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Effects of manganese on fat snook Centropomus parallelus (Carangaria: Centropomidae) exposed to different temperatures

ABSTRACT

This study evaluates the effects of exposure to manganese (Mn2+) for 96 hours at two different temperatures (24 and 27°C) on juveniles of Centropomus parallelus through the activities of glutathione S-transferase (GST) and catalase (CAT), micronuclei test (MN) and comet assay. The GST activity did not show any significant difference between the groups exposed to Mn2+ and the respective control groups; in contrast, a major increase in the CAT activity was observed at 27°C in the group exposed to Mn2+ compared to the control group. The genotoxic analyses showed that in all animals exposed to Mn2+, the number of red cells with micronuclei increased significantly compared to the respective control groups. There was also a significant increase in the incidence of DNA damage in the groups exposed to Mn2+. At a temperature of 24ºC, animals exposed to Mn2+ had more DNA damage than those at 27°C. It is likely that the increase in temperature can also induce oxidative stress. Thus, we conclude that manganese is toxic to the fat snook juveniles, causing genotoxic damage, and when associated with an increase in temperature, manganese can also provoke an increase in oxidative stress.

Keywords:
Biomarkers; DNA damage; Enzymes; Genotoxicity; Metal; Micronuclei

RESUMO

Este estudo avaliou os efeitos da exposição ao manganês (Mn2+), após 96 horas, a duas temperaturas (24 e 27°C) em juvenis de Centropomus parallelus por meio de análises bioquímicas (atividade das enzimas glutationa S-transferase (GST) e catalase (CAT)) e genotóxicas (teste do micronúcleo e ensaio cometa). A atividade da GST não mostrou diferença significativa entre os grupos expostos ao Mn2+ e os seus respectivos grupos controle, enquanto que um aumento significativo na atividade da CAT foi observado a 27°C no grupo exposto ao Mn2+, quando comparado ao grupo controle. As análises genotóxicas mostraram que os animais expostos ao Mn2+ tiveram aumento significativo na quantidade de células com micronúcleo em relação aos seus grupos de controles. Houve também aumento significativo na incidência de danos ao DNA nos grupos expostos a esse contaminante. Na temperatura de 24°C, os animais expostos ao Mn2+ tiveram maior quantidade de danos no DNA em relação a 27°C. É provável que o aumento da temperatura também possa induzir o estresse oxidativo. Assim, concluímos que o manganês é tóxico para os juvenis de robalo, causando dano genotóxico, e quando associado a um aumento da temperatura, também pode provocar um aumento no estresse oxidativo.

Palavras-chave:
Biomarcadores; Danos no DNA; Enzimas; Genotoxicidade; Metal; Micronúcleo

Introduction

Global climate change is one of the critical challenges that can affect ecosystem health and chemical safety. The current increases in sea surface temperature are considered as one of the new and significant threats to aquatic ecosystems (Daufresne et al., 2009Daufresne M, Lengfellner K, Sommer U. Global warming benefits the small in aquatic ecosystems. Proc Natl Acad Sci USA. 2009; 106(31):12788-93.; IPCC, 2013IPCC, Fifth Assessment Report on Climate Change 2013: The Physical Science Basis, Final Draft Underlying Scientific-Technical Assessment. Intergovernmental Panel on Climate Change. Working group 1, Geneva, 2013.). Water temperature by itself may act as a stressor since marine organisms inhabiting these environments are mostly ectotherms; as a result, the temperature affects the metabolic rate, thereby affecting energy metabolism and inducing various physiological changes (Eales, Brown, 1993Eales JG, Brown SB. Measurement and regulation of thyroidal status in teleost fish. Rev Fish Biol Fisher. 1993; 3:299-347.; Caissie, 2006Caissie D. The thermal regime of rivers: a review. Freshwater Biol. 2006; 51:1389-406.; Vergauwen et al., 2013Vergauwen L, Knapen D, Hagenaars A, Blust RR. Hypothermal and hyperthermal acclimation differentially modulate cadmium accumulation and toxicity in the zebrafish. Chemosphere . 2013; 91(4):521-29.). Furthermore, many aquatic environments are affected by the release of human contaminants, resulting in increased metal concentrations (Gomiero, Viarengo, 2014Gomiero A, Viarengo A. Effects of elevated temperature on the toxicity of copper and oxytetracycline in the marine model, Euplotes crassus: a climate change perspective. Environ Pollut . 2014; 94(1):262-71.). These facts can lead to severe impairments to aquatic organisms because the rise in water temperature may modify the chemistry of many pollutants (Schiedek et al., 2007Schiedek D, Sundelin B, Readman JW, Macdonald RW. Interactions between climate change and contaminants. Mar Pollut Bull . 2007; 54:1845-56.). These facts can also lead to increases in the bioavailability of pollutants in the environment and, consequently, the metal uptake rates and their toxicity (Bervoets et al., 1996Bervoets L, Blust R, Verheyen R. Effect of temperature on cadmium and zinc uptake by the midge larvae Chironomus riparius. Arch Environ Contam Toxicol. 1996; 31:502-11.; Vergauwen et al., 2013Vergauwen L, Knapen D, Hagenaars A, Blust RR. Hypothermal and hyperthermal acclimation differentially modulate cadmium accumulation and toxicity in the zebrafish. Chemosphere . 2013; 91(4):521-29.; Lee et al., 2014Lee S, Ji K, Choi K. Effects of water temperature on perchlorate toxicity to the thyroid and reproductive system of Oryzias latipes. Ecotox Environ Safe. 2014; 108(1):311-17.).

