Acute Exposure to Microcystin-Producing Cyanobacterium <i>Microcystis aeruginosa</i> Alters Adult Zebrafish (<i>Danio rerio</i>) Swimming Performance Parameters

Kist, Luiza Wilges; Piato, Angelo Luis; da Rosa, João Gabriel Santos; Koakoski, Gessi; Barcellos, Leonardo José Gil; Yunes, João Sarkis; Bonan, Carla Denise; Bogo, Maurício Reis

doi:https://doi.org/10.1155/2011/280304

Journal of Toxicology

On this page

Abstract Introduction Materials and Methods Results Discussion Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2011 | Article ID 280304 | https://doi.org/10.1155/2011/280304

Acute Exposure to Microcystin-Producing Cyanobacterium Microcystis aeruginosa Alters Adult Zebrafish (Danio rerio) Swimming Performance Parameters

Luiza Wilges Kist,^1,2Angelo Luis Piato,^2,3João Gabriel Santos da Rosa,⁴Gessi Koakoski,⁴Leonardo José Gil Barcellos,⁵João Sarkis Yunes,⁶Carla Denise Bonan,^2,3and Maurício Reis Bogo^1,2

Academic Editor: J. J. Stegeman

Received12 Aug 2011

Revised15 Sept 2011

Accepted22 Sept 2011

Published28 Dec 2011

Abstract

Microcystins (MCs) are toxins produced by cyanobacteria (blue-green algae), primarily Microcystis aeruginosa, forming water blooms worldwide. When an organism is exposed to environmental perturbations, alterations in normal behavioral patterns occur. Behavioral repertoire represents the consequence of a diversity of physiological and biochemical alterations. In this study, we assessed behavioral patterns and whole-body cortisol levels of adult zebrafish (Danio rerio) exposed to cell culture of the microcystin-producing cyanobacterium M. aeruginosa (MC-LR, strain RST9501). MC-LR exposure (100 μg/L) decreased by 63% the distance traveled and increased threefold the immobility time when compared to the control group. Interestingly, no significant alterations in the number of line crossings were found at the same MC-LR concentration and time of exposure. When animals were exposed to 50 and 100 μg/L, MC-LR promoted a significant increase (around 93%) in the time spent in the bottom portion of the tank, suggesting an anxiogenic effect. The results also showed that none of the MC-LR concentrations tested promoted significant alterations in absolute turn angle, path efficiency, social behavior, or whole-body cortisol level. These findings indicate that behavior is susceptible to MC-LR exposure and provide evidence for a better understanding of the ecological consequences of toxic algal blooms.

1. Introduction

Microcystis aeruginosa is a freshwater cyanobacteria, known producer of a family of toxins termed microcystins (MCs) [1, 2]. MCs are hepatotoxic cyclic heptapeptides released into water during or on senescence of cyanobacterial blooms [3]. The peptide rings of MCs contain five nonprotein amino acids, whereas the two-protein amino acids distinguish MCs from one another. MC-LR contains the amino acids leucine and arginine. MC-LR is one of the most commonly occurring [2, 4] and the most toxic microcystin [5]. The intact cells as well as the toxins released after cellular lysis can be responsible for the toxic effects observed in many organisms, from microalgae [6] to mammals [7] including human [8–10].

Exposure to toxic cyanobacteria or administration of MCs may cause hepatotoxic effects [11–13], oxidative stress [14], kidney damage [15, 16], growth inhibition [17, 18], reproductive injury [19], haematological and biochemical alterations [20–22], apoptosis [23], and even fish death [24].

Alterations in normal behavioral patterns may be the first line of defense when an animal is exposed to an environmental perturbation [25–28]. Additionally, studies have shown important interrelationships between hormones and behavior [29–33]. Thus, alteration in cortisol level may consequently alter normal fish behavior. The effects of MC on fish behavior are still unknown, but some issues have already been addressed. Baganz et al. [34, 35] reported changes in the spontaneous locomotor behavior of zebrafish (Danio rerio) and Leucaspius delineatus after MC-LR exposure, and Cazenave et al. [36] showed changes in swimming activity of Jenynsia multidentata fed with microcystin-RR (MC-RR). In addition, studies using different exposure routes (intraperitoneal injection, oral ingestion, or immersion) have demonstrated that MCs can accumulate in fish tissues, mainly in the liver [21, 36–39], intestine [37, 39–41], gills [42, 43], kidney [37, 39], muscle [40, 41, 44–46], gallbladder [47], blood [40, 41, 48], and brain [43]. Altogether, these findings indicate possible neurotoxic effects of MCs on fish, causing serious risks to the success of fish populations and changes in biodiversity, among other ecological consequences [36].

The zebrafish is rapidly becoming a popular model species in many areas of biological research. Its application includes the fields of developmental biology [49], toxicology [50], neurophysiology, biomedicine, drug discovery [51], human diseases [52–54], pharmacology and behavioral analysis [55–59]. These fish exhibit robust behavioral responses, well-characterized genome, neural and endocrine systems homologous to humans [60–62], and possess all of the “classical” vertebrate neurotransmitters [63, 64]. Additionally, zebrafish are an ideal animal model for laboratory research because they are inexpensive, require low maintenance, and produce abundant offspring [65]. Recently, this fish was also used for proteomic studies on the toxicity of MCs [66, 67].

In order to better understand the neurotoxic effects of MCs on fish and to improve the knowledge of mechanisms underlying the toxicity, the main goal of this study was to assess the effects of MC-LR on zebrafish behavioral parameters and endocrine (whole-body cortisol) response after toxin exposure.

2. Materials and Methods

2.1. Animals

Wild-type adult (<8 months old) zebrafish (Danio rerio) of both sexes were obtained from specialized supplier (Redfish Agroloja, RS, Brazil). Animals were kept in 50 L housing tanks with tap water previously treated with Tetra’s AquaSafe (to neutralize chlorine, chloramines, and heavy metals present in the water that could be harmful to fish) and continuously aerated (7.20 mg O₂/L) at °C, under a 14–10 h light/dark photoperiod in a density of up to five animals per liter. Animals were acclimated for at least two weeks before the experiments. They were fed three times a day with TetraMin Tropical Flake fish.

The procedures were previously approved by the Animal Ethics Committee of Pontifical Catholic University of Rio Grande do Sul (PUCRS) under the protocol number 10/00142-CEUA.

