Modulation of substrate-specific glutathione S-transferase activity in Daphnia magna with concomitant effects on toxicity tolerance

Glutathione S-transferase (GST) activity was measured in Daphnia magna and Ceriodaphnia reticulata using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA) as conjugation substrates. Levels of GST activity were comparable between species with CDNB; however, D. magna had nearly twice the GST activity with EA as compared to C. reticulata. GST activity with CDNB was elevated from exposure of daphnids to either CDNB or sodium pentachlorophenate (PCP), but not from exposure to EA. GST activity with EA could not be modulated from exposure to CDNB or EA. GST activity towards CDNB and EA was biochemically separated into different protein fractions suggesting the existence of two distinct isozymes. Preexposure of daphnids to CDNB or PCP increased the organisms' tolerance to the toxic effects of PCP, but not CDNB.     

Daphnia magna as a test animal in acute and chronic toxicity tests

Daphnia magna is a commonly used test animal in aquatic toxicology.Test procedures for acute and chronic test are described, together with the influence of several variables on their results.The practicability of the methods was checked with four model compounds, viz. 1,1,2-trichloroethane, dieldrin, pentachlorophenol and 3,4-dichloroaniline. Toxicity data of these compounds for Daphnia magna are given.     

The use of biomarkers in Daphnia magna toxicity testing

We investigated the effect of short-term exposure to cadmium and 2,4-dichlorophenoxy acetic acid on the digestive physiology of Daphnia magna and the consequences for the bioenergetics of the organism. In both cases, ingestion was more drastically reduced compared to digestive enzyme activity. Furthermore a differential shift in catabolism was noted: in general polysaccharidases were less affected than the enzymes responsible for protein and lipid digestion. Comparison of the ‘1 h in vivo fluorescence’ criterion (Janssen & Persoone, 1993) with the ingestion and digestive enzyme activity revealed that this rapid screening assay should be considered as a quantification of ingestion inhibition rather than a methodology assessing digestive enzyme inhibition. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of multiple glutathione S-transferases from Daphnia magna

1. Six anionic glutathione S-transferases (GST) were purified from the crustacean, Daphnia magna, by means of affinity chromatography, that are responsible for ca. 40% of cytosolic GST activity. 2. Electrophoresis in the presence of sodium dodecyl sulfate (SDS) revealed the presence of three proteins, with molecular weights of 27,500, 28,000, and 30,200. 3. Separation under nondenaturing conditions revealed six proteins, all of which exhibited GST activity, with molecular weights ranging from 55,000 to 61,700. 4. Ethacrynic acid is a competitive inhibitor of activity towards CDNB of all six GSTs, binding each with similar affinities. 5. Chlorinated phenols are also competitive inhibitors of the enzyme, with the degree of inhibition being directly correlated with the lipophilicity of the compounds. 6. Analysis of inhibition of separated isoforms reveals that form 4 is most strongly inhibited by these chlorinated phenols, with forms 5 and 6 being inhibited to a lesser degree.     

Tests with Daphnia magna: a new approach to prescreen toxicity of newly synthesized acetylcholinesterase reactivators

Reactivators of phosphorylated acetylcholinesterase (oximes) are substances used as a human antidotal therapy for organophosphate poisoning. The objective of our study was to examine if juveniles of freshwater microcrustacean Daphnia magna could be employed as test animals in early screen toxicity tests of those substances as a first step for further experiments with daphnids intoxicated by organophosphates. For this purpose, seven different oximes were investigated. It was found that toxicity of all tested oximes increased with time. Mono-quaternary oximes were approximately ten fold (EC50, 14.9 mg.l(-1)) more toxic in 24 hour tests and five fold (EC50 was 79.46 mg.l(-1)) more toxic in 48 hour tests than bis-quaternary oximes. Tests with daphnids were shown to be easy to carry out at low cost and provided valuable results which could be used as a starting point for further research.     

