Effects of ectoine on behavioural, physiological and biochemical parameters of Daphnia magna

Comparative Biochemistry and Physiology, Part C 168 (2015) 2–10

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc

Effects of ectoine on behavioural, physiological and biochemical parameters of Daphnia magna Adam Bownik a,⁎, Zofia Stępniewska b, Tadeusz Skowroński a a b

Department of Physiology and Ecotoxicology, Faculty of Biotechnology and Environmental Sciences, The John Paul II Catholic University of Lublin, Kontstantynow 1 “I”, 20-708 Lublin, Poland Department of Biochemistry Environmental Chemistry, Faculty of Biotechnology and Environmental Sciences, The John Paul II Catholic University of Lublin, Kontstantynow 1 “I”, 20-708 Lublin, Poland

a r t i c l e

i n f o

Article history: Received 4 September 2014 Received in revised form 5 November 2014 Accepted 7 November 2014 Available online 15 November 2014 Keywords: Ectoine Compatible solute Daphnia magna

a b s t r a c t Ectoine (ECT) is a compatible solute produced by soil, marine and freshwater bacteria in response to stressful factors. The purpose of our study was to determine the possible toxic influence of ECT on Daphnia magna. We determined the following endpoints: survival rate during exposure and recovery, swimming performance, heart rate, thoracic limb movement determined by image analysis, haemoglobin level by ELISA assay, catalase and nitric oxide species (NOx) by spectrophotometric methods. The results showed 80% survival of daphnids exposed to 50 mg/L of ECT after 24 h and 10% after 90 h, however lower concentrations of ECT were well tolerated. A concentrationdependent reduction of swimming velocity was noted at 24 and 48 h of the exposure. ECT (at 2.5 and 4 mg/L) induced an increase of heart rate and thoracic limb movement (at 2.5, 4 and 20 mg/L) after 24 h. After 10 h of the exposure to ECT daphnids showed a concentration-dependent increase of haemoglobin level synthesized and accumulated in the epipodite epithelia. After 24 h we noted a concentration-dependent decrease of haemoglobin level and its lowest value was found after 48 h of the exposure. ECT at a concentration of 20 and 25 mg/L slightly stimulated catalase activity after 24 h. NOx level was also increased after 10 h of the exposure to 20 and 25 mg/L of ECT reaching maximal activity after 24 h. Our results suggest that ECT possesses some modulatory potential on the behaviour, physiology and biochemical parameters in daphnids. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Selective intracellular accumulation of chemical compounds with low molecular weight, known as compatible solutes is one of various mechanisms of adaptation developed by organisms in order to survive in unfavourable conditions. These molecules protect whole cells and membrane proteins against stressful factors without interference with cellular processes (Brown, 1978; Foord and Leatherbarrow, 1998). As a result of accumulation of these solutes, cells maintain osmotic balance avoiding water loss and they also become more resistant to stress induced by changes of temperature, UV-radiation, high salinity and different oxidants (Pastor et al., 2013; Roessler and Müller, 2001). Compatible solutes can be divided into several structural groups: sugars (trehalose, sucrose), polyols (glycerol, sorbitol, mannitol, α-glucosyl-glycerol, mannosylglycerol, mannosyl-glyceramide), N-acetylated diamino acids (like N-acetylglutaminylglutamine amide), betaines (such as glycine betaine and derivatives), amino acids (proline, glutamate, glutamine, alanine, ectoine and hydroxyectoine) and derivatives (Pastor et al., 2013). Some organisms produce a variety of compatible solutes. For example, Escherichia coli was found to synthesize glycine, betaine and trehalose (Strøm, 1998). Most studies on the effects of compatible solutes were ⁎ Corresponding author. Tel.: +48 814545458. E-mail address: [email protected] (A. Bownik).

http://dx.doi.org/10.1016/j.cbpc.2014.11.001 1532-0456/© 2014 Elsevier Inc. All rights reserved.

performed on mammals, however some results suggesting their protective activity were obtained on tardigrades and brine shrimp enabling these animals to survive extreme conditions in dry state called anhydrobiosis (Westh and Ramlof, 1991; Yancey, 2004). Some results exist on the influence of compatible solutes on cladocerans. For example, trehalose was found to protect Daphnia magna and Triops against anhydrobiosis and freezing damage (Hengherr et al., 2011; Putman et al., 2012). There are several models explaining the mechanism of protective action of compatible solutes. “Preferential exclusion model” is a hypothesis which assumes that a compatible solute undergoes thermodynamic interactions with a protein (Timasheff, 2002). The protein repels the compatible solute from its surface to the bulk regions. As a result, the amount of the compatible solute close to the protein surface is lower but its concentration in the bulk regions increases. This state is caused by the accumulation of excess water molecules at the protein surface which results in preferential hydration of the macromolecule (Timasheff, 2002). The water molecules maintain the native protein protecting it from unfolding, particularly in stressful conditions. Ectoine (ECT) (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) is an amino acid produced by various bacteria species but mostly by aerobic, chemoheterotrophic, and halophilic bacteria (Galinski et al., 1985; Nagata and Wang, 2001). Microorganisms such as Marinococcus sp. ECT1 synthesize and accumulate intracellular ECT in response to osmotic stress in a hyperosmotic environment (Wei et al., 2011).