2.2. Treatments

The amount of MC-LR in the cell culture of M. aeroginosa (strain RST9501) was detected by a Quantitative Antibody Immunoassay (Elisa) against MC-LR provided by Envirologix (Portland, USA), within a range of detection from 0.05 to 2.5 μg/L MCs. A suitable dilution was applied to the culture sample to provide detection within the range. Zebrafish were distributed in three groups: the first group (controls) was exposed to water containing the culture medium of M. aeroginosa for 24 hours; the second and third groups were exposed to cell culture in a final MC-LR concentration of 50 μg/L and 100 μg/L during 24 hours, respectively. Immediately after the exposure, animals were tested in tank-diving behavioral test and social interaction. After behavioral tests animals were euthanized by decapitation.

The MC-LR concentrations and the time of exposure were chosen based on a previous study using J. multidentata [43]. Besides, such concentrations are commonly encountered in cyanobacterial bloom events [68, 69].

2.3. Tank-Diving Behavioral Test

Behavioral testing took place during the light phase between 10:00 AM and 4:00 PM The animals were individually transferred to a 2.7 L tank (24 cm L × 8 cm W × 20 cm H) with laterals and bottom white covered to avoid any visual disturbances and habituated to the tank for 30 s, as previously described [70]. There was no drug exposure during behavioral experiments. The locomotor activity of the animals was video-recorded using Logitech Quickcam PRO9000 for five minutes after the habituation period and further analyzed using the ANY-Maze recording software (Stoelting Co., Wood Dale, IL, USA). The tank was divided into equal sections with four vertical lines and one horizontal line, and the following behavior patterns were measured: distance traveled (meters), immobility time (seconds), number of crossings, absolute turn angle, path efficiency, and time (seconds) spent in the bottom portion. This task exploits the natural tendency for zebrafish to spend most of the time at the bottom when introduced into a novel environment and then gradually extend the swimming range, over a period of minutes, to include the upper portions of the test tank. A longer time spent in the bottom part of the tank indicates heightened anxiety [71].

2.4. Social Interaction

Zebrafish is a schooling fish that may exhibit preference for its conspecifics under certain circumstances. The social interaction analysis was based on Gerlai [72]. After 24 hours of exposition to 50 or 100 μg/L of MC-LR, fish were placed in groups of five in a small experimental tank (30 cm L × 15 cm H × 10 cm W). On one side of the experimental tank, an empty fish tank was placed, and, on the other side, there was a tank of identical size containing 15 conspecifics. The experimental fish were allowed to acclimate to the experimental tank for a 30 s period, after which their behavior was analyzed. The next 10 s of this test was analyzed as follows. The experimental tank was virtually divided into two equal sections with one vertical line. The time that all five experimental fish spent on the side of the tank closer to the conspecific school was measured using a stopwatch.

2.5. Acute Restraint Stress (ARS) Protocol

The ARS protocol was based on Piato et al. [73]. Following the habituation period, fish were submitted to the ARS protocol. This experiment consisted in keeping each animal enclosed into microcentrifuge plastic tubes of 2 mL with the cap closed and small openings in both ends to allow free water circulation inside the tube and completely avoid fish locomotion. After 90 min of confinement, animals were gently captured and immediately frozen in liquid nitrogen and stored at −80°C until cortisol extraction. Aeration (8 ppm, Labcom Test Camboriú, SC, Brazil) and water temperature (°C) were controlled throughout the test.

2.6. Measurement of Cortisol

The extraction and measurement of whole-body cortisol from zebrafish have been described in detail by Barcellos et al. [74]. Briefly, zebrafish were distributed in four groups: the first group, which consisted of zebrafish exposed to water containing the culture medium of M. aeroginosa for 24 hours, was considered the “negative control”; the second and third groups were exposed to cell culture in a final MC-LR concentration of 50 μg/L and 100 μg/L during 24 hours, respectively; in the fourth group, considered the “positive control,” zebrafish were submitted to the ARS protocol. After, zebrafish were captured and immediately frozen in liquid nitrogen and stored at −80°C until whole-body cortisol extraction. Each zebrafish was weighed, and a pool of three fish was minced and placed into a disposable stomacher bag with 2 mL of phosphate buffered saline (PBS, pH 7.4) for 6 min. The contents were transferred to a 10 mL screw top disposable test tube, and 5 mL of laboratory grade ethyl ether was added. The tube was vortexed for 1 min and centrifuged for 10 min at 3000 rpm. The tube was then immediately frozen at liquid nitrogen, and the unfrozen portion (ethyl ether containing cortisol) was decanted. The ethyl ether was transferred to a new tube and completely evaporated under a gentle stream of nitrogen for 2 h, yielding a lipid extract containing the cortisol. The extract was stored at −20°C until the ELISA was conducted on the samples suspended with 1 mL of PBS buffer. In order to prevent a possible stress response induced by manipulation, the time elapsed between capture and killing was less than 10 s. Whole-body cortisol was measured in duplicate samples of tissue extract with a commercially available high sensitivity salivary cortisol-enzyme immunoassay kit (Salimetrics, USA). The specificity of the test was evaluated by comparing the parallelism between the standard curve and serial dilutions of the tissue extracts in PBS (pH 7.4). The standard curve constructed with the human standards ran parallel to that obtained using serial dilutions of zebrafish tissue extracts. In the linear regression test, high positive correlation () was found between the curves. The intra-assay coefficient of variation was 3.33–3.65%.

2.7. Statistical Analysis

Data of the exploratory assessment, social interaction, and cortisol levels were expressed as mean ± SEM and analyzed by one-way ANOVA, followed by Newman-Keuls post hoc test. A significant difference was attributed to . All data were evaluated by SPSS 18.0 for Windows.

3. Results

Distinct parameters of zebrafish swimming activity were evaluated in the 5-min tank-diving behavioral test. MC-LR exposure at 100 μg/L significantly (one-way ANOVA/Newman-Keuls, , ) decreased the distance traveled ( meters) in relation to control animals ( meters) (Figure 1(a)) and significantly (one-way ANOVA/Newman-Keuls, , ) increased the immobility time ( seconds) when compared to the control group ( seconds) (Figure 1(b)) whereas MC-LR exposure at 50 μg/L did not alter both parameters. No differences in the number of line crossings, absolute turn angle, and path efficiency were observed in both concentrations tested (Figure 1(c), 1D and 1E, resp.).