Tests with Daphnia magna: A new approach to prescreen toxicity of newly synthesized acetylcholinesterase reactivators

Reactivators of phosphorylated acetylcholinesterase (oximes) are substances used as a human antidotal therapy for organophosphate poisoning. The objective of our study was to examine if juveniles of freshwater microcrustacean Daphnia magna could be employed as test animals in early screen toxicity tests of those substances as a first step for further experiments with daphnids intoxicated by organophosphates. For this purpose, seven different oximes were investigated. It was found that toxicity of all tested oximes increased with time. Mono-quaternary oximes were approximately ten fold (EC₅₀, 14.9 mg.l^{? 1}) more toxic in 24 hour tests and five fold (EC₅₀ was 79.46 mg.l^{? 1}) more toxic in 48 hour tests than bis-quaternary oximes. Tests with daphnids were shown to be easy to carry out at low cost and provided valuable results which could be used as a starting point for further research.     

Reproducibility of short-term and reproduction toxicity experiments with Daphnia magna and comparison of the sensitivity of Daphnia magna with Daphnia pulex and Daphnia cucullata in short-term experiments

Short-term toxicity tests with Daphnia magna are reasonably reproducible. The sensitivity of Daphnia pulex, Daphnia cucullata and Daphnia magna proved to be about the same in short-term tests with 15 different chemical compounds.Reproduction toxicity experiments with Daphnia magna can easily be carried out within three weeks. At least duplicate experiments are necessary to arrive at an approximate no-effect level.     

不同种类农药表面活性剂对大型溞的急性毒性

运用评价化学品对水生生物毒性的标准试验方法,比较了39种非离子型、6种阴离子型和3种阳离子型的常用代表性表面活性剂对大型溞的急性毒性.结果表明:3种阳离子型表面活性剂1427、1227及C8-10的急性毒性均为剧毒,其中1427毒性最高,EC50值为0.97×10-2 mg·L-1;非离子型表面活性剂中蓖麻油聚氧乙烯醚、吐温和斯潘系列乳化剂均为低毒,而烷基酚聚氧乙烯醚系列和脂肪醇聚氧乙烯醚系列表面活性剂的毒性稍偏高,而AEO-7和AEO-5的毒性达到高毒水平,EC50值分别为0.82和0.97 mg·L-1,且此类表面活性剂脂溶性越大,对大型溞的毒性越大;大部分阴离子表面活性剂的毒性为中毒,但NNO表现为高毒,EC50值为0.17 mg·L-1.     

The cholinesterase properties of Daphnia magna

It has been demonstrated that cholinesterase of Daphnia magna is capable of the hydrolysis of propionylthiocholine iodide at the highest rate as compared to the other substrates studied, the hydrolysis being inhibited by high concentrations of the substrate. The rate of splitting of acetylthiocholine iodide is similar to that of propionylthiocholine iodide, whereas the hydrolysis of butyrylthiocholine iodide is 3 times slower. Cholinesterase from D. magna is extremely sensitive to an organophosphorus inhibitor, DDVP. The value of bimolecular constant of the inhibition rate (kappa II) is equal to (1.60 +/- 0.20).10(8)1.mol-1.min-1.     

The oculomotor system of Daphnia magna

Summary The highly mobile cyclopic compound eye of Daphnia magna is rotated by six muscles arranged as three bilateral pairs. The three muscles on each side of the head share a common origin on the carapace and insert dorsally, laterally and ventrally on the eye. The dorsal and ventral muscles are each composed of two muscle fibers and the lateral muscle is composed of from two to five fibers, with three the most common number. Individual muscle fibers are spindle-shaped mononucleated cells with organized bundles of myofilaments. Lateral eye-muscle fibers are thinner than those of the other muscles but are otherwise similar in ultrastructure. Two motor neurons innervate each dorsal and each ventral muscle and one motor neuron innervates each lateral muscle. The cell bodies of the motor neurons are situated dorsally in the supraesophageal ganglion (SEG) and are ipsilateral to the muscles they innervate. The dendritic fields of the dorsal-muscle motor neurons are ipsilateral to their cell bodies; those of the ventral-muscle motor neurons are bilateral though predominantly contralateral. The central projections of the lateral-muscle motor neurons are unknown. In the dorsal and ventral muscles one motor axon synapses principally with one muscle fiber; in each lateral muscle the single motor axon branches to, and forms synapses with, all the fibers. The neuromuscular junctions, characterized by pre- and postsynaptic densities and clear vesicles, are similar in all the eye muscles.     

