despite keystone species in freshwater food webs, the molecular response (e.g. oxidative defense) of zooplankton in response to microplastics is still in its infancy. The thioredoxin (TRx) system has a vital function in cellular antioxidative defense via eliminating the excessive generation of reactive oxygen species (ROS). Therefore, it is necessary to investigate the effects of thioredoxin reductase (TRxR), due to its triggering the TRx catalysis cascade. The present study identified TRxR in Daphnia magna (Dm-TRxR) for the first time, and found that the full-length cDNA was 1862 bp long, containing an 1821-bp open reading frame. Homologous alignments showed the presence of conserved catalytic domain CVNVGC and the seleocysteine (SeCys) residue (U) located in the N- and C- terminal portions. Subsequently, the expression of Dm-TRxR, together with permease, arginine kinase (AK), was investigated by approach of quantitative real-time PCR after exposure to four (1.25-µm) polystyrene (PS) microbeads concentrations: 0 (control), 2, 4 and 8 mg L-1 for 10 days. Dm-TRxR, permease and AK mRNA were significantly upregulated after exposure to 2, 4 mg L-1 of PS, but then declined in the presence of 8 mg L-1 PS. The gene expression results suggested that oxidative defense, energy production and substance extra cellular transportation were significantly regulated by microplastic exposure. Collectively, the present study will advance our knowledge regarding the biological effects of microplastic pollution on zooplankton, and builds a foundation for freshwater environmental studies on mechanistic and biochemical responses to microplastics.

Multigenerational effects of carbendazim in Daphnia magna: From a subcellular to a population level.

Anthropogenic activities such as the use of pesticides may affect aquatic biota populations, due to potential agricultural runoffs or disposals. Carbendazim is one example of a widely used fungicide with a high potential to end up in aquatic ecosystems through runoff. Deleterious effects observed at the individual level are possibly explained by changes in homeostasis at the cellular level, and both factors can then be used to predict effects at the population level. In the present study, an isoclonal population of Daphnia magna (clone K6) was exposed to a concentration that mimics relevant levels of carbendazim in the environment over 12 generations. The effects of carbendazim were assessed in some generations using the following endpoints: biochemical biomarkers (cholinesterase, catalase, and glutathione-S-transferase), lipid peroxidation and energy-related parameters (carbohydrates, lipids, and proteins along with available energy and energy consumption), parental longevity, and population growth (r). Long-term exposure to carbendazim had no effect on the intrinsic rate of natural increase (r) of adult D. magna, but longevity was decreased at the F12 generation compared to that of control. Differences between the exposed and nonexposed populations were found for cholinesterase, glutathione-S-transferase, and lipid peroxidation. However, for catalase and energy-related parameters, no differences were observed between these 2 populations. Natural variability was seen throughout the test period, under control conditions, within the 12 generations. Overall, carbendazim induced some effects at the subcellular level that translated into changes in longevity but these later vanished in terms of population effects. Environ Toxicol Chem 2019;38:412-422. © 2018 SETAC.

The synergistic potential of azole fungicides does not directly correlate to the inhibition of cytochrome P450 activity in aquatic invertebrates.

The ability of azole fungicides to inhibit cytochrome P450 dependent metabolism is proposed to be the main mechanism for their synergizing effect on pyrethroid insecticide toxicity in aquatic invertebrates. This study investigates the correlation between inhibition strength and synergistic potential of azole fungicides in the crustacean Daphnia magna and the insect larvae Chironomus riparius. Inhibition strength was measured in vivo toward the cytochrome P450 catalysed conversion of 7-ethoxycoumarin to 7-hydroxycoumarin (ECOD). Synergistic potentials were determined as the ratio between predicted and observed toxicity of mixtures based on the model of concentration addition (CA) and independent action (IA). Azoles (n = 9-11) enhanced the toxicity of α-cypermethrin in D. magna (Synergy ratios CA: 0.8 - 16; IA: 1.1 - 22) and inhibited cytochrome P450 activity by different degrees (IC50: 0.0023 - 36 µM for D. magna and 0.08 - 24 µM for C. riparius). Inhibition strengths were strongly correlated in the two organisms (r: 0.937 p: 0.019 for triazoles and r: 0.903 p: 0.097 for imidazoles). Lipophilicity governed the inhibition strength of triazoles in both species (r > 0.9, p < 0.05). No correlation was observed between inhibition strengths and synergistic potentials. Several reasons for the apparent lack of correlation were discussed.