Manganese (Mn2+) is a constitutive element of a series of essential enzymes and cofactors that are fundamental to brain function, such as glutamine synthetase, superoxide dismutase and others (Yokel, 2009Yokel RA. Manganese flux across the blood-brain barrier. Neuromol Med. 2009; 11(4):297-310.), but it can be very toxic at concentrations above the optimal threshold level (Vieira et al., 2012Vieira MC, Torronteras R, Córdoba F, Canalejo A. Acute toxicity of manganese in gold fish Carassius auratus is associated with oxidative stress and organ specific antioxidant responses. Ecotoxicol Environ Safe . 2012; 78(1):212-17.). In natural waters, dissolved manganese from anthropogenic sources/influences associated with metal mining and other industrial activities may reach very high concentrations (McNeely et al., 1979McNeely RN, Neimanis VP, Dwyer L. Water Quality Sourcebook: a guide to water quality parameters. Ottawa: Environment Canada, Inland Waters Directorate, Water Quality Branch; 1979.; Morillo, Usero, 2008Morillo J, Usero J. Trace metal bioavailability in the waters of two different habitats in Spain: Huelva estuary and Algeciras Bay. Ecotoxicol Environ Safe . 2008; 71(1):851-59.). Recently, the Doce River basin (Minas Gerais, Brazil) suffered a severe environmental impact after the rupture of two dams controlled by a mining company (Escobar, 2015Escobar H. 2015. Mud tsunami wreaks ecological havoc in Brazil. Science 350:1138-39.), and a significant amount of metal was released into the water body; notably, Mn2+ was one of the metals that presented higher concentrations.

The deleterious effects of either chronic or acute exposure to Mn2+ depend on the species and within a species, depend on the tissue and ambient water chemistry (Fish, 2009Fish JT. Groundwater water treatment for iron and manganese reduction and fish rearing studies applied to the design of the Ruth Burnett Sport Fish Hatchery, Fairbanks, Alaska. Aquacult Eng. 2009; 41(2):97-108.; Arndt et al., 2012Arndt A, Borella, MI, Espósito BP. Toxicity of manganese metallodrugs toward Danio rerio. Chemosphere. 2014; 96(1):46-50.). Numerous studies have shown that the effects of manganese on fish include impaired functions of the gill epithelium, such as hydromineral imbalance (Gonzalez et al., 1990Gonzalez RJ, Grippo RS, Dunson WA. The disruption of sodium balance in brookchar, Salvelinus fontinalis (Mitchell), by manganese and iron. J Fish Biol . 1990; 37(1):765-74.) and histopathology of the gills (Dalzell, Macfarlane, 1999Dalzell DJB, Macfarlane NAA. The toxicity of iron to brown trout and effects on the gills: a comparison of two grades of iron sulphate. J Fish Biol. 1999; 55(2):301-15.; Hedayati et al., 2015Hedayati A, Hoseini SM, Ghelichpour M. Acute toxicity of waterborne manganese to Rutilus caspicus (Yakovlev, 1870) - gill histopathology, immune indices, oxidative condition and saltwater resistance. Toxicol Environ Chem. 2015; 96:1535-45.; Dolci et al., 2017Dolci GS, Rosa HZ, Vey LT, Pase CS, Barcelos RCS, Dias VT, Loebens L, Dalla Vecchia P, Bizzi CA, Baldisserotto B, Burger ME. Could hypoxia acclimation cause morphological changes and protect against Mn-induced oxidative injuries in silver catfish (Rhamdia quelen) even after reoxygenation? Environ Pollut. 2017; 224:466-75.). Impacts on hematology (Agrawal, Srivastava, 1980Agrawal SJ, Srivastava AK. Hematological responses in a freshwater fish to experimental manganese poisoning. Toxicology. 1980; 17:97-100.; Wepener et al., 1992Wepener V, VanVuren JHJ, Du Preez HH. Effect of manganese and iron at a neutral and acidic pH on the hematology of the banded tilapia (Tilapia sparrmanii). Bull Environ Contam Toxicol. 1992; 49(1):613-19.), immunomodulation (Cossarini-Dunier et al., 1988Cossarini-Dunier M, Demael A, Lepot D, Guerin V. Effect of manganese ions on the immune response of carp (Cyprinus carpio) against Yersinia ruckeri. Dev Comp Immunol. 1988; 12:573-79.; Hernroth et al., 2004Hernroth B, Baden SP, Holm K, Andre T, Soderhall I. Manganese induced immune suppression of the lobster, Nephrops norvegicus. Aquat Toxicol . 2004; 70:223-31.), and hormonal interference (Hoseini et al., 2004Hoseini SM, Aliakbar H, Ghelichpour M. Plasma metabolites, ions and thyroid hormones levels, and hepatic enzymes’ activity in Caspian roach (Rutilus rutiluscaspicus) exposed to waterborne manganese. Ecotoxicol Environ Safe . 2004; 107:84-89.) can also occur. Other damages on the metabolic system were also observed, such as impacts on carbohydrate metabolism (Nath, Kumar, 1987Nath K, Kumar N. Toxicity of manganese and its impact on some aspects of carbohydrate metabolism of a freshwater teleost, Colisa fasciatus. Sci Total Environ. 1987; 67:257-62.; Barnhoorn et al., 1999Barnhoorn I, van Vuren JHJ, du Preez HH. Sublethal effects of manganese on the carbohydrate metabolism of Oreochromis mossambicus after acute and chronic exposure. S Afr J Zool. 1999; 34:102-07.; Partridge, Lymbery, 2009Partridge GJ, Lymbery AJ. Effects of manganese on juvenile mulloway (Argyrosomus japonicus) cultured in water with varying salinity - implications for inland mariculture. Aquaculture. 2009; 290:311-16.) and alterations to the antioxidant system (Falfushynska et al., 2011Falfushynska HI, Gnatyshyna LL, Stoliar OB, Nam YK. Various responses to copper and manganese exposure of Carassius auratus gibelio from two populations. Comp Biochem Physiol C. 2011; 154(3):242-53.; Vieira et al., 2012Vieira MC, Torronteras R, Córdoba F, Canalejo A. Acute toxicity of manganese in gold fish Carassius auratus is associated with oxidative stress and organ specific antioxidant responses. Ecotoxicol Environ Safe . 2012; 78(1):212-17.; Dolci et al., 2013Dolci GS, Dias VT, Roversi K, Roversi K, Pase CS, Segat HJ, Teixeira AM, Benvegnu DM, Trevisol F, Barcelos RCS, Riffel APK, Nunes MAG, Dressler VL, Flores EMM, Baldisserotto B, Buerger ME. Moderate hypoxia is able to minimize the manganese-induced toxicity in tissues of silver catfish (Rhamdia quelen). Ecotoxicol Environ Safe . 2013; 91:103-07.; Gabriel et al., 2013Gabriel D, Riffel APK, Finamor IA, Saccol EMH, Ourique GM, Goulart LO, Kochhann D, Cunha MA, Garcia LO, Pavanato MA, Val AL, Baldisserotto B, Llesuy SF. Effects of subchronic manganese chloride exposure on tambaqui (Colossoma macropomum) tissues: oxidative stress and antioxidant defenses. Arch Environ Contam Toxicol . 2013; 64(1):659-67.). However, the use of biochemical and genotoxic endpoints to understand the interaction between water temperature and manganese exposure in fish has not yet been investigated.