A. Bownik et al. / Comparative Biochemistry and Physiology, Part C 168 (2015) 2–10

Production of ectoine was also found in bacteria temporarily inhabiting aquatic environments such as Vibrio cholerae that must adjust to changes in ionic composition and osmolarity (Pflughoeft et al., 2003). Accumulation of ECT inside the cell protects bacterial cell membranes, enzymes and nucleic acids against hyperthermia. ECT was demonstrated to enhance the stability of phytase, lactic dehydrogenase (LDH) and phosphofructokinase (PFK), enzymes sensitive to heating, urea, freezing and drying, and freeze–thaw treating (Göller and Galinski, 1999; Knapp et al., 1999; Lippert and Galinski, 1992; Zhang et al., 2006). It was proposed that ECT increases the hydration of the cell surface improving the mobility of the lipid head groups and fluidizing the lipid layer. The increased fluidity may be advantageous for cell membranes to cope with extreme conditions like temperature or osmotic pressure and may accelerate repair mechanisms in some cells (Harishchandra et al., 2010). A lot of microbial products are known to induce various effects both in the invertebrates and vertebrates. Some of them induce toxic changes and other, such as vitamins may be beneficial (Bownik, 2010; Rossi et al., 2011). It is known that compatible solutes are protective molecules, however some of them, such as dimethylsulfide produced by marine algae and acrylate may act as predator repellents (Van Alstyne and Houser, 2003; Wolfe, 2000). We selected D. magna for the experimental animal of the present study since it is a microcrustacean sensitive to various toxic compounds and the most popular invertebrate used as a model animal in ecotoxicology with good organ visibility due to its transparency. It has been commonly used to study the effects of bioactive compounds and pharmaceuticals (Campbell et al., 2004 Villegas-Navarro et al., 2003 Pirow et al., 2001). Our previous studies indicated the protective effects of ectoine on survival of D. magna during heat stress (Bownik et al., 2014), however there is a lack of knowledge on the influence of this amino acid on cladocerans. Therefore, we aimed to determine the effects of pure ectoine on the survival, swimming velocity, heart rate, thoracic limb movement, haemoglobin, catalase and nitric oxide species levels in D. magna. 2. Material and methods 2.1. Culture method ECT preparation and experimental setup D. magna were cultured for several generations in 6 L tanks with 5 L of aerated culture medium on the window ledge in a laboratory under a light:dark period of 16 h:8 h. Daphnia culture medium was prepared following the ASTM standards (American Society of Testing and Materials, 1986). The medium was synthetic freshwater (48 mg of NaHCO3, 30 mg of CaSO4·2H2O, 30 mg of MgSO4 and 2 mg of KCl per litre of deionized water adjusted to a pH of 7.4), with a temperature of 23 ± 2 °C. The number of cultured daphnids was about 30 animals per litre. The animals were fed once daily with a few drops of powdered Spirulina (2 mg/L water) per tank and supplemented with a few drops per tank of 10 mg/L stock suspension of baker's yeast. Ectoine standard (ECT) (purchased from Sigma-Aldrich) of ≥ 99% purity produced by Halomonas elongata was diluted in Daphnia culture medium and used at appropriate concentrations. Neonates b24 h old of the 2nd–5th clutches were treated with different solutions of ECT. Daphnids that were not treated with ECT and maintained in clean medium only were treated as the control. 2.2. Determination of survival rate Survival rate of daphnids exposed to 2.5, 4, 20, 25 and 50 mg/L of was determined after 24, 48, 72 and 96 h. 10 animals (in triplicate) were placed in 150 mL glass beakers with 100 mL of appropriate solution of ECT. During the exposure the animals were observed for not typical behaviour and mortality. The immobilized daphnids were transferred to a microscope slide and monitored for mortality under a light microscope. The animals were treated as dead when no heart activity was

3

noted. After 96 h of the exposure the alive daphnids were transferred to the clean medium for recovery. 2.3. Swimming velocity Swimming behaviour of D. magna neonates treated with, 2.5, 4, 20 and 25 mg/L of ECT was analysed after 10, 24 and 48 h according to the experimental setup described by Shimizu et al. (2002) with some modifications. 10 daphnids were transferred from the culture tanks to one of the observation dishes of 35 mm diameter containing 3 mL of the appropriate concentration of ECT. Each concentration of ECT was done in triplicate. Swimming velocity of unexposed daphnids maintained in clean medium only was also determined. Swimming behaviour of ECTexposed and unexposed (control) animals in all observation dishes was recorded for a minimum of 1 min with a digital camera Nikon D3100 (with a resolution of 30 frames/s) mounted on a stand over the observation dish and processed with motion analysis software, Tracker®, version 4.82. After the examination the daphnids were returned to the beaker for continuation of the exposure. Vertical movement of Daphnia was negligible because of the very small depth of the medium present in the observation dish. The video file with the recorded trajectories of swimming Daphnia was analysed frame-by-frame with Tracker®. By clicking with the cursor on Daphnia image in separate frames, the programme plotted the whole trail left by a single individual (interpreted by the programme as a mass point) measuring its maximal, minimal and mean velocity (v) expressed in millimetres per second (mm/s). Since the animals moved virtually only in two dimensions swimming behaviour analysis was based on the trajectory represented by x and y coordinates. The velocities of ten daphnids calculated by software were plotted in separate graphs which were then superimposed. Since swimming speed was not equal for all individuals in each experimental and control group, the mean velocity (v) of 10 daphnids from each experimental group was meaned and treated as one result. 2.4. Heart rate and thoracic limb activity 10 neonate daphnids were treated (in triplicate) with 2.5, 4, 20 and 25 mg/L of ECT. Optical measurement of physiological parameters: heart rate and thoracic limb movement was done after 24, 48, 72 and 96 h. A single daphnid was transferred in a 50 μL droplet of appropriate concentration of ECT or clean medium to a microscope slide. The daphnid movement on the slide was limited by cotton wool fibres. The microscopic view of the examined daphnid was recorded for at least 2 min (with the speed of 30 frames per second) with a digital camera Nikon D3100 mounted on a light microscope. The magnification (30–100×) and camera resolution allowed us to perform the analysis with a good visibility of the heart and thoracic limbs. Heart rate and thoracic limb movement were analysed with Tracker® software by a frame-by-frame method. 2.5. Haemoglobin level 10 neonate daphnids exposed to ECT at concentrations of 2.5, 4, 20, 25 mg/L and 10 unexposed individuals were taken for determination of haemoglobin level after 10, 24 and 48 h. Daphnids from each experimental and control group of unexposed daphnids were homogenized in a pestle homogenizer with 300 μL of PBS (Phosphate Buffered Saline, Dulbecco). The resulting suspension was sonicated with an ultrasonic homogenizer (Omni Ruptor 4000) to disrupt the remaining cells and cell membranes. Afterwards, homogenates were centrifuged at 5000 g for 5 min. The supernatants were taken for the analysis of haemoglobin with a spectrophotometric enzyme-linked immunosorbent assay (CEB409Hu, USCN, USA). The test was performed according to the manufacturer's manual. Briefly, 100 μL of experimental samples was added to the appropriate wells of a 96-well antibody coated microplate in triplicate. The plate was covered with the plate sealer and incubated