Heterologous expression and characterization of a novel serine protease from Daphnia magna: A possible role in susceptibility to toxic cyanobacteria.

Mass developments of toxin-producing cyanobacteria are frequently observed in freshwater ecosystems due to eutrophication and global warming. These mass developments can partly be attributed to cyanobacterial toxins, such as protease inhibitors (PIs), which inhibit digestive serine proteases of Daphnia, the major herbivore of phytoplankton and cyanobacteria. To date, mechanisms of this inhibition in the gut of the crustacean Daphnia magna are not known. Here, we characterize a single serine protease, chymotrypsin 448 (CT448), which is present in the gut of the crustacean D. magna. Sequence alignments with human serine proteases revealed that CT448 has a putative N-terminal pro-peptide which is extended compared to the mammalian homologs and within this pro-peptide two N-linked glycosylation motifs were found. CT448 was heterologously expressed in Sf21 insect cells using a baculovirus expression system for optimized protein production and secretion into the medium. The protein was purified via a one-step affinity chromatography, which resulted in a protein yield of 3.45 mg/l medium. The inactive precursor (zymogen) could be activated by tryptic digestion. This is the first example of a recombinant expression of an active crustacean serine protease, which functions in the gut of Daphnia. Proteomic identification of protease cleavage sites (PICS) and hydrolysation of various synthetic substrates showed that CT448 is a chymotrypsin-like elastase. In this study, we confirm that CT448 is a target of cyanobacterial protease inhibitors. Local evolutionary modifications of CT448 might render this proteolytic enzyme less susceptible against cyanobacterial secondary metabolites and might improve the fitness of Daphnia during cyanobacterial blooms.

Effects of Microcystis aeruginosa on the life history traits and SOD activity of Daphnia similoides sinensis.

With water eutrophication and global warming, cyanobacteria blooms have occurred frequently, and the interaction between M. aeruginosa and Daphnia has been widely paid attention by researchers. However, the effects of toxic M. aeruginosa on the SOD activity of Daphnia are poorly known. Six D. similoides sinensis clones collected from Lake Junshan and the offspring of two clones were employed. The effects of toxic M. aeruginosa on the life history traits and SOD activities of D. similoides sinensis in the mother and their offspring were studied. Toxic M. aeruginosa could significantly inhibit the life history traits (e.g., body lengths, offspring numbers at first reproduction, cumulative offspring numbers, and the intrinsic rate of population) and induce higher SOD activities of D. similoides sinensis. Compared with the mother, the effects of toxic M. aeruginosa on the life history traits and SOD activities of D. similoides sinensis in the offspring showed obvious differences. Moreover, the adaptability of the offspring to M. aeruginosa indicated also the differences between two clones. Our results suggested that the mother exposed to toxic M. aeruginosa could enhance the fitness of their offspring to Microcystis by maternal effect and was also affected by the D. similoides sinensis genotypes.

Carbonyl reductases from Daphnia are regulated by redox cycling compounds.