Eye movements in Daphnia magna

Summary Three types of behavior of the compound eye of Daphnia magna are characterized: flick, a transient rotation elicited by a brief flash of light; fixation, a maintained eye orientation in response to a stationary light stimulus of long-duration; tracking, the smooth pursuit of a moving stimulus. The magnitudes of the flick and fixation responses vary with stimulus position and are generally proportional to stimulus intensity, although at high intensities there is an attenuation of both behaviors. When the stimulus is placed at a position 80° dorsal to the eye axis, there is no response; this area is called the null region. For stationary stimuli in other positions, the direction of the response is such as to bring the stimulus closer to the null region. During tracking, the relative positions of the eye and stimulus change; the eye velocity is approximately half that of the moving stimulus. The regions of the eye in which these behaviors may be induced are different, being largest for flick and smallest for tracking. It is proposed that flick and fixation responses are a means for rotating the eye so that the stimulus is within the area surrounding the null region which is used for tracking.     

Sensitivity of bacterial vs. acute Daphnia magna toxicity tests to metals

The objectives of this study were to evaluate the sensitivity of two bacterial tests commonly used in metal toxicity screening — the Vibrio fischeri bioluminescence inhibition test and the Pseudomonas putida growth inhibition test — in comparison to the standard acute Daphnia magna test, and to estimate applicability of the selected methods to the toxicity testing of environmental samples. The D. magna acute test proved to be more sensitive to cadmium (Cd), zinc (Zn) and manganese (Mn) than the two bacterial assays, whereas P. putida seems to be the most sensitive species to lead (Pb). Manganese appears to be slightly toxic to D. magna and non-toxic to the two selected bacteria. This leads to the conclusion that even in regions with high background concentrations, manganese would not act as a confounding factor. Low sensitivity of V. fischeri to heavy metals questions its applicability as the first screening method in assessing various environmental samples. Therefore, it is not advisable to replace D. magna with bacterial species for metal screening tests. P. putida, V. fischeri and/or other bacterial tests should rather be applied in a complex battery of ecotoxicological tests, as their tolerance to heavy metals can unravel other potentially present toxic substances and mixtures, undetectable by metal-sensitive species.     

The effect of humic substances on the toxicity of copper and zinc for Daphnia magna]

The results of laboratory experiments of humic matter influence on toxic resistance of Daphnia magna to zinc and copper using water samples from a few Karelia water bodies are presented. The increasing of humic matter content in water samples has been shown to promote the binding of a part of metals in organic complexes that leads to decrease of their toxicity for hydrobionts.     

Calcium limitation in Daphnia magna

The role of ambient calcium concentrations on survival, moulting,growth and egg production was assessed in the cladoceran Daphniamagna. A threshold for survival was found in the range 0.1–0.5mg Ca l^–1, even when ionic strength of the medium waskept constant. Accumulated length and length specific dry weightwas retarded at low Ca (0.5–1.0 mg Ca l^–1) at foodconcentrations above incipient limiting level. For lower foodlevels, the effect of Ca on growth was less clear. The effectof low Ca on growth rate was most manifest during the firstdays after hatching, reflecting the higher Ca demands of theearly juveniles. Age-specific egg production was strongly reducedat Ca concentrations <10 mg Ca l^–1. This was partlyan indirect effect of reduced growth rates, but could also bean effect of higher energetic costs associated with Ca uptakein a Ca-poor medium. The high Ca demands in D.magna may notbe representative of other Daphnia species. Nevertheless, highspecific Ca content seems to be a common property of Daphniaspp. and Ca deficiency could be a major determinant of speciessuccess and community structure among crustacean zooplankton;it also puts constraints on carbon sequestration in the pelagicfood web of softwater lakes.     

Haemoglobin in male Daphnia magna

• 1.1. To define the respiratory function of haemoglobin in male Daphnia magna, the swimming activity, the depression of oxygen uptake by treatment with carbon monoxide and in vivo oxygenation of Hb at various oxygen pressures were investigated.
• 2.2. The P₅₀, values for the purified Hbs from male and female red animals were 2.0 and 2.7 torr in 0.1 M phosphate buffer (pH 7.2) at 20°C, respectively.
• 3.3. The isoelectric focusing patterns of the purified Hbs from male and female red animals showed only small differences in Hb components of high PI values.

