Habitat uncertainty explains variation in offspring provisioning strategies in a temporary pond crustacean

Along with the development of nanotechnology, an increase in production and application of nanosized magnetite (Fe₃O₄) is expected. Though magnetite is considered relatively safe, information concerning potential hazards of synthetic magnetite nanoparticles with unique physico-chemical characteristics to aquatic organisms is still limited. In this study, we evaluated the toxicity of nanosized (27.2 ± 9.8 nm) and bulk (144.2 ± 67.7 nm) magnetite particles to different life stages of the aquatic crustacean Daphnia magna. In addition, phytotoxicity of the magnetite was evaluated using duckweed Lemna minor. The study did not reveal any statistically significant differences between the biological effects of nanosized and bulk magnetite particles. Both forms of magnetite induced very low toxicity (EC50 > 100 ppm) to D. magna and L. minor in the standard acute assays. However, it was demonstrated that at acutely subtoxic magnetite concentrations (10 and 100 ppm), the number of neonates hatched from D. magna ephippia was decreased. Moreover, short-term (48 h) exposure of neonate daphnids to these concentrations may significantly affect the long-term survival and reproductive potential of daphnids. These results indicate that substantial contamination of aquatic ecosystems by magnetite may disrupt the stability of cladoceran populations.     

Setae thickening in <Emphasis Type="Italic">Daphnia magna</Emphasis> alleviates the food stress caused by the filamentous cyanobacteria

It is assumed that daphnids adjust the filter screen morphology in order to minimize the interference with cyanobacterial filaments. The aim of this study was to investigate the impact of filamentous cyanobacteria (Aphanizomenon gracile Lemmermann, Cylindrospermopsis raciborskii Woloszynska Seenaya et Subba Raju) on the thickness and length of setae of the third pair of thoracic limbs of Daphnia magna. The second objective was to assess whether the setae modifications could improve the performance of daphnids in the presence of cyanobacteria. Three clones of Daphnia magna Straus were cultured with: green algae; green algae with filaments of Cylindrospermopsis; and green algae with filaments of Aphanizomenon. The size and age of animals in the first reproduction cycle as well as the number of offspring were recorded. Setae thickness and length were measured in the central part of each endopodite. Additionally, we analyzed how the changes in setae morphology affect the fitness of experimental animals using the intrinsic rate of population increase calculated with the Euler–Lotka equation. The results showed that the thickness and length of setae increased in the presence of filamentous cyanobacteria. Moreover, cyanobacteria-induced setae thickening was positively correlated to the fitness of daphnids, which may indicate setae thickening as a phenotypic adaptation to cope with food stress caused by filamentous cyanobacteria.     

The use of biomarkers in the antioxidant responses of Daphnia magna to the acute and chronic exposure to no. 20 diesel oil and 2,4-dichlorophenol

Abstract

The effects of no. 20 diesel oil exposure, 2,4-dichlorophenol (2,4-DCP) exposure and combined exposure on the antioxidant defences of Daphnia magna have been studied systematically for the first time. Daphnia magna was exposed for 1 day or 10 days to several concentrations of 0, 0.005, 0.01, 0.05, 0.1, 0.5 or 1.0 mg L^?1 solutions. Antioxidant defences consisting of the levels of reduced glutathione (GSH) and glutathione disulfide (GSSG) and activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), selenium dependent glutathione peroxidase (Se-GPx) and glutathione S-transferase (GST) of daphnids were determined to evaluate their protective roles and to analyse the occurrence of oxidative stress. The possible antioxidant defence mechanisms are discussed. Furthermore, GST can be a potential biomarker and an early-warning index for the pollutants in waters in that GST responded sensitively to 1 day and 10 days of exposure to diesel oil and 2,4-DCP and 10 days of combined exposure. Crossover comparisons showed an antagonistic action about the no-observed-effect concentration (NOEC) against Daphnia magna, which needs further studies.     