4

A. Bownik et al. / Comparative Biochemistry and Physiology, Part C 168 (2015) 2–10

at 37 °C for 2 h. The liquid from each well was removed and 100 μL of detection reagent A (biotin-labelled haemoglobin) working solution was added to each well. The plate was incubated at 37 °C for 1 h after covering with the plate sealer. The solution was aspirated and washed with wash solution. The plate was then aspirated, inverted and blotted against the absorbent paper. 100 μL of detection reagent B (avidin conjugated to horseradish peroxidase) was added to each well and the plate was incubated for 30 min at 37 °C. Aspiration and wash were repeated 5 times and 90 μL of TMB (3,3′,5,5′-Tetramethylbenzidine) for horseradish peroxidase detection was added to each well and the microplate was incubated for 20 min at 37 °C with protection from light. Next, 50 μL of stop solution (sulfuric acid) was added to each well and the solution was mixed. The microplate was then read with a spectrophotometric microplate reader (Biorad 550) at 450 nm. The haemoglobin level was determined by referring the optical density of the samples of the experimental groups of daphnids exposed to ECT to those in the group of unexposed daphnids. 2.6. Catalase activity Catalase activity was determined with a spectrophotometric method described by Goth (1991) with our slight modification to a micromethod. Briefly, ten individuals from the experimental and control groups were taken after 10, 24, 48 h of exposure to 2.5, 4, 20 and 25 mg/L of ECT and homogenized in a pestle micro-homogenizer with 300 μL of phosphate buffer. The homogenized samples were centrifuged and 100 μL of supernatants was transfered to a 96-well microplate and incubated with 100 μL of 60 μM H2O2/60 mM sodium potassium buffer at room temperature for 60 s in a 96-well microtiter plate. The enzymatic reaction was stopped by the addition of 100 μL of ammonium molybdate (32 mM) and the absorbance of the yellow molybdate/hydrogen peroxide complex was measured with a spectrophotometric microplate reader (Biorad 550) at 415 nm. All samples were done in triplicate. A mixture of ammonium molybdate and the buffer was treated as blank. Catalase activity was determined by referring the optical density in the samples from the experimental groups of daphnids exposed to ECT to those in the group of unexposed daphnids.

using Develve® statistical software. Values were statistically significant when p b 0.05. 3. Results 3.1. Survival rate Survival rate of daphnids exposed to various concentrations of ECT is presented in Fig. 1. The decrease of survival occurred after 24 h of the exposure to 50 mg/L of ECT (80 ± 11%). Further incubation of experimental animals at that concentration resulted in increased mortality. 70 ± 9% and 60 ± 3% of living daphnids were noted after 48 and 72 h, respectively. 10 ± 1% survival was noted after 96 h. Daphnids exposed to lower concentrations showed 100% survival after 24 and 48 h of the exposure. However, a slight decrease of daphnid survival was observed at 25 mg/L after 72 (90 ± 10%) and 96 h (80 ± 8%) and 20 mg/L (90 ± 6%) after 96 h. After the exposure to different concentrations of ECT daphnids were transferred to clean medium for recovery. 10% of survived daphnids transferred from ECT at a concentration of 50 mg/L to clean medium were alive over the next 96 h. Also, 80 ± 9% survival of animals that were previously exposed to 20 mg/L of ECT was reduced during recovery to 60 ± 9% after 96 h. No mortality during recovery was observed in the group of daphnids previously exposed to 2.5 and 4 mg/L of ECT over 96 h. 3.2. Swimming velocity Swimming velocity of daphnids exposed to ECT and exemplary mean velocities of three randomly selected daphnids measured with Tracker® software are presented in Fig. 2. The most inhibited motility of daphnids was found at 25 mg/L of ECT after 24 and 48 h (1.71 ± 0.8 and 1.2 ± 0.6 mm/s, respectively). A significant, concentration- and time-dependent decrease of velocity was also seen at 20 and 4 mg/L

2.7. Nitric oxide species (NOx) Nitric oxide (NO) has a short half-life, therefore analysis of its derivative, NO2 was used which is a stable end product and a surrogate marker of nitric oxide metabolism. Briefly, ten daphnids from the experimental (exposed to ECT at a concentration of 2.5, 4, 20 and 25 mg/L) and control (unexposed to ECT) groups were taken after 10, 24 and 48 h and washed in artificial medium, then dried on a paper towel and homogenized in a pestle micro-homogenizer in 300 μL of PBS. The suspension was sonicated with an ultrasonic homogenizer (Omni Ruptor 4000) to disrupt the remaining cells and cell membranes. Afterwards, homogenates were centrifuged at 5000 g for 5 min. After centrifugation, 100 μL of supernatant was placed in a well of a 96-well microplate. The wells from each experimental and control group were done in triplicate. NOx level was measured by the Griess reaction (Griess, 1879; Smith et al., 2000) by adding 50 μL of 1% sulfanilamide in 5% H3PO4 to each well and subsequently 50 μL of naphthylethylenediamine dihydrochloride in distilled water. The microplate was incubated at room temperature for 15 min and the absorbance was read with a spectrophotometric microplate reader (Biorad 550) at 550 nm. NOx activity was determined by referring the optical density in the samples from the experimental groups of daphnids exposed to ECT to those in the group of unexposed daphnids. Results are presented as means ± standard deviation (SD). All data were assessed for homogeneity of variance for ANOVA assumptions. Experimental data were analysed using ANOVA followed by Tukey's test to detect differences among means. All analyses were completed

Fig. 1. Mean survival time of Daphnia magna exposed to different concentrations of ECT during exposure (panel a) and subsequent recovery (panel b). All points represent the mean values of three replicates ± respective standard deviations, n = 30.