The most commonly used biochemical tests in fish studies are assays of liver enzymes that are involved in the detoxification of xenobiotics and their respective metabolites, such as the glutathione S-transferase enzymes (GST) and catalase (CAT) (Teles et al., 2005Teles M, Pacheco M, Santos MA. Spa rus aurata L. liver EROD and GST activities, plasma cortisol, lactate, glucose and erythrocytic nuclear anomalies following short-term exposure either to17β-estradiol (E2) or E2 combined with 4-nonylphenol. Sci Total Environ . 2005; 336:57-69.; Halliwell, Gutteridge, 2006Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine, 4th ed. Oxford University Press; 2006.). The GST are phase II enzymes that play a major role in conjugation reactions and detoxification mechanisms by reducing organic hydroperoxides (ROOH). GST hydrolyzed lipophilic compounds are subsequently excreted as a water-soluble non-reactive conjugate (Simonato et al., 2011Simonato JD, Fernandes MN, Martinez CBR. Gasoline effects on biotransformation and antioxidant defenses of the freshwater fish Prochilodus lineatus. Ecotoxicology. 2011; 20:1400-10.; Azevedo et al., 2013Azevedo JS, Braga ES, Silva de Assis HC, Oliveira Ribeiro CA. Biochemical changes in the liver and gill of Cathorops spixii collected seasonally in two Brazilian estuaries under varying influences of anthropogenic activities. Ecotoxicol Environ Safe. 2013; 96:220-30.). Catalase is an essential enzyme that catalyzes the conversion of hydrogen peroxide (H2O2) into water and oxygen, thereby preventing its conversion to hydroxyl radicals and reducing oxidative stress levels (Gonçalves-Soares et al., 2012Gonçalves-Soares D, Zanette J, Yunes JY, Yepiz-Plascencia GM, Bainy ACD. Expression and activity of glutathione S-transferases and catalase in the shrimp Litopenaeus vannamei inoculated with a toxic Microcystis aeruginosa strain. Mar Environ Res . 2012; 75(1):54-61.). Catalase is usually regulated by complex and interconnected systems that are sensitive to the concentration of its substrates (Lushchak, 2011Lushchak VI. Environmentally induced oxidative stress in aquatic animals. Aquat Toxicol . 2011; 101(1):13-30.). Thus, these enzymes often show increased activities when the production of superoxide and peroxide anions increase up to a certain level (McCord, Fridovich, 1969McCord JM, Fridovich I. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem . 1969; 244(1):6049-55.; Beutler, 1975Beutler E. Red cell metabolism: a manual of biochemical methods. Grune & Stratton, New York; 1975.; Lushchak, 2011Lushchak VI. Environmentally induced oxidative stress in aquatic animals. Aquat Toxicol . 2011; 101(1):13-30.).

Among the available genotoxicity tests, the alkaline comet assay and the micronuclei test (MN) are the most commonly used and recognized due to their robustness, sensitivity and statistical power in the evaluation of DNA injuries. Thus, these analyses can be considered complementary biomarkers of DNA damage based on two different endpoints (Tice et al., 2000Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF. Single cell gel/comet assay, guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen . 2000; 35(3):206-21.; Udroiu, 2006Udroiu I. The micronucleus test in piscine erythrocytes. Aquat Toxicol . 2006; 79:201-204.; Heuser et al., 2008Heuser VM, de Andrade VM, Peres A, Braga LMGM, Chies AB. Influence of age and sex on the spontaneous DNA damage detected by Micronucleus test and Comet assay in mice peripheral blood cells. Cell Biol Int. 2008; 32:1223-29.). The MN test is a fast method of detecting structural and numerical chromosomal alterations that are induced by clastogenic and aneugenic agents (Heddle et al., 1991Heddle JA, Cimino MC, Hayashi M, Romanga F, Shelby MD, Tucker JD, Vanparys PH, MacGregor JT. Micronuclei as an index of cytogenetic damage: past, present and future. Environ. Mol Mutagen. 1991; 18:277-91.; Jha, 2008Jha AN. Ecotoxicological applications and significance of the comet assay. Mutagenesis 2008; 23:207-21.). By contrast, the comet assay measures strand breaks before the DNA repair systems intervene; these can include DNA single-and double-strand breaks, alkali-labile sites and excision-repair events caused by simple and bulky DNA adducts (Singh et al., 1988Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantification of low levels of DNA damage in individual cells. Exp Cell Res. 1998; 175:184-91.; Tice et al., 2000Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF. Single cell gel/comet assay, guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen . 2000; 35(3):206-21.).

The fat snook (Centropomus parallelus Poey, 1860) can inhabit coastal waters, estuaries and freshwater environments (Cerqueira, Tsuzuki, 2009Cerqueira VR, Tsuzuki EMY. A review of spawning induction, larviculture and juvenile rearing of the fat snook, Centropomus parallelus. Fish Physiol Biochem. 2009; 35(1):17-28.) that are commonly characterized by high anthropogenic pressure, including the occurrence of metal contamination. Moreover, because the fat snook is an ectothermic species, its susceptibility to metal toxicity can be modified by higher water temperatures through changes in its rates of biochemical and physiological processes and the stability of its biomolecules (Heugens et al., 2001Heugens EH, Hendriks AJ, Dekker T, van Straalen, NM, Admiraal W. A review of the effects of multiple stressors on aquatic organisms and analysis of uncertainty factors for use in risk assessment Crit Rev Toxicol. 2001; 31(3):247-84.; Lannig et al., 2006Lannig G, Flores JF, Sokolova IM. Temperature-dependent stress response in oysters Crassostrea virginica: pollution reduces temperature tolerance in oysters. Aquat Toxicol . 2006; 79(3):278-87.).

Thus, we conducted short-term thermal bioassays (96 h) to investigate the effects of different temperatures (24 and 27°C) on manganese toxicity in C. parallelus. To do this, we analyzed responses to the phase II biotransformation enzyme glutathione S-transferase (GST), the antioxidant enzyme catalase (CAT) and genotoxic analyses (i.e., alkaline comet assay and the micronuclei test).