(a)

(b)

(c)

(d)

(e)

(f)

Control animals spent 58.4% of time () in the bottom portion of the test tank. Animals exposed to 50 and 100 μg/L MC-LR significantly (one-way ANOVA/Newman-Keuls, , ) increased (93%) the time spent in the bottom portion of the test tank ( and , resp.) when compared with control group () (Figure 1(f)).

In relation to social interaction test, the results showed that 50 and 100 μg/L of MC-LR concentrations did not promote any alteration in the animals regarding this behavior (Figure 2).

Levels of whole-body cortisol also were measured. The ARS protocol (positive control) resulted in enhanced whole-body cortisol in relation to control group (one-way ANOVA/Newman-Keuls, , ; and , resp.). Zebrafish treated with both concentrations of MC-LR did not present altered levels of cortisol in relation to control group (Figure 3).

4. Discussion

Behavioral alterations reflect how an animal senses and responds to its environment and is the first line of defense when an animal is exposed to an environmental perturbation [28]. Since it was already demonstrated that the effects promoted by cyanobacterial crude extracts on aquatic organisms were either more pronounced or different from those observed using pure toxins [75, 76], we used cell culture of the microcystin-producing cyanobacterium M. aeruginosa (MC-LR) in order to evaluate the effects of MCs on zebrafish behavior.

The toxin concentration and time period of animals’ exposure were chosen based on previous studies that showed MCs accumulation in fish tissues [21, 37–48]. The results presented herein demonstrated that 100 μg/L MC-LR decreased the distance traveled and increased the immobility time. However, no significant alterations were found in the number of line crossings with both concentrations. When animals were exposed to 50 and 100 μg/L, MC-LR led to a significant increase in the time spent in the bottom portion. The results also showed that none of the MC-LR concentrations tested promoted significant alterations in the absolute turn angle, path efficiency, or social interaction.

Since behavior links physiological function with ecological processes for a given species, it might provide a useful indicator or biomarker for detecting harmful chemical pollutants [77]. The potential of the zebrafish as a model in neurobehavioral research has emerged only recently. Studies have examined behavior in zebrafish larvae [78–81], as well as their responses to different drugs, such as ethanol [82, 83] and fluoxetine [84]. Studies on adult zebrafish include social behavior [85–87], olfactory-related behaviors [88, 89], anxiety [74], addiction [90–92], sleep [93], learning and memory [94, 95].

There are still only few studies evaluating the effects of MCs on fish behavior. Baganz et al. [34] verified that exposure to MC-LR caused dose-effect-related changes in spontaneous locomotor activity in zebrafish. Whereas exposure to lower concentrations (0.5 and 5 μg/L) caused an increase in daytime mobility, elevated exposures (15 and 50 μg/L) led to significantly increased immobility. The highest exposure (50 μg/L) also reduced the spawning activity and reduced spawning success. In contrast to daytime activities, night-time swimming activity was significantly greater at the higher MC-LR exposures. In another study, Baganz et al. [35] showed changes in the spontaneous locomotor behavior of zebrafish and L. delineatus after exposure to MC-LR in concentrations of 0.5, 5, and 15 μg/L for 17 days and 50 μg/L for six days. During the daytime, the mobility of zebrafish as well as L. delineatus increased significantly by exposure to the lowest concentrations, whereas higher concentrations led to significantly decreased mobility. Influenced by MC-LR, the swimming time of L. delineatus reversed, going from a prominently diurnal activity to a nocturnal one; zebrafish remained active during the daytime. Additionally, Cazenave et al. [36] reported changes in the swimming activity of J. multidentata fed with contaminated food pellets containing MC-RR. Low levels (0.01 μg/g) increased the swimming activity, while the highest dose (1 μg/g) used produced significant changes with respect to control group (only since approximately 20 hours of exposure), when the swimming activity was decreased.

In this sense, our findings demonstrate that MC-LR at the highest concentration (100 μg/L) caused a decrease in the distance traveled and an increase in the immobility time in zebrafish. Interestingly, no significant alterations in the number of line crossings were found at the same MC-LR concentration and time of exposure, despite the tendency to decrease the number of crossings in greater concentration. It is important to emphasize that these findings are similar to the results published previously by Baganz et al. [35]. However, these authors have used purified toxin whereas a cell culture of the microcystin-producing cyanobacterium M. aeruginosa (MC-LR) was used in our experiments. Reduction in swimming capability, resulting in a reduction in the rate of activity, may decrease the ability to gather food and make the fish more vulnerable to predation [96]. Under natural conditions, this reduced overall level of activity will eventually cause disadvantages to the organisms in the ecosystem, and, therefore, influence the biocoenotic structures and functions [34].

Exposure to a novel environment evokes a robust anxiety response in zebrafish [97], as they dive to the bottom (geotaxis) until they feel safe to swim in the upper regions of the tank [58]. Here, MC-LR at 50 and 100 μg/L promoted an increase in the time spent in the bottom portion, suggesting an anxiety behavior.

The zebrafish is a social species and exhibits group preference as well as aggression. Shoaling behavior commences soon after hatching and fish reared in isolation quickly form shoals when placed together [98]. One study has demonstrated that exposure to nonylphenol over a 5-day period decreased shoaling tendency in juvenile rainbow trout (Oncorhynchus mykiss) [99]. Similarly, herbicide-exposed goldfish also showed a decreased aggregation [100]. Locomotor activity, aggressive behavior, and group preference of the male zebrafish and group preference of the females were clearly inhibited when zebrafish were exposed for 60 days to 100 μg/L nonylphenol concentration [101]. For this reason, we evaluated the effect of MC-LR exposure on zebrafish social interaction. However, no significant alteration was found between control and MC-LR-exposed animals.