A. Bownik et al. / Comparative Biochemistry and Physiology, Part C 168 (2015) 2–10

5

Fig. 2. Mean swimming velocity of Daphnia magna exposed to different concentrations of ECT for 10, 24 and 48 h. Panel a shows the mean velocity of 10 daphnids exposed to each concentration of ECT. Results are presented as means ± SD, * — statistical significance, p b 0.05. Panels b, c and d present exemplary swimming velocity of three randomly selected control ECT-free daphnids (b) and exposed to 20 (c) and 25 mg/L (d) for 24 h. The x axis is time (t) and y axis is velocity (v) of daphnids. Mean velocities of three randomly selected individuals were superimposed.

with the values of 1.93 ± 0.34 and 3.2 ± 1.02 mm/s after 24 h and 1.6 ± 0.21 and 3.01 ± 0.5 mm/s after 48 h, respectively. Slight, but statistically significant reduction of daphnid swimming speed was noted at 2.5 mg/L of ECT (3.7 ± 0.7 and 3.6 ± 0.9 mm/s after 24 and 48 h, respectively). The exemplary graphs b, c and d in Fig. 2 (that are images from Tracker® showing velocities of three daphnids during a 60 s video clip). demonstrate that the mean velocity of daphnids at 20 (panel c) and 25 mg/L (panel d) of ECT was reduced when compared to the control group (panel b).

3.3. Heart rate Effects induced by various concentrations of ECT on heart rate of D. magna are presented in Fig. 3. Optical measurement with a light microscope revealed that after a 10-hour exposure heart rate was not altered in any concentration of ECT. However, the microcrustaceans exposed to 2.5 and 4 mg/L showed increased heart activity (468 ± 15 and 500 ± 17 bpm, respectively) after 24 h of the exposure when compared to that in the control, unexposed daphnids (396 ± 13 bpm). A slight

6

A. Bownik et al. / Comparative Biochemistry and Physiology, Part C 168 (2015) 2–10

Fig. 3. Heart rate of Daphnia magna exposed for 10, 24 and 48 h to various concentrations of ectoine (ECT). Results are presented as means ± SD, * — statistical significance, p b 0.05, n = 30.

decrease of heart rate was observed at 20 and 25 mg/L of ECT with frequencies of 340 ± 9 and 320 ± 12 bpm, respectively. Similar heart rate was found after 48 h of the exposure. Increased values of frequency was observed at 2.5 and 4 mg/L of ECT (450 ± 16 and 450 ± 12 bpm, respectively), however slightly lower than those after 24 h. Heart rate of daphnids exposed to the highest concentrations of 20 and 25 mg/L of ECT (350 ± 11 and 340 ± 11 bpm, respectively) was still decreased after 48 h of the exposure. 3.4. Thoracic limb activity The effects of different concentrations of ECT are presented in Fig. 4. Thoracic limb activity of daphnids was not changed after a 10 hourexposure at any concentration of ECT. On the other hand, ECT at 20, 4 and 2.5 mg/L stimulated the daphnid limb activity to 270 ± 12, 265 ± 23 and 264 ± 11 bpm, respectively in comparison to the unexposed control group (203 ± 20 bpm). After 48 h of the exposure the limb activity was still increased at those concentrations when compared

to the control (191 ± 20 bpm) but it began to decrease to 243 ± 13, 250 ± 17 and 264 ± 14 bpm in daphnids exposed to 2.5, 4 and 20 mg/L, respectively. However, ECT at a concentration of 25 mg/L reduced the limb activity after 24 and 48 h to 172 ± 14 and 160 ± 17 bpm, respectively. 3.5. Haemoglobin level Haemoglobin level in daphnids exposed to various concentrations of ECT is presented in Fig. 5. Haemoglobin was detected in the homogenates of animals in each experimental group including the control daphnids, however its level was altered in each experimental group with ECT. Haemoglobin level determined by the ELISA assay was reverse proportional to OD values. Stimulation of haemoglobin amount was noted after 10 h of the exposure to 25 mg/L of ECT (OD = 0.3 ± 0.075) compared to the control sample with no haemoglobin (OD = 1.08 ± 0.089) and control unexposed group (0.44 ± 0.09). Examination of daphnids exposed to 25 mg/L of ECT under a light microscope showed

Fig. 4. Thoracic limb activity in Daphnia magna exposed for 10, 24 and 48 h to various concentrations of ectoine (ECT). Results are presented as means ± SD, * — statistical significance, p b 0.05, n = 30.

A. Bownik et al. / Comparative Biochemistry and Physiology, Part C 168 (2015) 2–10

7

Fig. 5. Haemoglobin level in neonates of Daphnia magna exposed to ectoine (ECT). Panel a) presents haemoglobin level in daphnids exposed for 10, 24 and 48 h to various concentrations of ECT. The haemoglobin level determined by ELISA assay is reverse proportional to optical density (OD). Panel b presents percentage of daphnids with haemoglobin synthesized and accumulated in the epipodites after 10-hour exposure. Results are presented as means ± SD, * — statistical significance, p b 0.05. Panel c presents microscopic images of an exemplary neonate daphnid with accumulated haemoglobin during 10-hour exposure to ECT at 25 mg/L. Reddish areas marked with arrows are epipodites with synthesized haemoglobin. Image d) shows a pale neonate after 24-hour exposure to ECT at 25 mg/L. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

reddish epipodites in 90 ± 10% of individuals suggesting stimulation of haemoglobin synthesis (Fig. 5 b, c and d). Production of haemoglobin was also observed at lower concentrations of ECT: 2.5 mg/L (in 10 ± 10% daphnids), 4 mg/L (in 20 ± 10% daphnids) and 20 mg/L (in 80 ± 20% daphnids). No reddish epipodites were found in the control daphnids. An increase of haemoglobin level was also found in daphnids exposed to ECT at 2.5, 4 and 20 mg/L with OD values of 0.38 ± 0.078, 0.37 ± 0.017 and 0.35 ± 0.012, respectively. Continuation of exposure

to 25 mg/L of ECT resulted in a decrease of haemoglobin level (OD = 0.67 ± 0.13) after 24 h in comparison to the unexposed control. A less significant decrease of haemoglobin level was observed at and 2.5, 4 and 20 mg/L with OD values of 0.44 ± 0.078, 0.49 ± 0.018 and 0.50 ± 0.011, respectively. After 48 h of the experiment further reduction of haemoglobin level at concentrations of 2.5, 4, 20 and 25 mg/L of ECT was noted with OD values of 0.744 ± 0.021, 0.767 ± 0.011, 0.741 ± 0.019 and 0.783 ± 0.07, respectively.

Fig. 6. Catalase level in Daphnia magna neonates exposed for 10, 24 and 48 h to various concentrations of ectoine (ECT). Results are presented as means ± SD, * — statistical significance, p b 0.05.