Material and Methods

Animals and acclimation. The fat snook, C. parallelus juveniles (Voucher # MBML 12877) were obtained from a hatchery and transferred to the laboratory. For temperature acclimatization, 72 fish (2.46 ± 0.54 g and 7.12 ± 0.52 cm) were equally divided into two clean tanks (300 L; fish density = 0.3 g L-1) with water temperatures of either 24°C (similar conditions of seawater) or 27°C (approximately a 2.5°C increase in water temperature as devised by the moderate Intergovernmental Panel on Climate Change (IPCC) emission scenario). The water temperature was gradually increased by 1.0 ± 0.1°C/day using a heater coupled to a thermostat (Full Gauge, TIC-17RGT). The tanks were filled with aerated seawater that was biologically filtered and experienced a 12:12 h (light:dark) photoperiod. During the acclimatization period (three weeks), the fish were fed daily to satiation with commercial fish food that was composed of 1.2 mm pellets with 60% protein (NRD INVE, Belgium), and the seawater was replaced twice each week with seawater of the same temperature. Feeding was suspended 24 h before the animals were transferred into the test aquaria. The physicochemical parameters were monitored daily using a multiparameter YSI (model 85, Yellow Springs Inc. Ohio, United States). The water quality parameters for tanks with water at 24ºC and 27ºC were as follows: temperature (24.2 ± 0.3 and 27.1 ± 0.2°C, respectively), dissolved oxygen (5.8 ± 0.5 and 6.0 ± 0.4 mg L-1, respectively), salinity (25.4 ± 0.1 and 25.3 ± 0.1 ppt, respectively) and conductivity (25.4 ± 0.1 and 25.3 ± 0.1 µS cm-1, respectively). The pH (8.1 ± 0.1 and 8.1 ± 0.1, respectively) was monitored with a pH meter YSI 100 (Yellow Springs Inc., OH, USA). The total ammonia (0.63 ± 0.3 and 0.63 ± 0.4, respectively) and total nitrites (0.3 ± 0.4 and 0.3 ± 0.3, respectively) were monitored according to APHA guidelines (2005APHA. Standard methods for the examination of water and waste water, 21st ed. American Public Health Association, Washington, DC; 2005.).

Short-term toxicity test. After acclimatization, groups of six fish were transferred to 12 polyethylene aquaria containing 20 L of seawater (fish density = 0.75 g L-1; salinity = 32) under constant aeration, and temperature was controlled by a heater coupled to a digital thermostat (0.1°C precision). Fish were kept under the respective temperatures they were acclimated to (i.e., 24.2 ± 0.3°C and 27.1 ± 0.2°C). For each temperature, the fish groups were divided into two treatments: i) control (no contaminant addition) or ii) exposed to a nominal manganese concentration (3.18 mg L-1 or 26.426 nM Mn2+ as MnCl2·4H2O), and the experiment lasted for 96 h. A total of four treatments were conducted (i.e., the two temperatures versus the two Mn2+ concentrations) (three replicates for each treatment; 18 fish per treatment). After contamination, the concentration of dissolved Mn2+ was measured in the water of all aquaria at the start of the experiment, and the average values were 2.269 mg L-1 (treatment group) and 0.00 mg L-1 (control group). Values of 0.00 were considered as the control group since the Mn2+ measurements in this group were below of the limit of quantification of AAS. The manganese treatments were chosen according to previous studies conducted with Lithobates catesbeianus (Veronez et al., 2016Veronez ACS, Salla RV, Baroni VD, Barcarolli IF, Bianchini A, Martinez CBR, Chippari-Gomes AR. Genetic and biochemical effects induced by iron ore, Fe and Mn exposure in tadpoles of the bullfrog Lithobates catesbeianus. Aquat Toxicol . 2016; 174 (1):101-108.). During the experiment, one-third of the water (of the appropriate temperature and contaminant level) was renewed after 48 h. No mortality was observed during the experiment. All procedures performed in the present study were approved by the Ethics Committee for Animal Use of the Universidade Vila Velha (CEUA-UVV), number 198-2011.

Biological material sampling. Immediately after removing the fish from the aquaria, they were anesthetized with benzocaine (0.1 g L-1), and blood samples were taken from the caudal vein using heparinized syringes; blood samples were used in the alkaline comet assay and the micronuclei test (MN). Then, animals were weighed, measured and euthanized by cervical sectioning. A fragment of the liver from each animal was removed and stored at -80°C until enzymatic analyses.

Enzyme assays. Assays of the activity of the biotransformation enzyme, glutathione S-transferase (GST - EC 2.5.1.18), and the antioxidant enzyme, catalase (CAT - EC 1.11.1.6), were carried out according to well-established protocols. Initially, the frozen liver samples were weighed and homogenized (1:4 - w/v) in 20 mM Tris buffer (pH 7.4) with 0.5 mM sucrose, 0.15 mM KCl and 1 mM protease inhibitor (PMSF). The homogenates were then centrifuged at 10,000 g for 20 min at 4°C, and the enzyme assays were run on a SpectraMax Plus 384 spectrophotometer (Molecular Devices). The GST activity was determined using a phosphate buffer solution (pH 7.0) containing 1-chloro-2,4-dinitrobenzene (CDNB; 1 mM) and glutathione (GSH; 1 mM) as substrate. Enzyme activity was determined based on the extinction coefficient of CDNB (Habig et al., 1974Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem. 1974; 249(22):7130-39.; Habig, Jakoby, 1981Habig WH, Jakoby WB. Assays for differentiation of glutathione S-transferases. Methods Enzymol. 1981;77:398-405.). Catalase activity was determined by the continuous evaluation of the decrease in the concentration of hydrogen peroxide (H2O2) (Aebi, 1984Aebi H. Catalase in vitro. Method Enzymol. 1984; 105(2):121-26.). The reaction medium was prepared with a buffer solution (1 M Tris HCl and 5 mM EDTA) containing hydrogen peroxide (10 mM). The results are reported as µmol min-1 mg protein-1. The protein quantification of all samples was determined at a controlled room temperature (25°C), according to Lowry et al. (1951Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem . 1951; 193(1):265-75.).