Studies have shown important interrelationships between stress hormones and behavior [29–33]. An elevated plasma cortisol level is a primary indicator of a stress response in fish [102]. Zebrafish, like humans, employ cortisol as a primary stress response hormone [74]. Considering this, we measured whole-body cortisol in zebrafish to verify if MC could elicit a stress response in treated fish. However, no significant alterations were found in whole-body cortisol levels in animals exposed to both concentrations of MC-LR compared to the control group. Barcellos et al. [74] demonstrated that whole-body cortisol level of zebrafish increases after visual contact with a predator species. Cortisol levels were significantly higher in zebrafish submitted to unpredictable chronic stress (UCS) protocol when compared to control group [103]. Bury et al. [104] reported a significant increase in plasma cortisol levels of the brown trout (Salmo trutta) after 1 h and returned to the control level after 24 h of exposure to lysed toxic Microcystis aeruginosa cells. Crucian carp (Carassius auratus) exposed to sublethal and lethal doses (150 μg/kg and 600 μg/kg, resp.) of Microcystis extracts exhibited a significant acute increase in plasma cortisol levels, which suggested that MC elicited a stress response in treated fish. The profiles of cortisol changes in fish treated with MC appeared to be dose dependent, indicating that fish in the high-dose group experienced greater MC-induced disturbance [105].

In summary, behavioral response of fish may be a promising biomarker of sublethal toxicity and water contamination. Several behavioral endpoint measurements, especially locomotor activity and the time spent in the bottom portion, may provide an effective assessment of MCs in aquatic ecosystem.

Acknowledgments

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Proc. 472298/2010-0) and DECIT/SCTIEMS through CNPq and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) (Proc. FAPERGS 10/0036-5-PRONEX). L. W. Kist, J. G. S. da Rosa, and G. Koakoski were recipient of fellowships from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