8

A. Bownik et al. / Comparative Biochemistry and Physiology, Part C 168 (2015) 2–10

3.6. Catalase activity The results on catalase activity of D. magna exposed to ECT at different concentrations are presented in Fig. 6. A 10-hour exposure even to the highest concentration of ECT resulted in no significant changes in catalase activity when compared to the control ECT-free group. However, an increase of catalase activity was observed in daphnids after 24 h of the exposure to 20 mg/L (OD = 0.199 ± 0.045) and 25 mg/L (OD = 0.21 ± 0.032) of ECT when compared to the control (OD = 0.165 ± 0.03). Slight stimulation of catalase activity to OD values of 0.189 ± 0.045 and 0.19 ± 0.034 was also observed in daphnids exposed to concentrations of 2.5 and 4 mg/L of ECT, respectively, however, the differences between the experimental and control groups were insignificant. After 48 h of exposure catalase activity returned to the control values. 3.7. NOx production The production of NOx in daphnids exposed to different concentrations of ECT is presented in Fig. 7. A 10-hour exposure to ECT at 20 and 25 mg/L resulted in the increased level of NOx (OD = 0.153 ± 0.012 and 0.16 ± 0.011, respectively) when compared to the control (OD = 0.13 ± 0.15). No changes were noted at 2.5 and 4 mg/L of ECT. Further stimulation of NOx production was seen after 24 h of the exposure in the animals treated with 20 and 25 mg/L of ECT (OD = 0.172 ± 0.014 and 0.174 ± 0.012, respectively). The NOx level in daphnids exposed to ECT at lower concentrations of 2.5 and 4 mg/L (OD = 0.123 ± 0.013 and 0.122 ± 0.015, respectively) was similar to that in the control, unexposed daphnids (OD = 0.13 ± 0.013) and changes had no statistical significance. After 48 h, the animals exposed to 20 and 25 mg/L of ECT still showed increased levels of NOx (OD = 0.154 ± 0.015 and 165 ± 0.013, respectively), however they were slightly lower than those after 24 h. 4. Discussion Compatible solutes are molecules which do not interfere with cellular processes, even when they are accumulated at high concentrations. Most results on the biological activity of compatible solutes, for example taurine, glycine or trehalose are associated with protection of animals against various stressors (Chen et al., 2012; Churchill et al., 1995; Luyckx and Baudouin, 2011; Wang et al., 2008). Ectoine, which is known to be an amino acid with osmoprotective activity, may reach high concentrations inside the bacterial cell without inducing detrimental

effects. Its protective influence and low toxicity were also described in isolated mammalian cells in the in vivo and in vitro conditions (Graf et al., 2008), which implicated its application in the treatment of atopic dermatitis and atopic eczemas, and gastrointestinal and pulmonary diseases (Abdel-Aziz et al., 2013; Marini et al., 2014; Sydlik et al., 2009). Although ectoine is a water-binding molecule which may affect the biology of aquatic organisms, the knowledge on its effects on aquatic organisms is very scarce. Our preliminary toxicity tests (Bownik and Stępniewska, unpublished) showed no significant differences in the protective effects of 95% pure and 99% pure ECT in daphnids subjected to high salinities. This suggests that the protective effects of 95% pure ECT on heatstressed daphnids reported by Bownik et al. (2014) were indeed due to ectoine itself and not to any impurity. Nevertheless, the present study was performed with 99% pure ECT to obtain results for our further research in which combinatorial exposure together with other bioactive factors would be used. Such studies would require chemicals of very high purity. To our best knowledge this is the first study showing the influence of ECT on the behavioural, physiological and biochemical levels in D. magna. ECT, particularly at lower concentrations used in the study is well-tolerated by daphnids. The two lowest concentrations of ECT did not decrease the survival of experimental animals, on the other hand, its highest concentration turned out be lethal after 24 h and the survival was decreasing proportionally to time of exposure. Currently, it is not possible to explain the cause of daphnid mortality induced by ECT at high concentrations since very little is known of the mechanisms of toxicity of ECT and metabolism of this amino acid in cladocerans. However it should not be linked to oxidative stress, since catalase activity was not significantly increased. Since ECT turned out to be lethal at 50 mg/L, further behavioural, physiological and biochemical assays were performed with the use of lower concentrations. Swimming behaviour of D. magna has been used as a reliable biomarker of effects induced by various stressful factors, particularly those which affect the neuromuscular system (Oliveira et al., 2013; Shimizu et al., 2002; Silva et al., 2013). There are many endpoints of swimming behaviour, however mean velocity is usually chosen as the most reliable one. Our study revealed that ECT inhibits the swimming velocity of daphnids in a concentration-dependent manner. Reduction of the velocity may be linked to reduced neuromotor activity in daphnids exposed to the highest concentrations of ECT. Some other compatible solutes, amino acids like taurine and glycine are also found to depress neuromuscular transmission in some invertebrates (Finger, 1983). The crustaceans exposed to the highest concentration of ECT showed abnormal swimming after 24 h characterized by temporal

Fig. 7. NOx level in Daphnia magna exposed for 10, 24 and 48 h to different concentrations of ectoine (ECT). Results are presented as means ± SD, * — statistical significance, p b 0.05.