Bacterial epibionts of Daphnia: a potential route for the transfer of dissolved organic carbon in freshwater food webs

The identification of interacting species and elucidation of their mode of interaction may be crucial to understand ecosystem-level processes. We analysed the activity and identity of bacterial epibionts in cultures of Daphnia galeata and of natural daphnid populations. Epibiotic bacteria incorporated considerable amounts of dissolved organic carbon (DOC), as estimated via uptake of tritiated leucine: three times more tracer was consumed by microbes on a single Daphnia than in 1 ml of lake water. However, there was virtually no incorporation if daphnids were anaesthetised, suggesting that their filtration activity was essential for this process. Microbial DOC uptake could predominantly be assigned to microbes that were located on the filter combs of daphnids, where the passage of water would ensure a continuously high DOC supply. Most of these bacteria were Betaproteobacteria from the genus Limnohabitans. Specifically, we identified a monophyletic cluster harbouring Limnohabitans planktonicus that encompassed sequence types from D. galeata cultures, from the gut of Daphnia magna and from daphnids of Lake Zurich. Our results suggest that the epibiotic growth of bacteria related to Limnohabitans on Daphnia spp. may be a widespread and rather common phenomenon. Moreover, most of the observed DOC flux to Daphnia in fact does not seem to be associated with the crustacean biomass itself but with its epibiotic microflora. The unexplored physical association of daphnids with heterotrophic bacteria may have considerable implications for our understanding of carbon transfer in freshwater food webs, that is, a trophic ‘shortcut'' between microbial DOC uptake and predation by fish.     

Effect of single and repeated heat stress on chemical signals of heat shock response cascade in the rat’s heart

Exposure to sublethal heat stress activates a complex cascade of signaling events, such as activators (NO), signal molecules (PKCε), and mediators (HSP70 and COX-2), leading to implementation of heat preconditioning, an adaptive mechanism which makes the organism more tolerant to additional stress. We investigated the time frame in which these chemical signals are triggered after heat stress (41?±?0.5°С/45 min), single or repeated (24 or 72 h after the first one) in heart tissue of male Wistar rats. The animals were allowed to recover 24, 48 or 72 h at room temperature. Single heat stress caused a significant increase of the concentration of HSP70, NO, and PKC level and decrease of COX-2 level 24 h after the heat stress, which in the next course of recovery gradually normalized. The second heat stress, 24 h after the first one, caused a significant reduction of the HSP70 levels, concentration of NO and PKC?, and significant increase of COX-2 concentration. The second exposure, 72 h after the first heat stress, caused more expressive changes of HSP70 and NO in the 24 h-recovery groups. The level of PKC? was not significantly changed, but there was significantly increased COX-2 concentration during recovery. Serum activity of AST, ALT, and CK was reduced after single exposure and increased after repeated exposure to heat stress, in both time intervals. In conclusion, a longer period of recovery (72 h) between two consecutive sessions of heat stress is necessary to achieve more expressive changes in mediators (HSP70) and triggers (NO) of heat preconditioning.     

ERK1/2 regulates heat stress-induced lactate production via enhancing the expression of HSP70 in immature boar Sertoli cells

Lactate produced by Sertoli cells plays an important role in spermatogenesis, and heat stress induces lactate production in immature boar Sertoli cells. Extracellular signaling regulated kinase 1 and 2 (ERK1/2) participates in heat stress response. However, the effect of ERK1/2 on heat stress-induced lactate production is unclear. In the present study, Sertoli cells were isolated from immature boar testis and cultured at 32 °C. Heat stress was induced in a 43 °C incubator for 30 min. Proteins and RNAs were detected by western blotting and RT-PCR, respectively. Lactate production and lactate dehydrogenase (LDH) activity were detected using commercial kits. Heat stress promoted ERK1/2 phosphorylation, showing a reducing trend with increasing recovery time. In addition, heat stress increased heat shock protein 70 (HSP70), glucose transporter 3 (GLUT3), and lactate dehydrogenase A (LDHA) expressions, enhanced LDH activity and lactate production at 2-h post-heat stress. Pretreatment with U0126 (1?×?10^?6 mol/L), a highly selective inhibitor of ERK1/2 phosphorylation, reduced HSP70, GLUT3, and LDHA expressions and decreased LDH activity and lactate production. Meanwhile, ERK2 siRNA1 reduced the mRNA level of ERK2 and weakened ERK1/2 phosphorylation. Additionally, ERK2 siRNA1 reduced HSP70, GLUT3, and LHDA expressions decreased LDH activity and lactate production. Furthermore, HSP70 siRNA3 downregulated GLUT3 and LDHA expressions and decreased LDH activity and lactate production. These results show that activated ERK1/2 increases heat stress-induced lactate production by enhancing HSP70 expression to promote the expressions of molecules related to lactate production (GLUT3 and LDHA). Our study reveals a new insight in reducing the negative effect of heat stress in boars.     