Alkaline comet assay. Blood samples were diluted 1:120 (v/v) in RPMI 1640 medium (RPMI-Roswell Park Memorial Institute) and used immediately. The alkaline comet assay was performed as described by Tice et al. (2000Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF. Single cell gel/comet assay, guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen . 2000; 35(3):206-21.) and Andrade et al. (2004Andrade VM, De Freitas TRO, Da Silva J. Comet assay using mullet (Mugil sp.) and sea catfish (Netuma sp.) erythrocytes for the detection of genotoxic pollutants in aquatic environment. Mutat Res . - Genet Toxicol Environ Mutagen. 2004; 560, 57-67.), with some modifications. Briefly, 5 μl of each diluted blood sample was added to 95 μl of 0.75% (w/v) molten low-melting-point agarose, and an aliquot of the mixture was spread on a microscope slide that was pre-coated with 1.5% (w/v) normal melting point agarose and topped with a coverslip. After agarose solidification, coverslips were removed, and the slides were immersed in a lysis solution (2.5 M NaCl,100 mM EDTA and 10 mM Tris, pH 10.0-10.5) containing 1% Triton X-100 and 20% DMSO. Slides were maintained in this lysis solution (4°C) and kept in the dark for 2-3 h. Slides were then incubated in a freshly prepared alkaline buffer solution (300 mM NaOH and 1 mM EDTA, pH ≥13) for 20 min for DNA unwinding. Electrophoresis (15 min at 300 mA and 25 V) was performed using the same buffer solution. Each step was performed under indirect yellow light. After electrophoresis, slides were neutralized in a Tris solution (400 mM; pH 7.5), rinsed three times with distilled water, and dried overnight at room temperature. Slides were fixed for 10 min in trichloroacetic acid (15% w/v), zinc sulfate (5% w/v), and glycerol (5% v/v), and then rinsed three times with distilled water and dried at 37°C for 2 h. Dry slides were rehydrated for 5 min in distilled water and stained under constant shaking for 35 min using a solution containing sodium carbonate (5% w/v), ammonium nitrate (0.1% w/v), silver nitrate (0.1% w/v), tungstosilicic acid (0.25%), and formaldehyde (0.15% w/v), which was freshly prepared in the dark. Stained slides were rinsed twice with distilled water, submerged in the stop solution (acetic acid 1%), rinsed again with distilled water, and immediately coded for analysis. A total of 100 cells from each replicate (i.e., 50 from each duplicate slide) were randomly analyzed under an optical microscope (100× magnification) to measure the length of the comet’s tail. The analysis of the slides involved 100 cells/fish using a visual classification based on the degree of DNA fragment migration from the nucleus. Cells were classified into class 0 (no damage), class 1 (little damage - when the tail length was smaller than the nucleus), class 2 (medium damage - when the tail length was between 1 and 2 times the nucleus diameter), class 3 (extensive damage - when the tail length was over 2 times the nucleus diameter), and class 4 (presence of apoptosis) (Kobayashi et al., 1995Kobayashi H, Suguyama C, Morikawa Y, Hayashi M, Sofuni T. A comparison between manual microscopic analysis and computerized image analysis in the single cell gel electrophoresis. MMS Commun. 1995; 2(3):103-15.; Speit, Hartmann, 1999Speit G, Hartmann A. The comet assay (single-cell gel test): a sensitive genotoxicity test for the detection of DNA damage and repair. Methods Mol Biol. 1999; 113(1):203-12.). The DNA damage index (DI) was calculated for each fish as the sum of the number of nucleoids that were observed for each damage class multiplied by the value of its respective damage class (0, 1, 2, 3 or 4). The results were expressed as the mean DNA damage index for each experimental group, where 0 represented the absence of damage and 400 indicated the highest damage score.

Micronuclei (MN) frequency. Peripheral blood samples were obtained from the caudal vein and smeared onto clean slides. The slides were air-dried after fixation in pure ethanol for 20 min, and the smears were stained with a 10% Giemsa solution for 25 min. Each MN was identified according to the following criteria: spherical cytoplasmic inclusions with a sharp contour, a diameter smaller than one-third the diameter of the nucleus, a color and texture resembling the nucleus, and no contact with the nucleus (Al-Sabti, Metcalfe, 1995Al-Sabti K, Metcalfe CD. Fish micronuclei for assessing genotoxicity in water. Mutat Res. 1995; 343(2-3):121-35.; Kirsch-Volders et al., 2003Kirsch-Volders M, Sofuni T, Aardema M, Albertini S, Eastmond D, Fenech M, Ishidate MJr, Kirchner E, Lorge E, Morita T, Norppa H, Surrales J, Vanhauwaert A, Wakata A. Report from the in vitro micronucleus assay working group. Environ Mol Mutagen. 2003; 540(2):153-63.). A total of 2000 erythrocytes per fish were examined under an Olympus optical microscope (1000× magnification), and the mean frequencies of the MN found in each experimental group were calculated and expressed per 1000 cells (‰). Only intact cells with distinct nuclear and cellular membranes were scored.

Statistical analyses. The acute toxicity test data were analyzed for normality and homoscedasticity through Shapiro-Wilk and Levene’s test, respectively. Data were analyzed by two-way ANOVA to determine significant differences in Mn2+ exposure, temperatures and the interaction between these two factors, followed by the Bonferroni test for post hoc comparisons. All data are expressed as the mean ± standard error of the mean (SEM), and the differences were considered significant at p ≥ 0.05. GraphPad Prisma 5.0 software was used for statistical analyses.

Results

Enzyme activities. The biotransformation enzyme GST activity did not show any significant changes as a result of the heat stress treatments (P=0.905; F=0.01) and Mn2+ exposure (Fig. 1a; P=0.178; F=1.85). In addition, there was no significant interaction between the two factors (temperature and Mn2+) (Fig. 1a; P=0.7611; F=0.09).

For antioxidant enzyme CAT activity, temperature did not affect the activity of this enzyme (P=0.1154; F=2.55), and CAT activity was not affected by exposure to Mn at 24°C (Fig. 1b; P=0.1246; F=2.42). However, at 27°C, the enzymatic activity of CAT was significantly higher in fat snook exposed to Mn2+ compared to the respective control (Fig. 1b; P=0.0034; F=4.67).