A. Bownik et al. / Comparative Biochemistry and Physiology, Part C 168 (2015) 2–10

long periods without movement. This suggests that high concentrations of ECT may induce neuromuscular transmission inhibition. Some authors link swimming velocity with the level of endogenous nitric oxide which is a signalling molecule involved in the regulation of swimming performance of animals and human sperm cells (Martínez, 1995) mediated by the NO/cGMP signalling pathway (Miraglia et al., 2011; Moroz et al., 2004). However, it seems that the decrease of swimming velocity in ECT-exposed D. magna was rather not NOx-mediated since its level at lower concentrations of ECT was similar to that in the unexposed daphnids and the velocity was significantly decreased when compared to that in the unexposed group. Nitric oxide is not stable in the in vitro conditions, therefore its surrogate species such as NO2− and NO3 are used as its biomarkers. The present study showed that ECT increased the production of NOx after a 10 hour exposure to the highest concentrations of ECT reaching maximal values after 24 h. In spite of further exposure the level of NOx was slightly decreased. Nitric oxide when it is present at lower concentration may act as an antioxidant scavenging free radicals, however at higher concentrations it may be a prooxidant (Albrecht et al., 2003; Mohanakumar et al., 2002). The increase of NOx induced by the highest concentration of ECT was not significant, therefore, it can be concluded that this amino acid may induce some antioxidative effects during oxidative stress in crustaceans. Heart rate and thoracic limb movement of D. magna are common physiological endpoints that have been used by some authors for the assessment of the toxic action of some chemicals and pharmaceuticals (Campbell et al., 2004; Pirow et al., 2001; Villegas-Navarro et al., 2003). We found that ECT affects the heart rate and thoracic limb activity of daphnids. Lower concentrations of this amino acid slightly stimulated heart beat frequency but its higher concentrations induced the opposite effect. The mechanism of positive inotropic effects of ECT at lower concentrations is not known, however it may be similar to that of taurine, associated with modulation of ion pump functioning and alteration of Ca2+ current in the heart muscle. The increase of heart rate may enhance the haemolymph flow, better the distribution of nutrients and improve gas exchange. Currently, no data exist on the effects of ECT or other compatible solutes on the heart rate in D. magna but similar effects on mammalian heart functioning were induced by taurine which was described to stimulate the heart rate in mammals (Azuma et al., 1985; Baskin and Finney, 1979). It is generally accepted that taurine, plays a very important role in mammalian heart muscle contraction (Schaffer et al., 2010). In normal conditions this amino acid is accumulated in cardiomyocytes and impaired heart contractions are usually associated with its decreased levels (Kocsis et al., 1976). The results from our studies and those obtained by other authors seem to confirm the hypothesis that some compatible solutes may affect the heart rate both in vertebrates and in invertebrates. ECT turned out to have a modulatory influence on thoracic limb activity. Stimulation of beating rate was found at its lower concentrations and inhibitory effects were noted at the highest concentration of the amino acid. Movement of thoracic appendages is an important physiological parameter indicating food acquisition and gas exchange in cladocerans (Fryer, 1991; Pirow et al., 1999). On the basis of our findings we may conclude that ECT at lower concentrations seems to be a slight enhancer of thoracic limb activity and, in a consequence, may enhance food uptake and ventilation in daphnids. The mechanisms of stimulation of thoracic limb movement probably associated with alteration of ion currents require further research. D. magna may increase haemoglobin production by stimulation with various factors, such as terpenoid hormones or in response to hypoxia (Rider and LeBlanc, 2006). The present study revealed a transient increase of haemoglobin level after 10 h of exposure to ECT at the highest concentration and its accumulation in the epipodites. The microscopic observations showed that daphnids turned reddish in the lumen of the epipodites where haemoglobin is synthesized (Smaridge, 1956). The temporal increase of haemoglobin level was probably a result of

9

the adaptation of daphnids to ECT, however the molecular mechanism of this induction is not known. Interestingly, after 24 h daphnids turned pale showing a decrease of haemoglobin level which was lower than that in the group of unexposed daphnids. After 48 h the level was even lower at each concentration of ECT by two-fold compared to the daphnids that were not exposed to ECT. This observation raised further questions, for example whether ECT may enhance the use of oxygen in daphnids or whether it may increase water aeration. Further investigations are also needed to explain the mechanism of ECT-induced haemoglobin synthesis and assess its detrimental or beneficial effects for crustaceans. Catalase is one of the most important antioxidants increasing its activity during oxidative stress in most organisms, also in D. magna (Alberto et al., 2011; Kim et al., 2010). Our previous studies showed that ECT acts as a heat stress-protectant without the induction of catalase during heat stress (Bownik et al., 2014) but no data could be found on the effects of ECT on this antioxidant without concomitant stressful factors on catalase activity in the invertebrates. Although a variety of bacterial products such as toxins are shown to induce oxidative stress and increase the levels of different antioxidants, we found that ECT is not a potent catalase inducer in cladocerans. Only the two highest concentrations used were able to induce a slight increase of the enzyme activity after 24 h. Despite stimulation of catalase, it does not suggest a response of daphnids to oxidative stress because of its little difference when compared to its level in the control, unexposed daphnids. It may be also possible that the transient increase of catalase activity (and also other parameters such as haemoglobin) found in our study might be a result of a temporal reaction of daphnids to slight stress (not oxidative) associated with their transfer from stock culture to ECT solution with altered density. Results from the other studies showed that catalase level may be altered without oxidative stress but by temperature changes and oxygen decrease (Becker et al., 2011). Temporal alteration of catalase activity was demonstrated to occur in ectothermal animals during seasonal changes (Radovanovic et al., 2010). Protective properties of ECT towards catalase and other enzymes have been used in some antioxidative cosmetic formulations (Patent, EP, 1100453 A1). Modulatory effects on catalase activity and its excretion in mammals were also observed after the application of another compatible solute, taurine (Ward et al., 2001). The results of our study suggest that ECT shows some level of toxicity to D. magna but only at the highest concentrations. ECT turned out to be a very effective agent in the alteration of behaviour, and physiological and biochemical parameters of daphnids even at lower concentrations without the induction of toxicity, perhaps due to its water binding ability and specific interaction with cell membranes. References Abdel-Aziz, H., Wadie, W., Abdallah, D.M., Lentzen, G., Khayyal, M.T., 2013. Novel effects of ectoine, a bacteria-derived natural tetrahydropyrimidine, in experimental colitis. Phytomedicine 20, 585–591. Alberto, A.C., Rocío, O.B., Fernando, M.J., 2011. Age effect on the antioxidant activity of Daphnia magna (Anomopoda: Daphniidae): does younger mean more sensitivity? J. Environ. Biol. 32, 481–487. Albrecht, E., Stegeman, C., Heeringa, P., Hanning, R., van Goor, H., 2003. Protective role of endothelial nitric oxide synthase. J. Pathol. 199, 8–17. American Society of Testing and Materials, 1986. Standard practice for conducting static acute toxicity tests on wastewaters with Daphnia. Annual Book of ASTM Standardsvol vol. 11.04. ASTM International, Philadelphia, pp. D4229–D4284. Azuma, J., Sawamura, A., Awata, N., Ohta, H., Hamaguchi, T., Harada, H., Takihara, K., Hasegawa, H., Yamagami, T., Ishiyama, T., Iwata, H., Kishimoto, S., 1985. Therapeutic effect of taurine in congestive heart failure: a double-blind crossover trial. Clin. Cardiol. 8, 276–282. Baskin, S.I., Finney, C.M., 1979. Effects of taurine and taurine analogues on the cardiovascular system. Sulfur Containing Amino Acids 2, 1. Becker, D., Brinkmann, B.F., Zeis, B., Paul, R.J., 2011. Acute changes in temperature or oxygen availability induce ROS fluctuations in Daphnia magna linked with fluctuations of reduced and oxidized glutathione, catalase activity and gene (haemoglobin) expression. Biol. Cell. 103, 351–363. Bownik, A., 2010. Harmful algae: effects of alkaloid cyanotoxins on animal and human health. Toxin Rev. 29, 99–114. Bownik, A., Stępniewska, Z., Skowronski, T., 2014. Protective effects of ectoine in heatstressed Daphnia magna. J. Comp. Physiol. B 184, 961–976.