Three-Dimensional Analysis of the Swimming Behavior of Daphnia magna Exposed to Nanosized Titanium Dioxide

Due to their surface characteristics, nanosized titanium dioxide particles (nTiO₂) tend to adhere to biological surfaces and we thus hypothesize that they may alter the swimming performance and behavior of motile aquatic organisms. However, no suitable approaches to address these impairments in swimming behavior as a result of nanoparticle exposure are available. Water fleas Daphnia magna exposed to 5 and 20 mg/L nTiO₂ (61 nm; polydispersity index: 0.157 in 17.46 mg/L stock suspension) for 96 h showed a significantly (p<0.05) reduced growth rate compared to a 1-mg/L treatment and the control. Using three-dimensional video observations of swimming trajectories, we observed a treatment-dependent swarming of D. magna in the center of the test vessels during the initial phase of the exposure period. Ensemble mean swimming velocities increased with increasing body length of D. magna, but were significantly reduced in comparison to the control in all treatments after 96 h of exposure. Spectral analysis of swimming velocities revealed that high-frequency variance, which we consider as a measure of swimming activity, was significantly reduced in the 5- and 20-mg/L treatments. The results highlight the potential of detailed swimming analysis of D. magna for the evaluation of sub-lethal mechanical stress mechanisms resulting from biological surface coating and thus for evaluating the effects of nanoparticles in the aquatic environment.     

Prevalence of kairomone-induced diapause in Daphnia magna from habitats with and without fish

The prevalence of the diapause response of the freshwater crustacean Daphnia magna to chemical cues on fish predation was evaluated in 35 clones originating from 14 European water bodies—inhabited or not inhabited by fish. Clonal lineages of experimental animals were cultured for 4 weeks in the presence or absence of water to which an extract of faeces of crucian carps (Carassius carassius), that were fed with Daphnia, was added. The proportion of females producing diapausing forms during the experimental period was used as a measure of the diapause response. A positive diapause response to fish predation cues was observed in 43% of investigated clones that originated from 71% of the water bodies tested. This indicated that the diapause response to fish scent is a common phenomenon in Daphnia magna from various locations in Europe. Surprisingly, no signs of a stronger diapause reaction to the tested cues were found in Daphnia originating from water bodies inhabited by fish compared with those from fish-free habitats.     

Direct and transgenerational impact on Daphnia magna of chemicals with a known effect on DNA methylation

The purpose of this study is to investigate (1) the induction of epigenetic effects in the crustacean Daphnia magna using DNA methylation as an epigenetic mark and (2) the potential stable transfer of such an epigenetic effect to non-exposed subsequent generations. Daphnids were exposed to chemical substances known to affect DNA methylation in mammals: vinclozolin, 5-azacytidine, 2′-deoxy-5-azacytidine, genistein and biochanin A. Effects on overall DNA cytosine methylation, body length and reproduction were evaluated in 21 day experiments. Using a multi-generational experimental design these endpoints were also evaluated in the F₁ and F₂ generation of both exposed and non-exposed offspring from F₀ daphnids exposed to 5-azacytidine, genistein or vinclozolin. A reduction in DNA methylation was consistently observed in daphnids exposed to vinclozolin and 5-azacytidine. Only in organisms exposed to 5-azacytidine was this effect transferred to the two subsequent non-exposed generations. A concurrent reduction in body length at day 7 was observed in these treatments. For the first time, exposure to environmental chemicals was shown to affect DNA methylation in the parental generation of D. magna. We also demonstrated a transgenerational alteration in an epigenetic system in D. magna, which indicates the possibility of transgenerational inheritance of environment-induced epigenetic changes in non-exposed subsequent generations.     