     

Phylogeography of Daphnia magna in Europe

The phylogeographical structure of the water flea Daphnia magna in Europe was analysed using a 609-bp fragment of the cytochrome oxidase subunit I gene. Only moderate overall genetic divergence was detected within Europe. We detected four genetically and, to a large extent, geographically distinct phylogroups within Europe. Our results suggest that these groups recolonized large parts of Europe around 100,000 BP from different refugia. Overall, the pattern suggests a high degree of provincialism with a patchy occurrence of specific lineages, thus confirming the highly subdivided genetic structure usually observed in freshwater zooplankton populations. Although the region around the Mediterranean Sea was only sampled patchily, we obtained strong indications for the occurrence of more divergent genetic lineages in this region. Comparing our European samples to samples from North America and Japan revealed a higher level of differentiation, reflecting limited intercontinental dispersal.     

Thermal avoidance and preference in Daphnia magna

1. Water fleas (Daphnia magna) bred at 23°C were non-responsive to temperatures between 13 and 25°C.

2. At the lower (11°C) and upper limits (30°C) their klinokinetic avoidance behaviour showed a larger intraindividual than interindividual variation.

3. Thermal sensitivity for avoidance responses in D. magna was about 1.5°C.

4. For D. magna bred for one parthenogenetic generation at 14°C heat avoidance temperature was about 8°C lower, and cold avoidance temperature was about 1°C higher than in D. magna from 23°C.

5. In group experiments the animals showed some preference for the acclimation temperature.

6. Cold induced stenothermy and warm induced eurythermy in D. magna were related to the mode of reproduction.

Evaluation of a high throughput toxicity biosensor and comparison with a Daphnia magna bioassay

A high throughput toxicity biosensor has been designed and constructed using recombinant Escherichia coli cells, containing stress specific promoters (recA, fabA, or katG) or constitutive promoters (lac) fused to luciferase genes originating from Vibrio fisheri. These genetically engineered cells were immobilized in 96 well plates. By optimizing cell immobilization conditions and the strains' response specificity to toxic chemicals, bioluminescent outputs decreased or increased dose-dependently upon adding test chemicals. However, to date the toxicity data obtained using this biosensor have not been compared with the results of other toxicity tests. Phenolics were chosen to evaluate the correlation between the LD50 and the EC50 (GC2) or EC120 (DPD2540) of Daphnia magna and E. coli, respectively. Toxicity data obtained from constitutive strains by bioluminescent level decrements were compared with the results from D. magna as a standard. LD50 values were used as parameters of D. magna toxicity and EC50 of EC120 values were used for the immobilized biosensor. In the DPD2540 test, phenolics, membrane damaging toxic chemicals, for testing immobilized stress specific bacterial strains trigger dose-dependant bioluminescence increase within specific concentration. Although the stress specific responsiveness from the strains could not be compared with D. magna's LD50 values, these responses offer additional information, such as upon the mode of toxic action in the sample, in addition to the cellular toxicity results as indicated by the EC50. This novel high throughput toxicity biosensor can be implemented to investigate the toxicity of any other soluble materials, and can be used as a standardization tool for the evaluation of toxicity.     

Intersex and male development in Daphnia magna

Sex induction is environmentally stimulated in Daphnia and involves a cue-dependent response for sex determination. Somatic growth was shown to be similar in males and females during juvenile instars, but divergent due to a reduction in male somatic growth, at about the time that females produce ovaries. At this time, males appeared morphologically adult with respect to secondary sex characteristics. Intersex was rare and is unlikely to be important in natural populations. Intersex could be induced in both sexes, and observed more frequently after longer exposure to high temperature, or in the second generation following a temperature change. This indicates an impact on the ability of mothers to determine the sex of the offspring. It may be possible to use intersex characteristics for manipulative investigation of sex-determination mechanisms in Daphnia. Although sex-determination is initiated before birth, intersex occurrence suggests that development of male characters requires an additional process, probably involving hormone activity, during juvenile development and maturation.