10

A. Bownik et al. / Comparative Biochemistry and Physiology, Part C 168 (2015) 2–10

Brown, A.D., 1978. Compatible solutes and extreme water stress in eukaryotic microorganisms. Adv. Microb. Physiol. 17, 181–242. Campbell, A.K., Wann, K.T., Matthews, S.B., 2004. Lactose causes heart arrhythmia in the water flea Daphnia pulex. Comp. Biochem. Physiol. B 139, 225–234. Chen, G., Nan, C., Tian, J., Jean-Charles, P., Li, Y., Weissbach, H., Huang, X.P., 2012. Protective effects of taurine against oxidative stress in the heart of MsrA knockout mice. J. Cell. Biochem. 113, 3559–3566. Churchill, T.A., Green, C.J., Fuller, B.J., 1995. Protective properties of amino acids in liver preservation: effects of glycine and a combination of amino acids on anaerobic metabolism and energetics. J. Hepatol. 23, 720–726. EP 1100453 A1 Patent., 1991. Cosmetic formulation containing ectoine derivatives, enzymes, vitamins and/or vitamin derivatives. Bünger, J., Driller, H-J. Finger, W., 1983. Effects of glycine on the crayfish neuromuscular junctions. Pflugers Arch. 397, 128–134. Foord, R.L., Leatherbarrow, R.J., 1998. Effect of osmolytes on the exchange rates of backbone amide protons in proteins. Biochemistry 37, 2969–2978. Fryer, G., 1991. Functional morphology and the adaptive radiation of the Daphniidae (Branchiopoda: Anomopoda). Philos. Trans. R. Soc. Lond. B 331, 1–99. Galinski, E.A., Pfeiffer, H.P., Truper, H.G., 1985. 1, 4, 5, 6-Tetrahydro-2-methyl-4pyrimidinecarboxylic acid. A novel cyclic amino acid from halophilic phototrophic bacteria of the genus Ectothiorhodospira. Eur. J. Biochem. 149, 135–139. Göller, K., Galinski, E.A., 1999. Protection of a model enzyme (lactate dehydrogenase) against heat, urea and freeze–thaw treatment by compatible solute additives. J. Mol. Catal. B 7, 37–45. Goth, L., 1991. A simple method for determination of serum catalase activity and revision of reference range. Clin. Chim. Acta 196, 143–151. Graf, R., Anzali, S., Buenger, J., Pfluecker, F., Driller, H., 2008. The multifunctional role of ectoine as a natural cell protectant. Clin. Dermatol. 26, 326–333. Griess, P., 1879. Bemerkungen zu der abhandlung der H.H. Weselsky und Benedikt “Ueber einige azoverbindungen”. Chem. Ber. 12, 426–428. Harishchandra, R.K., Wulff, S., Lentzen, G., Neuhaus, T., Galla, H.J., 2010. The effect of compatible solute ectoines on the structural organization of lipid monolayer and bilayer membranes. Biophys. Chem. 150, 37–46. Hengherr, S., Heyer, A.G., Brümmer, F., Schill, R.O., 2011. Trehalose and vitreous states: desiccation tolerance of dormant stages of the crustaceans Triops and Daphnia. Physiol. Biochem. Zool. 84, 147–153. Kim, J., Kim, S., An, K.W., Choi, C.Y., Lee, S., Choi, K., 2010. Molecular cloning of Daphnia magna catalase and its biomarker potential against oxidative stresses. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 152, 263–269. Knapp, S., Ladenstein, R., Galinski, E.A., 1999. Extrinsic protein stabilization by the naturally occurring osmolytes b-hydroxyectoine and betaine. Extremophiles 3, 191–198. Kocsis, J.J., Kostos, V.J., Baskin, S.I., 1976. Taurine levels in the heart tissues of various species. In: Huxtable, R., Barbeau, A. (Eds.), Taurine. Raven Press, pp. 145–153. Lippert, K., Galinski, E.A., 1992. Enzyme stabilization by ectoine-type compatible solutes: protection against heating, freezing and drying. Appl. Microbiol. Biotechnol. 37, 61–65. Luyckx, J., Baudouin, C., 2011. Trehalose: an intriguing disaccharide with potential for medical application in ophthalmology. Clin. Ophthalmol. 5, 577–581. http://dx.doi. org/10.2147/OPTH.S18827 (Epub 2011 May 10). Marini, A., Reinelt, K., Krutmann, J., Bilstein, A., 2014. Ecotine-containing cream in the treatment of mild to moderate atopic dermatitis: a randomised, comparatorcontrolled, intra-individual double-blind, multi-center trial. Skin Pharmacol. Physiol. 27, 57–65. Martínez, A., 1995. Nitric oxide synthase in invertebrates. Histochem. J. 27, 770–776. Miraglia, E., De Angelis, F., Gazzano, E., Hassanpour, H., Bertagna, A., Aldieri, E., Revelli, A., Ghigo, D., 2011. Nitric oxide stimulates human sperm motility via activation of the cyclic GMP/protein kinase G signaling pathway. Reproduction 141, 47–54. Mohanakumar, K., Thomas, B., Sharma, S., Muralikrishnan, D., Chowdhury, R., Chiueh, C., 2002. Nitric oxide: an antioxidant and neuroprotector. Ann. N. Y. Acad. Sci. 962, 389–401. Moroz, L.L., Meech, R.W., Sweedler, J.V., Mackie, G.O., 2004. Nitric oxide regulates swimming in the jellyfish Aglantha digitale. J. Comp. Neurol. 471, 26–36. Nagata, S., Wang, Y.B., 2001. Accumulation of ectoine in the halotolerant Brevibacterium sp. JCM6894. J. Biosci. Bioeng. 91, 288–293. Oliveira, C., Almeida, J.R., Guilhermino, L., Soares, A.M., Gravato, C., 2013. Swimming velocity, avoidance behavior and biomarkers in Palaemon serratus exposed to fenitrothion. Chemosphere 90, 936–944.