References

W. W. Carmichael, “Cyanobacteria secondary metabolites—the cyanotoxins,” Journal of Applied Bacteriology, vol. 72, no. 6, pp. 445–459, 1992.
View at: Google Scholar
R. M. Dawson, “The toxicology of microcystins,” Toxicon, vol. 36, no. 7, pp. 953–962, 1998.
View at: Publisher Site | Google Scholar
C. Malbrouck and P. Kestemont, “Effects of microcystins on fish,” Environmental Toxicology and Chemistry, vol. 25, no. 1, pp. 72–86, 2006.
View at: Publisher Site | Google Scholar
M. F. Watanabe, K. I. Harada, W. W. Carmichael, and H. Fujiki, Toxic Microcystis, CRC Press, Boca Raton, Fla, USA, 1996.
K. L. Rinehart, M. Namikoshi, and B. W. Choi, “Structure and biosynthesis of toxins from blue-green algae (cyanobacteria),” Journal of Applied Phycology, vol. 6, no. 2, pp. 159–176, 1994.
View at: Publisher Site | Google Scholar
K. D. Kearns and M. D. Hunter, “Toxin-producing Anabaena flos-aquae induces settling of Chlamydomonas reinhardtii, a competing motile alga,” Microbial Ecology, vol. 42, no. 1, pp. 80–86, 2001.
View at: Google Scholar
B. Puschner, F. D. Galey, B. Johnson et al., “Blue-green algae toxicosis in cattle,” Journal of the American Veterinary Medical Association, vol. 213, no. 11, pp. 1605–1607, 1998.
View at: Google Scholar
Y. Ueno, S. Nagata, T. Tsutsumi et al., “Detection of microcystins, a blue-green algal hepatotoxin, in drinking water sampled in Haimen and Fusui, endemic areas of primary liver cancer in China, by highly sensitive immunoassay,” Carcinogenesis, vol. 17, no. 6, pp. 1317–1321, 1996.
View at: Publisher Site | Google Scholar
W. W. Carmichael, S. M. Azevedo, J. S. An et al., “Human fatalities form cyanobacteria: chemical and biological evidence for cyanotoxins,” Environmental Health Perspectives, vol. 109, no. 7, pp. 663–668, 2001.
View at: Google Scholar
J. Chen, P. Xie, L. Li, and J. Xu, “First identification of the hepatotoxic microcystins in the serum of a chronically exposed human population together with indication of hepatocellular damage,” Toxicological Sciences, vol. 108, no. 1, pp. 81–89, 2009.
View at: Publisher Site | Google Scholar
P. F. Solter, G. K. Wollenberg, X. Huang, F. S. Chu, and M. T. Runnegar, “Prolonged sublethal exposure to protein phosphatase inhibitor microcystin-LR results in multiple dose-dependent hepatotoxic effects,” Toxicological Sciences, vol. 44, no. 1, pp. 87–96, 1998.
View at: Publisher Site | Google Scholar
L. Li, P. Xie, and J. Chen, “In vivo studies on toxin accumulation in liver and ultrastructural changes of hepatocytes of the phytoplanktivorous bighead carp i.p.-injected with extracted microcystins,” Toxicon, vol. 46, no. 5, pp. 533–545, 2005.
View at: Publisher Site | Google Scholar
U. S. Gupta and S. Guha, “Microcystin toxicity in a freshwater fish, Heteropneustes fossilis (Bloch),” Current Science, vol. 91, no. 9, pp. 1261–1271, 2006.
View at: Google Scholar
J. Ángeles, S. Pichardo, A. I. Prieto et al., “Toxic cyanobacterial cells containing microcystins induce oxidative stress in exposed tilapia fish (Oreochromis sp.) under laboratory conditions,” Aquatic Toxicology, vol. 72, no. 3, pp. 261–271, 2005.
View at: Publisher Site | Google Scholar
B. G. Kotak, S. Semalulu, D. L. Fritz, E. E. Prepas, S. E. Hrudey, and R. W. Coppock, “Hepatic and renal pathology of intraperitoneally administered microcystin-LR in rainbow trout (Oncorhynchus mykiss),” Toxicon, vol. 34, no. 5, pp. 517–525, 1996.
View at: Publisher Site | Google Scholar
W. J. Fischer and D. R. Dietrich, “Pathological and biochemical characterization of microcystin-induced hepatopancreas and kidney damage in carp (Cyprinus carpio),” Toxicology and Applied Pharmacology, vol. 164, no. 1, pp. 73–81, 2000.
View at: Publisher Site | Google Scholar
N. R. Bury, F. B. Eddy, and G. A. Codd, “The effects of the cyanobacterium Microcystis aeruginosa, the cyanobacterial hepatotoxin microcystin-LR, and ammonia on growth rate and ionic regulation of brown trout,” Journal of Fish Biology, vol. 46, no. 6, pp. 1042–1054, 1995.
View at: Publisher Site | Google Scholar
M. Zhao, S. Xie, X. Zhu, Y. Yang, L. Gan, and L. Song, “Effect of inclusion of blue-green algae meal on growth and accumulation of microcystins in gibel carp (Carassius auratus gibelio),” Journal of Applied Ichthyology, vol. 22, no. 1, pp. 72–78, 2006.
View at: Publisher Site | Google Scholar
X. S. Ding, X. Y. Li, H. Y. Duan, I. K. Chung, and J. A. Lee, “Toxic effects of Microcystis cell extracts on the reproductive system of male mice,” Toxicon, vol. 48, no. 8, pp. 973–979, 2006.
View at: Publisher Site | Google Scholar
V. Vajcova, S. Navrátil, and M. Palíková, “The effect of intraperitoneally applied pure microcystin LR on haematological, biochemical and morphological indices of silver carp (Hypophthalmichthys molitrix Val.),” Acta Veterinaria, vol. 67, no. 4, pp. 281–287, 1998.
View at: Google Scholar
C. Malbrouck, G. Trausch, P. Devos, and P. Kestemont, “Hepatic accumulation and effects of microcystin-LR on juvenile goldfish Carassius auratus L,” Comparative Biochemistry and Physiology: C, vol. 135, no. 1, pp. 39–48, 2003.
View at: Publisher Site | Google Scholar
X. Zhang, P. Xie, D. Li, and Z. Shi, “Hematological and plasma biochemical responses of crucian carp (Carassius auratus) to intraperitoneal injection of extracted microcystins with the possible mechanisms of anemia,” Toxicon, vol. 49, no. 8, pp. 1150–1157, 2007.
View at: Publisher Site | Google Scholar
C. M. McDermott, C. W. Nho, W. Howard, and B. Holton, “The cyanobacterial toxin, microcystin-LR, can induce apoptosis in a variety of cell types,” Toxicon, vol. 36, no. 12, pp. 1981–1996, 1998.
View at: Publisher Site | Google Scholar
T. Lindholm, P. Öhman, K. Kurki-Helasmo, B. Kincaid, and J. Meriluoto, “Toxic algae and fish mortality in a brackish-water lake in Aland, SW Finland,” Hydrobiologia, vol. 397, pp. 109–120, 1999.
View at: Publisher Site | Google Scholar
E. Scherer, “Behavioral responses as indicators of environmental alterations, approaches, results, developments,” Journal of Applied Ichthyology, vol. 8, no. 1–4, pp. 122–131, 1992.
View at: Google Scholar
E. A. Paul and H. A. Simonin, “Effects of Naled, synergized,and non-synergized resmethrin on the swimming performance of young trout,” Bulletin of Environmental Contamination and Toxicology, vol. 57, no. 3, pp. 495–502, 1996.
View at: Publisher Site | Google Scholar