Fig. 1
Activities of glutathione S-transferase (GST) (a) and catalase (CAT) (b) in liver of Centropomus parallelus, after 96 h exposure to manganese (Mn2+) (grey bars) at two different temperatures, and their respective control groups (white bars). Values represent the means ± standard error. Each treatment was performed in triplicate (n = 6 per aquarium).

Comet assay. The result of the DNA damage or fragmentation, measured as the damage index (DI), demonstrated that Mn2+ provoked damage on DNA in both temperatures compared with the respective control group (Fig. 2a; P<0.0001; F=56.06). The temperature increase from 24 to 27°C, by itself, did not interfere with the results since the control groups were similar. However, there was a significant interaction between the two factors, Mn2+ and temperature (Fig. 2a; P=0.0435; F=4.24). At 27ºC, it was observed that there was a decrease in the amount of DNA damage caused by exposure to Mn2+ compared to the group acclimated to 24°C.

Micronuclei (MN) test. The fat snook exposed to Mn2+ showed a significant increase in the frequency of micronuclei in the erythrocytes of the fat snook acclimatized in two temperatures (Fig. 2b; P<0.0001; F=48.61) when compared to their respective control groups. In addition, it was observed that there was a significant interaction between the two factors, Mn2+ and temperature (Fig. 2b; P=0.0073; F=7.64), since the fish acclimated to 27°C and exposed to Mn2+ showed more micronuclei in relation to fish acclimated to 24°C and exposed to Mn2+.

Fig. 2
Damage index (DI) (a) and Micronuclei frequency (b) in erythrocytes of Centropomus parallelus, after a 96 h exposure to manganese (Mn2+) (grey bars) at two different temperatures, and their respective control groups (white bars). Values represent the means ± standard error. Different lowercase letters indicate significant differences (p ≥ 0.05) between the temperature treatments, asterisks mark (*) indicate significant differences (p ≥ 0.05) in relation to controls. Each treatment was performed in triplicate (n = 6 per aquarium).

Discussion

In the present study, the interaction between short-term exposure to a sublethal concentration of Mn2+ and an increase in water temperature did not affect the GST activity. Some GST changes were expected in fish exposed to Mn2+ since GST plays a significant role in metabolism, acting on the detoxification of some electrophilic compounds, and changes in the activities of this enzyme directly reflect metabolic disturbances and cell damage in specific organs of fish (Carvalho-Neta, Abreu-Silva, 2013Carvalho-Neta RNF, Abreu-Silva AL. Glutathione S-Transferase as biomarker in Sciades herzbergii (Siluriformes:Ariidae) for environmental monitoring: the case study of São Marcos Bay, Maranhão, Brazil. Lat Am J Aquat Res. 2013; 41:217-25.); however, this response was not verified in this study. The present results might indicate that the metabolism of Mn2+ occurs by another biotransformation pathway. Instead, the biochemical responses of biotransformation found in this study are in accordance with the previous investigations of thermal stress by other authors. These studies reported that the levels of GSH-dependent antioxidant enzymes, such as glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST), in the different tissues of Carassius auratus (at 3 and 23°C) (Bagnyukova et al., 2007Bagnyukova TV, Lushchak OV, Storey KB, Lushchak VI. Oxidative stress and antioxidant defense responses by goldfish tissues to acute change of temperature from 3 to 23 °C. J Therm Biol. 2007; 32(1):227-34.), Morone saxatilis (at 7 and 25°C) (Grim et al., 2013Grim JM, Simonik EA, Semones MC, Kuhn DE, Crockett EL. The glutathione dependent system of antioxidant defense is not modulated by temperature acclimation in muscle tissues from striped bass, Morone saxatilis. Comp Biochem Physiol A. 2013; 164(2):383-90.) and Notothenia coriiceps (at 0 and 8°C) (Machado et al., 2014Machado C, Zaleski C, Rodrigues E, Carvalho CS, Cadena SMSC, Gozzi GJ, Krebsbach P, Rios FS, Donatti L. Effect of temperature acclimation on the liver antioxidant defense system of the Antarctic nototheniids Notothenia coriiceps and Notothenia rossii. Comp. Biochem. Physiol. B. 2014; 172-173:21-28.) were slightly affected by the increase in temperature.