Pastor, J.M., Bernal, V., Salvador, M., Argandoña, M., Vargas, C., Csonka, L., Sevilla, Á., Iborra, J.L., Nieto, J.J., Cánovas, M., 2013. Role of central metabolism in the osmoadaptation of the halophilic bacterium Chromohalobacter salexigens. J. Biol. Chem. 288, 17769–17781. Pflughoeft, K.J., Kierek, K., Watnick, P.I., 2003. Role of ectoine in Vibrio cholerae osmoadaptation. Appl. Environ. Microbiol. 69, 5919–5927. Pirow, R., Wollinger, F., Paul, R.J., 1999. Importance of the feeding current for oxygen uptake in the water flea Daphnia magna. J. Exp. Biol. 202, 553–562. Pirow, R., Bäumer, C., Paul, R.J., 2001. Benefits of haemoglobin in the cladoceran crustacean Daphnia magna. J. Exp. Biol. 204, 3425–3441. Putman, A., Panis, B., Vanvlasselaer, E., De Meester, L., 2012. Cryopreservation of Daphnia magna genotypes. Cryobiology 65, 348. Radovanovic, T.B., Borkovic, Mitic, S.S., Perendija, B.R., Despotovic, S.G., Pavlovic, S.Z., Cakic, P.D., Saicic, Z.S., 2010. Superoxide dismutase and catalase activities in the liver and muscle of barbel (Barbus barbus) and its intestinal parasite (Pomphoryinchus laevis) from the Danube river, Serbia. Arch. Biol. Sci. 62, 97–105. Rider, C.V., LeBlanc, G.A., 2006. Atrazine stimulates hemoglobin accumulation in Daphnia magna: is it hormonal or hypoxic? Toxicol. Sci. 93, 443–449. Roessler, M., Müller, V., 2001. Osmoadaptation in bacteria and archaea: common principles and differences. Environ. Microbiol. 3, 743–754. Rossi, M., Amaretti, A., Raimondi, S., 2011. Folate Production by Probiotic Bacteria Toxicol. 17, 441–448. Schaffer, S.W., Jong, C.J., Ramila, K.C., Azuma, J., 2010. Physiological roles of taurine in heart and muscle. J. Biomed. Sci. 17 (Suppl. 1), S2. Shimizu, N., Ogino, C., Kawanishi, T., Hayashi, Y., 2002. Fractal analysis of Daphnia motion for acute toxicity bioassay. Environ. Toxicol. 17, 441–448. Silva, C., Oliveira, C., Gravato, C., Almeida, J.R., 2013. Behaviour and biomarkers as tools to assess the acute toxicity of benzo(a)pyrene in the common prawn Palaemon serratus. Mar. Environ. Res. 90, 39–46. Smaridge, M.W., 1956. Distribution of iron in Daphnia in relation to hemoglobin synthesis and breakdown. Q. J. Microsc. Sci. 97, 205–214. Smith, K.L., Galloway, T.S., Depledge, M.H., 2000. Neuro-endocrine biomarkers of pollution-induced stress in marine invertebrates. Sci. Total Environ. 262, 185–190. Strøm, A.R., 1998. Osmoregulation in the model organism Escherichia coli: genes governing the synthesis of glycine betaine and trehalose and their use in metabolic engineering of stress tolerance. J. Biosci. 23, 437–445. Sydlik, U., Gallitz, I., Albrecht, C., Abel, J., Krutmann, J., Unfried, K., 2009. The compatible solute ectoine protects against nanoparticle-induced neutrophilic lung inflammation. Am. J. Respir. Care Crit. Care Med. 180, 29–35. Timasheff, S.N., 2002. Protein hydration, thermodynamic binding, and preferential hydration. Biochemistry 41, 13473–13482. Van Alstyne, K.L., Houser, L.T., 2003. Dimethylsulfide release during macroinvertebrate grazing and its role as an activated chemical defense. Mar. Ecol. Prog. Ser. 250, 175–181. Villegas-Navarro, A., Rosas-L, E., Reyes, J.L., 2003. The heart of Daphnia magna: effects of four cardioactive drugs. Comp. Biochem. Physiol. C 136, 127–134. Wang, L., Zhang, L., Yu, Y., Wang, Y., Niu, N., 2008. The protective effects of taurine against early renal injury in STZ-induced diabetic rats, correlated with inhibition of renal LOX-1-mediated ICAM-1 expression. Ren. Fail. 30, 763–771. Ward, R.J., Kest, W., Bruyeer, P., Lallemand, F., De Witte, P., 2001. Taurine modulates catalase, aldehyde dehydrogenase, and ethanol elimination rates in rat brain. Alcohol Alcohol. 36, 39–43. Wei, Y.H., Yuan, F.W., Chen, W.C., Chen, S.Y., 2011. Production and characterization of ectoine by Marinococcus sp. ECT1 isolated from a high-salinity environment. J. Biosci. Bioeng. 111, 336–342. http://dx.doi.org/10.1016/j.jbiosc.2010.11.009 (Epub 2010 Dec 15). Westh, P., Ramlof, H., 1991. Trehalose accumulation in the tardigrade Androrybiotus coronifer during anhydrobiosis. J. Exp. Zool. 258, 303–311. Wolfe, G.V., 2000. The chemical defense ecology of marine unicellular plankton: constraints, mechanisms, and impacts. Biol. Bull. 198, 225–244. Yancey, P.H., 2004. Compatible and counteracting solutes: protecting cells from the Dead Sea to the deep sea. Sci. Prog. 87, 1–24. Zhang, L., Wang, Y., Zhang, C., Wang, Y., Zhu, D., Wang, C., Nagata, S., 2006. Supplementation effect of ectoine on thermostability of phytase. J. Biosci. Bioeng. 102, 560–563.