H. A. Campbell, R. D. Handy, and D. W. Sims, “Increased metabolic cost of swimming and consequent alterations to circadian activity in rainbow trout (Oncorhynchus mykiss) exposed to dietary copper,” Canadian Journal of Fisheries and Aquatic Sciences, vol. 59, no. 5, pp. 768–777, 2002.
View at: Publisher Site | Google Scholar
M. L. Begout Anras and J. P. Lagardère, “Measuring cultured fish swimming behaviour: first results on rainbow trout using acoustic telemetry in tanks,” Aquaculture, vol. 240, no. 1–4, pp. 175–186, 2004.
View at: Publisher Site | Google Scholar
R. F. Oliveira, V. C. Almada, and A. V. M. Canario, “Social modulation of sex steroid concentrations in the urine of male cichlid fish Oreochromis mossambicus,” Hormones and Behavior, vol. 30, no. 1, pp. 2–12, 1996.
View at: Publisher Site | Google Scholar
W. M. Contreras-Sánchez, C. B. Schreck, M. S. Fitzpatrick, and C. B. Pereira, “Effects of stress on the reproductive performance of rainbow trout (Oncorhynchus mykiss),” Biology of Reproduction, vol. 58, no. 2, pp. 439–447, 1998.
View at: Publisher Site | Google Scholar
T. G. Pottinger and T. R. Carrick, “Stress responsiveness affects dominant-subordinate relationships in rainbow trout,” Hormones and Behavior, vol. 40, no. 3, pp. 419–427, 2001.
View at: Publisher Site | Google Scholar
K. A. Sloman, N. B. Metcalfe, A. C. Taylor, and K. M. Gilmour, “Plasma cortisol concentrations before and after social stress in rainbow trout and brown trout,” Physiological and Biochemical Zoology, vol. 74, no. 3, pp. 383–389, 2001.
View at: Publisher Site | Google Scholar
Ø. Øverli, T. G. Pottinger, T. R. Carrick, E. Øverli, and S. Winberg, “Differences in behaviour between rainbow trout selected for high- and low-stress responsiveness,” Journal of Experimental Biology, vol. 205, no. 3, pp. 391–395, 2002.
View at: Google Scholar
D. Baganz, G. Staaks, and C. Steinberg, “Impact of the cyanobacteria toxin, microcystin-LR on behaviour of zebrafish, Danio rerio,” Water Research, vol. 32, no. 3, pp. 948–952, 1998.
View at: Publisher Site | Google Scholar
D. Baganz, G. Staaks, S. Pflugmacher, and C. E. W. Steinberg, “Comparative study of microcystin-LR-induced behavioral changes of two fish species, Danio rerio and Leucaspius delineatus,” Environmental Toxicology, vol. 19, no. 6, pp. 564–570, 2004.
View at: Publisher Site | Google Scholar
J. Cazenave, M. L. Nores, M. Miceli, M. P. Díaz, D. A. Wunderlin, and M. A. Bistoni, “Changes in the swimming activity and the glutathione S-transferase activity of Jenynsia multidentata fed with microcystin-RR,” Water Research, vol. 42, no. 4-5, pp. 1299–1307, 2008.
View at: Publisher Site | Google Scholar
D. E. Williams, M. L. Kent, R. J. Andersen, H. Klix, and C. F. B. Holmes, “Tissue distribution and clearance of tritium-labeled dihydromicrocystin-LR epimers administered to Atlantic salmon via intraperitoneal injection,” Toxicon, vol. 33, no. 2, pp. 125–131, 1995.
View at: Publisher Site | Google Scholar
V. O. Sipiä, H. T. Kankaanpää, J. Flinkman, K. Lahti, and J. A. O. Meriluoto, “Time-dependent accumulation of cyanobacterial hepatotoxins in flounders (Platichthys flesus) and mussels (Mytilus edulis) from the Northern Baltic Sea,” Environmental Toxicology, vol. 16, no. 4, pp. 330–336, 2001.
View at: Publisher Site | Google Scholar
Z. A. Mohamed, W. W. Carmichael, and A. A. Hussein, “Estimation of microcystins in the freshwater fish Oreochromis niloticus in an Egyptian fish farm containing a Microcystis bloom,” Environmental Toxicology, vol. 18, no. 2, pp. 137–141, 2003.
View at: Publisher Site | Google Scholar
L. Xie, P. Xie, K. Ozawa, T. Honma, A. Yokoyama, and H. D. Park, “Dynamics of microcystins-LR and -RR in the phytoplanktivorous silver carp in a sub-chronic toxicity experiment,” Environmental Pollution, vol. 127, no. 3, pp. 431–439, 2004.
View at: Publisher Site | Google Scholar
L. Xie, P. Xie, L. Guo, L. Li, Y. Miyabara, and H. D. Park, “Organ distribution and bioaccumulation of microcystins in freshwater fish at different trophic levels from the eutrophic Lake Chaohu, China,” Environmental Toxicology, vol. 20, no. 3, pp. 293–300, 2005.
View at: Publisher Site | Google Scholar
C. R. Carbis, J. A. Simons, P. Grant, G. F. Mitchell, J. W. Anderson, and I. McCauley, “A study of feral carp, Cyprinus carpio L., exposed to Microcystis aeruginosa at Lake Mokoan, Australia, and possible implications for fish health,” Journal of Fish Diseases, vol. 20, no. 2, pp. 81–91, 1997.
View at: Google Scholar
J. Cazenave, D. A. Wunderlin, M. D. L. A. Bistoni et al., “Uptake, tissue distribution and accumulation of microcystin-RR in Corydoras paleatus, Jenynsia multidentata and Odontesthes bonariensis: a field and laboratory study,” Aquatic Toxicology, vol. 75, no. 2, pp. 178–190, 2005.
View at: Publisher Site | Google Scholar
N. R. Bury, A. D. Newlands, F. B. Eddy, and G. A. Codd, “In vivo and in vitro intestinal transport of 3H-microcystin-LR, a cyanobacterial toxin, in rainbow trout (Oncorhynchus mykiss),” Aquatic Toxicology, vol. 42, no. 2, pp. 139–148, 1998.
View at: Publisher Site | Google Scholar
V. F. Magalhães, R. Moraes Soares, and S. M. F. O. Azevedo, “Microcystin contamination in fish from the Jacarepaguá Lagoon (Rio de Janeiro, Brazil): ecological implication and human health risk,” Toxicon, vol. 39, no. 7, pp. 1077–1085, 2001.
View at: Publisher Site | Google Scholar
V. F. Magalhães, M. M. Marinho, P. Domingos et al., “Microcystins (cyanobacteria hepatotoxins) bioaccumulation in fish and crustaceans from Sepetiba Bay (Brasil, RJ),” Toxicon, vol. 42, no. 3, pp. 289–295, 2003.
View at: Publisher Site | Google Scholar
A. Sahin, F. G. Tencalla, D. R. Dietrich, and H. Naegeli, “Biliary excretion of biochemically active cyanobacteria (blue-green algae) hepatotoxins in fish,” Toxicology, vol. 106, no. 1–3, pp. 123–130, 1996.
View at: Publisher Site | Google Scholar
F. Tencalla and D. Dietrich, “Biochemical characterization of microcystin toxicity in rainbow trout (Oncorhynchus mykiss),” Toxicon, vol. 35, no. 4, pp. 583–595, 1997.
View at: Publisher Site | Google Scholar
L. I. Zon and R. T. Peterson, “In vivo drug discovery in the zebrafish,” Nature Reviews Drug Discovery, vol. 4, no. 1, pp. 35–44, 2005.
View at: Publisher Site | Google Scholar
J. M. Spitsbergen and M. L. Kent, “The state of the art of the zebrafish model for toxicology and toxicologic pathology research—advantages and current limitations,” Toxicologic Pathology, vol. 31, pp. 62–87, 2003.