Oxidative stress is a major source of reactive carbonyl compounds that can damage cellular macromolecules, leading to so-called carbonyl stress. Aside from endogenously formed carbonyls, including highly reactive short-chain aldehydes and diketones, air pollutants derived from diesel exhaust like 9,10-phenanthrenequinone (PQ) can amplify oxidative stress by redox cycling, causing tissue damage. Carbonyl reductases (CRs), which are inducible in response to ROS, represent a fundamental enzymatic defense mechanism against oxidative stress. While commonly two carbonyl reductases (CBR1 and CBR3) are found in mammalian genomes, invertebrate model organisms like Drosophila melanogaster express no CR but a functional homolog to human CBR1, termed sniffer. The microcrustacean Daphnia is an ideal model organism to investigate the function of CRs because of its unique equipment with even four copies of the CR gene (CR1, CR2, CR3, CR4) in addition to one sniffer gene. Cloning and catalytic characterization of two carbonyl reductases CR1 and CR3 from D. magna and D. pulex arenata revealed that both proteins reductively metabolize aromatic dicarbonyls (e.g., menadione, PQ) and aliphatic α-diketones (e.g., 2,3-hexanedione), while sugar-derived aldehydes (methylglyoxal, glyoxal) and lipid peroxidation products such as acrolein and butanal were poor substrates, indicating no physiological function in the metabolism of short-chain aldehydes. Treatment of D. magna with redox cyclers like menadione and the pesticide paraquat led to an upregulation of CR1 and CR3 mRNA, suggesting a role in oxidative stress defense. Further studies are needed to investigate their potential to serve as novel biomarkers for oxidative stress in Daphnia.

Gene expression and activity of digestive enzymes of Daphnia pulex in response to food quality differences.

Food quality is an important factor influencing organisms' well-being. In freshwater ecosystems, food quality has been studied extensively for the keystone herbivore genus Daphnia, as they form the critical trophic link between primary producers and higher order consumers such as fish. For Daphnia, the edible fraction of phytoplankton in lakes (consisting mostly of unicellular algae and cyanobacteria) is extraordinarily diverse. To be able to digest different food particles, Daphnia possess a set of digestive enzymes that metabolize carbohydrates, lipids and proteins. Recent studies have found a connection between gene expression and activity of single digestive enzyme types of Daphnia, i.e. lipases and proteases, and transcriptome studies have shown that a variety of genes coding for gut enzymes are differentially expressed in response to different food algae. However, never before has a set of digestive enzymes been studied simultaneously both on the gene expression and the enzyme activity level in Daphnia. Here, we investigated several digestive enzymes of Daphnia pulex in a comparison between a high-quality (green algal) and a low-quality (cyanobacterial) diet. Diet significantly affected the expression of all investigated digestive enzyme genes and enzyme activity was altered between treatments. Furthermore, we found that gene expression and enzyme activity were significantly correlated in cellulase, triacylglycerol lipase and ß-glucosidase when switched from high to low-quality food. We conclude that one of the factors causing the often observed low biomass and energy transfer efficiency from cyanobacteria to Daphnia is probably the switch to a cost-effective overall increase of gene expression and activity of digestive enzymes of this herbivore.

Racemic, R-, and S-tebuconazole altered chitinase and chitobiase activity of Daphnia magna.

Tebuconazole is a chiral trizole fungicide and widely used in many crops for controlling disease. Tebuconazole is potential toxic to some aquatic organisms but relative information of its isomers is scarce. To detect the endocrine disrupting effects and difference of rac-, R-, and S-tebuconazole, the chitinase activity in Daphnia magna and chitobiase activity in each test medium were used as biomonitors after a 14-day exposure. Results showed that chitinase activity was significantly reduced by rac-, R-, and S-tebuconazole. The chitobiase activity in the test medium was reduced by rac- and R-tebuconazole before day 10, and only one peak was observed at day 10 or day 12 compared with two obvious peaks in the control group (days 6 and 12). S-tebuconazole delayed and reduced the reproduction of D. magna, but did not delay the first chitobiase activity peak, whereas the second peak could not be characterized as the exposure concentration and time increased. Compared with chitinase activity, chitobiase activity can still be used as a rudimentary model for identifying molt-interfering xenobiotics, and further studies should focus on the analysis of correlations between these parameters.