On the other hand, the 27°C treatment resulted in a stimulation of the CAT activity, which was not observed at 24°C; it is possible that the combination of both increased temperature and Mn2+ exposure could be related to changes in the oxidative stress levels. According to Daoud et al. (2007Daoud D, Chabot O, Audet C, Lambert Y. Temperature induced variation in oxygen consumption of juvenile and adult stages of the northern shrimp, Pandalus borealis. J Exp Mar Biol Ecol. 2007; 347(1):30-40.), increased CAT activity is usually associated with rising temperatures, which, in turn, further accelerate the metabolic rate of the organisms. In the present study, this was not observed, since the CAT activity of fat snook acclimated to 27°C did not differ from those acclimated to 24°C (control groups); however, when Mn2+ was added to the water at 27°C, CAT activity increased significantly in the group exposed to Mn2+. These results suggest that the interaction between elevated temperature and Mn2+ influences CAT activity. Nevertheless, there are several reports concerning patterns of CAT expression under oxidative-stress-inducing conditions, such as chemical toxicity and thermal stress. These antioxidant defenses may be increased (Khessiba et al., 2005Khessiba A, Romeo M, Aissa P. Effects of some environmental parameters on catalase activity measured in the mussel (Mytilus galloprovincialis) exposed to lindane. Environ Pollut. 2005; 133(2):275-81.; Vinagre et al., 2012Vinagre C, Madeira D, Narciso L, Cabral H, Diniz M. Effect of temperature on oxidative stress in fish: lipid peroxidation and catalase activity in the muscle of juvenile seabass Dicentrarchus labrax. Ecol Ind. 2012; 23(1):274-79., 2014Vinagre C, Madeira D, Mendonça V, Dias M, Roma J, Diniz M, Effect of temperature on multiple biomarkers of oxidative stress in coastal shrimp. J Therm Biol . 2014; 41(1):38-42.), inhibited (Kaur et al., 2011Kaur M, Atif F, Ansari RA, Ahmad F, Raisuddin S. The interactive effect of elevated temperature on deltamethrin-induced biochemical stress responses in Channa punctata Bloch. Chemico-Biol Interact. 2011; 193(1):216-24.; Sabatini et al., 2011Sabatini SE, Rocchetta I, Nahabedian DE, Luquet CM, Eppis MR, Bianchi L, Ríos de Molina MC. Oxidative stress and histological alterations produced by dietary copper in the fresh water bivalve Diplodon chilensis. Comp Biochem Physiol. C . 2011; 154:391-98.) or unaffected (Mueller et al., 2012Mueller IA, Devor DP, Grim JM, Beers JM, Crockett EL, O’Brien KM. Exposure to critical thermal maxima increases oxidative stress in hearts of white but not red-blooded Antarctic notothenioid fishes. J Exp Biol. 2012; 215(20):3655-64.; Gabriel et al., 2013Gabriel D, Riffel APK, Finamor IA, Saccol EMH, Ourique GM, Goulart LO, Kochhann D, Cunha MA, Garcia LO, Pavanato MA, Val AL, Baldisserotto B, Llesuy SF. Effects of subchronic manganese chloride exposure on tambaqui (Colossoma macropomum) tissues: oxidative stress and antioxidant defenses. Arch Environ Contam Toxicol . 2013; 64(1):659-67.; Machado et al., 2014Machado C, Zaleski C, Rodrigues E, Carvalho CS, Cadena SMSC, Gozzi GJ, Krebsbach P, Rios FS, Donatti L. Effect of temperature acclimation on the liver antioxidant defense system of the Antarctic nototheniids Notothenia coriiceps and Notothenia rossii. Comp. Biochem. Physiol. B. 2014; 172-173:21-28.) by agent stressors. The occurrence of one type of response or another depends on the intensity and duration of the stress applied, the susceptibility of the species that are exposed and/or the route of exposure (Bebianno et al., 2005Bebianno MJ, Company R, Serafim A, Cosson RP, Fiala-Medoni A. Antioxidant systems and lipid peroxidation in Bathymodiolus azoricus from Mid-Atlantic Ridge hydrothermal vent fields. Aquat Toxicol. 2005; 75(4):354-73.; Sanchez et al., 2005Sanchez W, Palluel O, Meunier L, Coquery M, Porcher JM, Ait-Aissa S. Copper-induced oxidative stress in three-spined stickleback: relationship with hepatic metal levels. Environ Toxicol Pharmacol . 2005; 19(1):177-83.). Furthermore, CAT activity may be species-specific (Fonseca et al., 2011Fonseca VF, França S, Serafim A, Company R, Lopes B, Bebianno MJ, Cabral HN. Multi-biomarker responses to estuarine habitat contamination in three fish species: Dicentrarchus labrax, Solea senegalensis and Pomatoschistus microps. Aquat Toxicol . 2011; 102(3-4):216-27.; Madeira et al., 2013Madeira D, Narciso L, Cabral HN, Vinagre C, Diniz MS, Influence of temperature in thermal and oxidative stress responses in estuarine fish. Comp Biochem Physiol. A. 2013; 166(2):237-43., 2014Madeira D, Narciso L, Cabral H, Diniz M, Vinagre C, Role of thermal niche in the cellular response to thermal stress: lipid peroxidation and HSP70 in coastal crabs. Eco Ind. 2014; 36(1):601-06.) and tissue-specific (Viera et al., 2012Vinagre C, Madeira D, Narciso L, Cabral H, Diniz M. Effect of temperature on oxidative stress in fish: lipid peroxidation and catalase activity in the muscle of juvenile seabass Dicentrarchus labrax. Ecol Ind. 2012; 23(1):274-79.; Vinagre et al., 2014Vinagre C, Madeira D, Mendonça V, Dias M, Roma J, Diniz M, Effect of temperature on multiple biomarkers of oxidative stress in coastal shrimp. J Therm Biol . 2014; 41(1):38-42.). In the present study, it can be observed that the association between the two conditions (temperature and Mn2+) to which the fat snook specimens were exposed induced the activation of the catalase enzyme, and therefore, induced the activation of the oxidation system.

In this study, we also elucidated the modulatory effects on genotoxicity of an increase in temperature associated with Mn2+ exposure in C. parallelus through the alkaline comet assay and the micronuclei test. Both assays are useful biomarkers of environmental genotoxicity testing (Barsiene et al., 2012Baršiene J, Rybakovas A, Lang T, Grygiel W, Andreikenaite L, Michailovas A. Risk of environmental genotoxicity in the Baltic Sea over the period of 2009-2011 assessed by micronuclei frequencies in blood erythrocytes of flounder (Platichthys flesus), herring (Clupea harengus) and eelpout (Zoarces viviparus). Mar Environ Res. 2012; 77(1):35-42.; Dar et al., 2015Dar SA, Yousuf AR, Balkhi MH, Ganai FA, Bhat FA. Assessment of endosulfan induced genotoxicity and mutagenicity manifested by oxidative stress pathways in freshwater cyprinid fish crucian carp (Carassius carassius L.). Chemosphere . 2015; 120(1):273-83.). Our results indicated that the exposure of C. parallelus to sublethal concentrations of Mn2+, combined with the increase in water temperature, induced significantly higher DNA damage in the temperature treatments than in the control; thus, these results indicated the genotoxic potential of these experimental conditions. Earlier studies, which also used the comet assay method, have associated thermal stress with genotoxic effects in aquatic organisms, such as freshwater fish (Carassius auratus), crayfish (Astacus leptodactylus) and mussels (Dreissena polymorpha) (Anitha et al., 2000Anitha B, Chandra N, Gopinath PM, Durairaj G. Genotoxicity evaluation of heat shock in gold fish (Carassius auratus). Mutat Res . 2000; 469(1):1-8.; Buschini et al., 2003Buschini A, Carboni P, Martino A, Poli P, Rossi C. Effects of temperature on baseline and genotoxicant-induced DNA damage in hemocytes of Dreissena polymorpha. Mutat Res . 2003; 537(1):81-92.; Malev et al., 2010Malev O, Šrut M, Maguire I, Štambuk A, Ferrero EA, Lorenzon S, Klobučar GIV. Genotoxic, physiological and immunological effects caused by temperature increase, air exposure or food deprivation in freshwater crayfish Astacus leptodactylus. Comp Biochem Physiol. C. 2010; 152:433-43.).