View at: Google Scholar
A. L. Rubinstein, “Zebrafish: from disease modeling to drug discovery,” Current Opinion in Drug Discovery and Development, vol. 6, no. 2, pp. 218–223, 2003.
View at: Google Scholar
K. Dooley and L. I. Zon, “Zebrafish: a model system for the study of human disease,” Current Opinion in Genetics and Development, vol. 10, no. 3, pp. 252–256, 2000.
View at: Publisher Site | Google Scholar
G. J. Lieschke and P. D. Currie, “Animal models of human disease: zebrafish swim into view,” Nature Reviews Genetics, vol. 8, no. 5, pp. 353–367, 2007.
View at: Publisher Site | Google Scholar
J. D. Best and W. K. Alderton, “Zebrafish: an In vivo model for the study of neurological diseases,” Neuropsychiatric Disease and Treatment, vol. 4, no. 3, pp. 567–576, 2008.
View at: Google Scholar
R. J. Egan, C. L. Bergner, P. C. Hart et al., “Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish,” Behavioural Brain Research, vol. 205, no. 1, pp. 38–44, 2009.
View at: Publisher Site | Google Scholar
J. Cachat, P. Canavello, M. Elegante et al., “Modeling withdrawal syndrome in zebrafish,” Behavioural Brain Research, vol. 208, no. 2, pp. 371–376, 2010.
View at: Publisher Site | Google Scholar
C. Maximino, T. M. de Brito, C. A. Dias, A. Gouveia, and S. Morato, “Scototaxis as anxietylike behavior in fish.,” Nature Protocols, vol. 5, no. 2, pp. 209–216, 2010.
View at: Publisher Site | Google Scholar
A. Stewart, F. Kadri, and J. DiLeo, “The developing utility of zebrafish in modeling neurobehavioral disorders,” International Journal of Comparative Psychology, vol. 23, pp. 104–121, 2010.
View at: Google Scholar
K. Wong, M. Elegante, B. Bartels et al., “Analyzing habituation responses to novelty in zebrafish (Danio rerio),” Behavioural Brain Research, vol. 208, no. 2, pp. 450–457, 2010.
View at: Publisher Site | Google Scholar
J. Kaslin and P. Panula, “Comparative anatomy of the histaminergic and other aminergic systems in zebrafish (Danio rerio),” Journal of Comparative Neurology, vol. 440, no. 4, pp. 342–377, 2001.
View at: Publisher Site | Google Scholar
J. T. Shin and M. C. Fishman, “From zebrafish to human: modular medical models,” Annual Review of Genomics and Human Genetics, vol. 3, pp. 311–340, 2002.
View at: Publisher Site | Google Scholar
J. P. Gabriel, R. Mahmood, A. Kyriakatos et al., “Serotonergic modulation of locomotion in zebrafish: endogenous release and synaptic mechanisms,” Journal of Neuroscience, vol. 29, no. 33, pp. 10387–10395, 2009.
View at: Publisher Site | Google Scholar
T. Mueller, P. Vernier, and M. F. Wullimann, “The adult central nervous cholinergic system of a neurogenetic model animal, the zebrafish Danio rerio,” Brain Research, vol. 1011, no. 2, pp. 156–169, 2004.
View at: Publisher Site | Google Scholar
P. Panula, V. Sallinen, M. Sundvik et al., “Modulatory neurotransmitter systems and behavior: towards zebrafish models of neurodegenerative diseases,” Zebrafish, vol. 3, no. 2, pp. 235–247, 2006.
View at: Publisher Site | Google Scholar
R. Gerlai, V. Lee, and R. Blaser, “Effects of acute and chronic ethanol exposure on the behavior of adult zebrafish (Danio rerio),” Pharmacology Biochemistry and Behavior, vol. 85, no. 4, pp. 752–761, 2006.
View at: Publisher Site | Google Scholar
G. Li, J. Chen, P. Xie, Y. Jiang, L. Wu, and X. Zhang, “Protein expression profiling in the zebrafish (Danio rerio) embryos exposed to the microcystin-LR,” Proteomics, vol. 11, no. 10, pp. 2003–2018, 2011.
View at: Google Scholar
Y. Zhao, Q. Xiong, and P. Xie, “Analysis of microRNA expression in embryonic developmental toxicity induced by MC-RR,” PLoS One, vol. 6, no. 7, Article ID e22676, 2011.
View at: Google Scholar
P. J. Oberholster, J. G. Myburgh, D. Govender, R. Bengis, and A. M. Botha, “Identification of toxigenic Microcystis strains after incidents of wild animal mortalities in the Kruger National Park, South Africa,” Ecotoxicology and Environmental Safety, vol. 72, no. 4, pp. 1177–1182, 2009.
View at: Publisher Site | Google Scholar
L. C. Backer, S. V. McNeel, T. Barber et al., “Recreational exposure to microcystins during algal blooms in two California lakes,” Toxicon, vol. 55, no. 5, pp. 909–921, 2010.
View at: Publisher Site | Google Scholar
R. Gerlai, M. Lahav, S. Guo, and A. Rosenthal, “Drinks like a fish: zebra fish (Danio rerio) as a behavior genetic model to study alcohol effects,” Pharmacology Biochemistry and Behavior, vol. 67, no. 4, pp. 773–782, 2000.
View at: Publisher Site | Google Scholar
E. D. Levin, Z. Bencan, and D. T. Cerutti, “Anxiolytic effects of nicotine in zebrafish,” Physiology & Behavior, vol. 90, no. 1, pp. 54–58, 2007.
View at: Publisher Site | Google Scholar
R. Gerlai, “Zebra fish: an uncharted behavior genetic model,” Behavior Genetics, vol. 33, no. 5, pp. 461–468, 2003.
View at: Publisher Site | Google Scholar
A. L. Piato, D. B. Rosemberg, K. M. Capiotti et al., “Acute restraint stress in zebrafish: behavioral parameters and purinergic signaling,” Neurochemical Research, vol. 36, no. 10, pp. 1876–1886, 2011.
View at: Google Scholar
L. J. G. Barcellos, F. Ritter, L. C. Kreutz et al., “Whole-body cortisol increases after direct and visual contact with a predator in zebrafish Danio rerio,” Aquaculture, vol. 272, no. 1–4, pp. 774–778, 2007.
View at: Publisher Site | Google Scholar
A. Oberemm, J. Becker, G. A. Codd, and C. Steinberg, “Effects of cyanobacterial toxins and aqueous crude extracts of cyanobacteria on the development of fish and amphibians,” Environmental Toxicology, vol. 14, no. 1, pp. 77–88, 1999.
View at: Google Scholar
C. Pietsch, C. Wiegand, M. V. Amé, A. Nicklisch, D. Wunderlin, and S. Pflugmacher, “The effects of a cyanobacterial crude extract on different aquatic organisms: evidence for cyanobacterial toxin modulating factors,” Environmental Toxicology, vol. 16, no. 6, pp. 535–542, 2001.
View at: Publisher Site | Google Scholar
S. M. Zala and D. J. Penn, “Abnormal behaviours induced by chemical pollution: a review of the evidence and new challenges,” Animal Behaviour, vol. 68, no. 4, pp. 649–664, 2004.
View at: Publisher Site | Google Scholar
L. Saint-Amant and P. Drapeau, “Time course of the development of motor behaviors in the zebrafish embryo,” Journal of Neurobiology, vol. 37, no. 4, pp. 622–632, 1998.
View at: Google Scholar
L. Saint-Amant and P. Drapeau, “Synchronization of an embryonic network of identified spinal interneurons solely by electrical coupling,” Neuron, vol. 31, no. 6, pp. 1035–1046, 2001.