Gene Silencing of Selenoprotein K Induces Inflammatory Response and Activates Heat Shock Proteins Expression in Chicken Myoblasts

In the present study, specific small interfering RNA (siRNA) for selenoprotein K (Selk) gene was designed and transfected into chicken myoblasts. Then, the expressions of inflammatory factors (including induced nitric oxide synthase [iNOS], nuclear factor-kappa B [NF-κB], heme-oxygenase-1 [HO-1], cyclooxygenase-2 [COX-2], and prostaglandin E synthase [PTGEs]), inflammation-related cytokines (including interleukin [IL]-1β, IL-6, IL-7, IL-8, IL-17, and interferon [IFN]-γ), and heat shock proteins (HSPs) (including HSP27, HSP40, HSP60, HSP70, and HSP90) were examined at 24 and 72 h after transfection. The results showed that messenger RNA (mRNA) expressions of iNOS, NF-κB, HO-1, COX-2, IL-6, IL-7, IL-8, HSP 27, HSP 40, HSP 60, HSP 70, and HSP 90 were significantly increased (p < 0.05) at 24 and 72 h after siRNA transfection, and the mRNA expressions of PTGEs, IL-1β, IL-17, and IFN-γ were significantly increased and decreased (p < 0.05) at 24 and 72 h after siRNA transfection. The results also showed that the protein expressions of iNOS, NF-κB, HO-1, COX-2, HSP60, HSP70, and HSP90 were significantly increased (p < 0.05) at 24 and 72 h after siRNA transfection. The correlation analysis and principal component analysis (PCA) showed that PTGEs, IL-1β, IL-17, IFN-γ, HSP40, and HSP90 might play special roles in response to Selk silencing in chicken myoblasts. These results indicated that Selk silencing induced inflammation response by affecting the expression levels of inflammatory factors and inflammation-related cytokines, and the heat shock proteins might play protective roles in this response in chicken myoblasts.     

Effect of Different Levels of Selenium Supplementation on Growth Rate,Nutrient Utilization,Blood Metabolic Profile,and Immune Response in Lambs

The nutritional role of silver for the freshwater crustacean, Daphnia magna, was examined through four generations of deprivation. Silver inclusion in animal media was set at a nominal zero (employing chemicals of the highest available purity). Both reproduction (−60%) and life span (−40%) were negatively affected when compared to animals reared in the presence of 0.4 ng g⁻¹ Ag. These results strongly suggest a nutritional requirement of silver for daphnids at nanomolar concentrations.     

Final thermal preference in parthenogenetic females of Daphnia magna straus (Crustacea: Cladocera) acclimated to various temperatures

The final thermal preference FTP) range in parthenogenetic females of cladoceran Daphnia magna was assessed by “acute” and “chronic” methods. The first method included 4-month acclimation to different temperatures in the range of 14.2 ± 0.7 to 27.1 ± 0.3°C; the “chronic” method was characterized by long-term acclimation to +20°C. Two ranges of FTP were found for D. magna, 13.3–15.4°C and 20.2–26.2°C. The thermal preference of daphnids and the temperature of acclimation were correspondingly linearly. The range of FTP was independent of the season. The searching activity of D. magna rose in April, when the FTP range increased, and the FTP was less pronounced.     

Seasonal variation in HSP70 expression and oxidative stress in skin of zebu (Tharparkar) and crossbred (Karan Fries) cattle under tropical climate

The study aimed to evaluate inducible HSP70 (HSP70.1 and HSP70.2) gene expression and oxidative stress status in skin of cattle during different seasons. Ten each of Tharparkar (zebu) and Karan Fries (crossbred) heifers were selected from NDRI herd, Karnal. Animals were maintained under standard managemental practices followed at the farm. Skin biopsies were aseptically collected from each animal during winter, spring, and summer. Real time PCR was performed to examine HSP70 expression. Reactive oxygen species (ROS) and antioxidant enzymes (SOD and CAT) were determined by ELISA. In both the breeds, significantly higher (p < 0.05) levels of HSP70 expression, ROS, caspases, and antioxidant enzymes were observed during summer followed by winter and spring. Breeds showed no significant difference during winter and spring. During summer, HSP70 expression, ROS, and antioxidant enzymes were higher (p < 0.05) in Karan Fries than Tharparkar, whereas caspases levels were higher in Tharparker than Karan Fries. The study concludes that levels of HSP70 expression, ROS, caspases, and antioxidant enzymes in skin of cattle were strongly affected by seasonal change in temperature. Differences exist in skin tissue thermotolerance of Tharparkar and Karan Fries cattle. This might be an efficient and centrally important mechanism for better adaptability of zebu cattle to heat stress.     