In the present study, although a significant increase in the DI was achieved in both temperature treatments compared to the control, we also observed a decrease in the DI values as the water temperature increased. Such depletion could be explained by the cytotoxic potential exerted by thermal stress, leading to changes in the blood cell kinetics and erythrocyte replacement (Çavas, Ergene-Gözükara, 2003Çavas T, Ergene-Gozukara S. Micronuclei, nuclear lesions and interphase silver-stained nucleolar organizer regions (AgNORs) as cyto-genotoxicity indicators in Oreochromis niloticus exposed to textile mill effluent. Mutat Res . 2003; 534(1):93-99.). This observation finds support in the literature (Polard et al., 2011Polard T, Jean SLG, Laplanche C, Merlina G, Sánchez-Pérez JM, Pinelli E. Mutagenic impact on fish of runoff events in agricultural areas in south-west France. Aquat Toxicol . 2011; 101(1):126-34.; Vera-Candioti et al., 2013Vera-Candioti V, Soloneski S, Larramendy ML. Single-cell gel electrophoresis assay in the ten spotted live-bearer fish, Cnesterodon decemmaculatus (Jenyns, 1842), as bioassay for agrochemical-induced genotoxicity. Ecotoxicol Environ Safe . 2013; 98(1):368-73.); nevertheless, the decrease in DI values might be caused by other cytotoxic agents that also affected circulating blood cell populations.

The alkaline comet assay detects DNA damage, such as DNA single-strand breaks, DNA double-strand breaks or DNA-DNA/DNA-protein cross-linking (Guilherme et al., 2012Guilherme S, Gaivão I, Santos MA, Pacheco M. DNA damage in fish (Anguilla anguilla) exposed to a glyphosate-based herbicide-Elucidation of organ-specificity and the role of oxidative stress. Mutat Res . 2012; 743(1-2):1-9.), that could have originated from the interaction between the free radicals formed as a result of the oxidative stress and the DNA of the blood cells (Azqueta et al., 2011Azqueta A, Meier S, Priestley C, Gutzkow KB, Brunborg G, Sallette J, Soussa-line F, Collins AR. The influence of scoring method on variability in results obtained with the comet assay. Mutagenesis. 2011; 26(3):393-99.; Nwani et al., 2013Nwani CD, Nagpure NS, Kumar R, Kushwaha B, Lakra WS. DNA damage and oxidative stress modulatory effects of glyphosate-based herbicide in freshwater fish, Channa punctatus. Environ Toxicol Pharmacol. 2013; 36(2):539-47.). Moreover, unlike the micronuclei induction, the DNA damage detected by the alkaline comet assay is relatively minor and often transient (Dixon et al., 2002Dixon DR, Pruski AM, Dixon LRJ, Jha AN, Marine invertebrate ecogenotoxicity: a methodological overview. Mutagenesis 2002; 17(6):495-507.). Furthermore, we also may suggest that the enhancement of CAT activity allows a significant decrease in DI values as the water temperature increases; repairs damaged macromolecules, such as DNA, and alleviates oxidative stress.

The micronuclei test is considered to be a sensitive and informative marker of cytogenetic damage caused by mutagenic compounds (Arslan et al., 2015Arslan ÖÇ, Boyacioglu M, Parlak H, Katalay S, Karaaslan MA. Assessment of micronuclei induction in peripheral blood and gill cells of some fish species from Aliag a Bay Turkey. Mar Pollut Bull. 2015; 94(1-2):48-54.) and has been applied to identify the adverse potential of various genotoxic agents (Barsiene et al., 2012Baršiene J, Rybakovas A, Lang T, Grygiel W, Andreikenaite L, Michailovas A. Risk of environmental genotoxicity in the Baltic Sea over the period of 2009-2011 assessed by micronuclei frequencies in blood erythrocytes of flounder (Platichthys flesus), herring (Clupea harengus) and eelpout (Zoarces viviparus). Mar Environ Res. 2012; 77(1):35-42.), both in freshwater and marine sentinel species (Guidi et al., 2010Guidi P, Frenzilli G, Benedetti M, Bernardeschi M, Falleni A, Fattorini D, Regoli F, Scarcelli V, Nigro M. Antioxidant, genotoxic and lysosomal biomarkers in the freshwater bivalve (Unio pictorum) transplanted in a metal polluted river basin. Aquat Toxicol . 2010; 100(1):75-83.). The increase in MN frequency was observed in both treatment groups compared to the control. The increase in MN frequency is an indirect marker of numeric and structural chromosomal irregularities in the cells caused by many agents (Arslan et al., 2015Arslan ÖÇ, Boyacioglu M, Parlak H, Katalay S, Karaaslan MA. Assessment of micronuclei induction in peripheral blood and gill cells of some fish species from Aliag a Bay Turkey. Mar Pollut Bull. 2015; 94(1-2):48-54.); thus, this indicates the clastogenic and/or aneugenic capacity of Mn2+ exposure combined with increased temperature. Furthermore, it is well documented that temperature modulates aquatic organisms’ sensitivity to metals by affecting their physiological tolerance, energy demand, oxygen supply, and/or mitochondrial biogenesis (Sokolova, Lanning, 2008Sokolova IM, Lanning G. Interactive effects of metal pollution and temperature on metabolism in aquatic ectotherms: implications of global climate change. Climate Res. 2008; 37(1):181-201.).

The results show that the combination of Mn2+ exposure and thermal stress caused significant DNA damage in C. parallelus, and the oxidative system can also be induced. Our findings indicate that the identified endpoints provide useful information for biomonitoring studies; additionally, they underline the need for comprehensive research on the possible influence of increased temperatures on the mechanism of Mn2+ and heavy metal toxicity in fish.

Acknowledgments

This work was supported by a research grant from FAPES (Proc. # 61902861). B.L.L Tuzuki was a master fellow from FAPES. F.A.C. Delunardo is a Ph.D. fellow from FAPES (Proc. # 61636509/2013). The authors thank the Instituto Nacional da Mata Atlântica (INMA) for accepting the deposit of voucher specimen in the Museu de Biologia Professor Mello Leitão.

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Publication Dates

  • Publication in this collection
    2017

History

  • Received
    30 June 2017
  • Accepted
    14 Nov 2017
Sociedade Brasileira de Ictiologia Neotropical Ichthyology, Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura, Universidade Estadual de Maringá., Av. Colombo, 5790, 87020-900, Phone number: +55 44-3011-4632 - Maringá - PR - Brazil
E-mail: neoichth@nupelia.uem.br