View at: Publisher Site | Google Scholar
M. A. Borla, B. Palecek, S. Budick, and D. M. O'Malley, “Prey capture by larval zebrafish: evidence for fine axial motor control,” Brain, Behavior and Evolution, vol. 60, no. 4, pp. 207–229, 2002.
View at: Publisher Site | Google Scholar
J. Watkins, A. Miklosi, and R. J. Andrew, “Early asymmetries in the behaviour of zebrafish larvae,” Behavioural Brain Research, vol. 151, no. 1-2, pp. 177–183, 2004.
View at: Publisher Site | Google Scholar
E. Loucks and M. J. Carvan, “Strain-dependent effects of developmental ethanol exposure in zebrafish,” Neurotoxicology and Teratology, vol. 26, no. 6, pp. 745–755, 2004.
View at: Publisher Site | Google Scholar
S. Damodaran, C. A. Dlugos, T. D. Wood, and R. A. Rabin, “Effects of chronic ethanol administration on brain protein levels: a proteomic investigation using 2-D DIGE system,” European Journal of Pharmacology, vol. 547, no. 1–3, pp. 75–82, 2006.
View at: Publisher Site | Google Scholar
M. J. Airhart, D. H. Lee, T. D. Wilson, B. E. Miller, M. N. Miller, and R. G. Skalko, “Movement disorders and neurochemical changes in zebrafish larvae after bath exposure to fluoxetine (PROZAC),” Neurotoxicology and Teratology, vol. 29, no. 6, pp. 652–664, 2007.
View at: Publisher Site | Google Scholar
M. Pyron, “Female preferences and male-male interactions in zebrafish (Danio rerio),” Canadian Journal of Zoology, vol. 81, no. 1, pp. 122–125, 2003.
View at: Publisher Site | Google Scholar
R. E. Engeszer, M. J. Ryan, and D. M. Parichy, “Learned social preference in zebrafish,” Current Biology, vol. 14, no. 10, pp. 881–884, 2004.
View at: Publisher Site | Google Scholar
S. L. S. Bass and R. Gerlai, “Zebrafish (Danio rerio) responds differentially to stimulus fish: the effects of sympatric and allopatric predators and harmless fish,” Behavioural Brain Research, vol. 186, no. 1, pp. 107–117, 2008.
View at: Publisher Site | Google Scholar
K. D. Mann, E. R. Turnell, J. Atema, and G. Gerlach, “Kin recognition in juvenile zebrafish (Danio rerio) based on olfactory cues,” The Biological Bulletin, vol. 205, no. 2, pp. 224–225, 2003.
View at: Google Scholar
A. Vitebsky, R. Reyes, M. J. Sanderson, W. C. Michel, and K. E. Whitlock, “Isolation and characterization of the laure olfactory behavioral mutant in the zebrafish, Danio rerio,” Developmental Dynamics, vol. 234, no. 1, pp. 229–242, 2005.
View at: Publisher Site | Google Scholar
T. Darland and J. E. Dowling, “Behavioral screening for cocaine sensitivity in mutagenized zebrafish,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 20, pp. 11691–11696, 2001.
View at: Publisher Site | Google Scholar
J. Ninkovic and L. Bally-Cuif, “The zebrafish as a model system for assessing the reinforcing properties of drugs of abuse,” Methods, vol. 39, no. 3, pp. 262–274, 2006.
View at: Publisher Site | Google Scholar
L. J. Kily, Y. C. Cowe, O. Hussain et al., “Gene expression changes in a zebrafish model of drug dependency suggest conservation of neuro-adaptation pathways,” Journal of Experimental Biology, vol. 211, no. 10, pp. 1623–1634, 2008.
View at: Publisher Site | Google Scholar
C. Cirelli and G. Tononi, “Differential expression of plasticity-related genes in waking and sleep and their regulation by the noradrenergic system,” Journal of Neuroscience, vol. 20, no. 24, pp. 9187–9194, 2000.
View at: Google Scholar
F. E. Williams, D. White, and W. S. Messer, “A simple spatial alternation task for assessing memory function in zebrafish,” Behavioural Processes, vol. 58, no. 3, pp. 125–132, 2002.
View at: Publisher Site | Google Scholar
R. M. Colwill, M. P. Raymond, L. Ferreira, and H. Escudero, “Visual discrimination learning in zebrafish (Danio rerio),” Behavioural Processes, vol. 70, no. 1, pp. 19–31, 2005.
View at: Publisher Site | Google Scholar
I. Plaut, “Effects of fin size on swimming performance, swimming behaviour and routine activity of zebrafish Danio rerio,” Journal of Experimental Biology, vol. 203, no. 4, pp. 813–820, 2000.
View at: Google Scholar
R. Blaser and R. Gerlai, “Behavioral phenotyping in zebrafish: comparison of three behavioral quantification methods,” Behavior Research Methods, vol. 38, no. 3, pp. 456–469, 2006.
View at: Google Scholar
R. Spence, G. Gerlach, C. Lawrence, and C. Smith, “The behavior and ecology of the zebrafish, Danio rerio,” Biological Reviews of the Cambridge Philosofical Society, vol. 83, no. 1, pp. 13–34, 2008.
View at: Google Scholar
A. J. W. Ward, A. J. Duff, and S. Currie, “The effects of the endocrine disrupter 4-nonylphenol on the behaviour of juvenile rainbow trout (Oncorhynchus mykiss),” Canadian Journal of Fisheries and Aquatic Sciences, vol. 63, no. 2, pp. 377–382, 2006.
View at: Publisher Site | Google Scholar
P. Saglio and S. Trijasse, “Behavioral responses to atrazine and diuron in goldfish,” Archives of Environmental Contamination and Toxicology, vol. 35, no. 3, pp. 484–491, 1998.
View at: Publisher Site | Google Scholar
J. Xia, C. Niu, and X. Pei, “Effects of chronic exposure to nonylphenol on locomotor activity and social behavior in zebrafish (Danio rerio),” Journal of Environmental Sciences, vol. 22, no. 9, pp. 1435–1440, 2010.
View at: Publisher Site | Google Scholar
B. A. Barton, “Stress in fishes: a diversity of responses with particular reference to changes in circulating corticosteroids,” Integrative and Comparative Biology, vol. 42, no. 3, pp. 517–525, 2002.
View at: Google Scholar
A. L. Piato, K. M. Capiotti, A. R. Tamborski et al., “Unpredictable chronic stress model in zebrafish (Danio rerio): behavioral and physiological responses,” Progress in Neuro-Psychopharmacology & Biological Psychiatry, vol. 35, no. 2, pp. 561–567, 2011.
View at: Google Scholar
N. R. Bury, F. B. Eddy, and G. A. Codd, “The stress responses of brown trout, Salmo truffa L, to the cyanobacterium, Microcystis aeruginosa,” Environmental Toxicology and Water Quality, vol. 11, no. 3, pp. 187–193, 1996.
View at: Google Scholar
D. Li, P. Xie, and X. Zhang, “Changes in plasma thyroid hormones and cortisol levels in crucian carp (Carassius auratus) exposed to the extracted microcystins,” Chemosphere, vol. 74, no. 1, pp. 13–18, 2008.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2011 Luiza Wilges Kist et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Views

2774

Downloads

1533

Citations