Proteomic analysis reveals significant elevation of heat shock protein 70 in patients with chronic heart failure due to arrhythmogenic right ventricular cardiomyopathy

As proteins are the ultimate biological determinants of phenotype of disease, we screened altered proteins associated with heart failure due to arrhythmogenic right ventricular cardiomyopathy (ARVC) to identify biomarkers potential for rapid diagnosis of heart failure. By 2-dimensional gel electrophoresis and mass spectrometry, we identified five commonly altered proteins with more than 1.5 fold changes in eight ARVC failing hearts using eight non-failing hearts as reference. Noticeably, one of the altered proteins, heat shock protein 70 (HSP70), was increased by 1.64 fold in ARVC failing hearts compared with non-failing hearts. The increase of cardiac HSP70 was further validated by Western blot, immunochemistry, and enzyme-linked immunosorbent assay (ELISA) in failing hearts due to not only ARVC, but also dilated (DCM, n = 18) and ischemic cardiomyopathy (ICM, n = 8). Serum HSP70 was also observed to be significantly increased in heart failure patients derived from the three forms of cardiomyopathies. In addition, we observed hypoxia/serum depletion stimulation induced significantly elevation of intracellular and extracellular HSP70 in cultured neonatal rat cardiomyocytes. For the first time to our knowledge, we revealed and clearly demonstrated significant up-regulation of cardiac and serum HSP70 in ARVC heart failure patients. Our results indicate that elevated HSP70 is the common feature of heart failure due to ARVC, DCM, and ICM, which suggests that HSP70 may be used as a biomarker for the presence of heart failure due to cardiomyopathies of different etiologies and may hold diagnostic/prognostic potential in clinical practice.     

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.     

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.

     

A proliferative disorder of possible hemic origin in the common cockle,Cerastoderma edule

A study is described concerning the infestation of a laboratory-reared Daphnia magna culture by the fungus Aphanomyces daphniae. The origin of this fungus is Lake Huron, Michigan, the water from which is used in the laboratory to culture daphnids and maintain fishes for toxicological testing. Infestations originate from the Lake during the autumnal and vernal homothermal periods. The fungus is apparently maintained in the laboratory in conjunction with the fishes. Onset of fungal infestations in zooplankton culture apparently is initiated by three types of stress: rising temperatures, declining dissolved oxygen values, and poor nutrition. These infections have been eliminated by separating the daphnid culturing facility from the fish facility, substituting an algal diet for a synthetic one, sterilizing all water, air, and glassware connected with zooplankton culture, and maintaining the daphnids in a constant temperature room with controlled dissolved oxygen levels in the culturing vessels.     

The expression of DAMP proteins HSP70 and cancer-testis antigen SPAG9 in peripheral blood of patients with HCC and lung cancer

There are different views of how the immune system participates in the reaction to cancer. Here, we evaluated expression of DAMP proteins HSP70 and cancer-testis antigen SPAG9 in patients with hepatocellular carcinoma (HCC) and lung cancer to explore tumor immunity. Our analysis showed that levels of HSP70 and SPAG9 antibody were significantly higher in the serum of lung cancer and HCC patients than in the serum of healthy subjects (P < 0.001), but there were no differences in levels of HSP70 antibody in patients and controls. Levels of serum SPAG9 antibody in newly diagnosed lung cancer patients were significantly higher than in treated lung cancer patients (P < 0.05), but there were no differences in levels of HSP70 or HSP70 antibody. Levels of serum HSP70 and SPAG9 antibody, but not HSP70 antibody, were also higher in hepatitis/cirrhosis patients than in healthy subjects (P = 0.005, P < 0.001). Levels of serum SPAG9 antibody were significantly higher in HCC patients than in hepatitis/cirrhosis patients, but there were no differences in HSP70 or HSP70 antibody levels. Finally, levels of serum HSP70 and SPAG9 antibody were significantly higher in HCC patients than in lung cancer patients (P < 0.05, P < 0.001). These results indicate that cancer-testis antigen SPAG9 induces a strong humoral immune response in cancer patients but HSP70 does not. These results show that SPAG9 has potential as a tumor-specific biomarker.