Harmful Algae 49 (2015) 135–146

Growth and antioxidant response of Microcystis aeruginosa(Cyanobacteria) exposed to anatoxin-a

Mathias Ahii Chia a, Micheline Kezia Cordeiro-Araujo a,b,Maria do Carmo Bittencourt-Oliveira a,*a Department of Biological Sciences, Luiz de Queiroz College of Agriculture, University of Sao Paulo, Av. Padua Dias, 11, Sao Dimas, CEP 13418-900 Piracicaba,

SP, Brazil1

b Botany Graduate Program, Rural and Federal University of Pernambuco, R. Dom Manoel de Medeiros, S/N, Dois Irmaos, CEP 52171-030 Recife, PE, Brazil2

A R T I C L E I N F O

Article history:

Received 19 February 2015

Received in revised form 21 September 2015

Accepted 21 September 2015

Keywords:

Neurotoxins

Anatoxin-a

Phytotoxicity

Antioxidant enzyme activities

Microalgae

Cyanobacteria

A B S T R A C T

Changing global climatic conditions are expected to stimulate excessive proliferation of toxic and non-

toxic cyanobacteria. Under these changing conditions, toxin production by different toxic cyanobacteria

may increase. However, the effects of most of these cyanotoxins including anatoxin-a (ATX) on the

growth and physiological response of phytoplankton species have been poorly investigated. The present

study investigated the effect of ATX at environmentally relevant concentrations (5, 10, 25 and 50 mg L�1)

on Microcystis aeruginosa (Kutzing) Kutzing. Cell density of M. aeruginosa did not significantly vary

between the different ATX concentrations, while chlorophyll a content decreased. Total proteins and

total microcystins (MCs) content declined in cells exposed to increasing ATX concentrations, while

antioxidant enzyme (catalase, peroxidase, superoxide dismutase and glutathione S-transferase)

activities were significantly increased under the same conditions. Furthermore, internal hydrogen

peroxide (H2O2) formation increased as ATX concentrations were increased. Significant positive

correlations were observed between antioxidant enzyme activities, hydrogen peroxide production and

ATX concentrations. The lack of significant growth inhibition by ATX may partly explain the co-

occurrence, and sometimes formation of multispecies cyanobacterial blooms by MCs and ATX producing

genera.

� 2015 Elsevier B.V. All rights reserved.

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Harmful Algae

jo u rn al h om epag e: ww w.els evier .c o m/lo cat e/ha l

1. Introduction

Toxic cyanobacterial blooms constitute a serious environmen-tal and public health problem. This is due to synthesis and releaseof toxins such as microcystins, saxitoxins, cylindrospermopsinsand anatoxins into the environment by cyanobacteria (Codd et al.,1997; Pawlik-Skowronska et al., 2004; Gugger et al., 2005; Al-Sammak et al., 2014). These toxins contaminate drinking water,may bioaccumulate in aquatic food chains (Pflugmacher et al.,

Abbreviations: ATX, anatoxin-a; MCs, microcystins; CAT, catalase; POD, peroxidase;

GST, glutathione S transferase; SOD, superoxide dismutase; H2O2, hydrogen

peroxide; BSA, bovine serum albumin; CDNB, 1-chloro-2,4-dinitrobenzene; ROS,

reactive oxygen species; O2�, superoxide radical; GSH, reduced glutathione.

* Corresponding author. Tel.: +55 1934294128; fax: +55 19 3434 8295.

E-mail address: mbitt@usp.br (M.d.C. Bittencourt-Oliveira).1 Tel.: +55 19 3429 4128; fax: +55 19 3434 82952 Tel.: +55 81 3320 6361.

http://dx.doi.org/10.1016/j.hal.2015.09.006

1568-9883/� 2015 Elsevier B.V. All rights reserved.

2001; Mitrovic et al., 2005), and when exposed to them, humansmay suffer from acute neurotoxicity, liver damage, gastrointestinaldiseases and cancers (Falconer and Humpage, 1996; Zegura et al.,2003).

Available evidence suggests that global temperatures andnutrient enrichment of aquatic ecosystems will continue toincrease (Houghton et al., 2001; Davis et al., 2009; IPCC, 2014),which will encourage a shift in phytoplankton community towarddominance by cyanobacteria (Paerl and Huisman, 2008; IPCC,2014). This shift in species dominance is accompanied by variousdirect interspecific and resource competition interactions betweenaquatic macrophytes and phytoplankton (Leu et al., 2002; Chiaet al., 2011, 2012), and cyanobacteria and microalgae (Sedmak andElersek, 2005; Bittencourt-Oliveira et al., 2015). During interspe-cific interactions in aquatic ecosystems, phytoplankton speciessynthesize and release bioactive substances, that may be classifiedas allelochemicals and/or cyanotoxins (Leflaive and Tem-Hage,2007).

Fig. 1. Cell density (cells mL�1) of Microcystis aeruginosa exposed to different anatoxin-a concentrations. Error bars are plus/minus standard error for n = 3. Arrows represent

the point of anatoxin-a addition to the cultures.

Fig. 2. Changes in specific growth rate (d�1) of Microcystis aeruginosa after exposure

to different anatoxin-a concentrations. Error bars are plus/minus standard

deviation for n = 3.

M.A. Chia et al. / Harmful Algae 49 (2015) 135–146136

The cyanotoxin anatoxin-a (ATX), also known as Very FastDeath Factor (VFDF), is a neurotoxin with a semi-rigid bicyclicsecondary amine alkaloid, and is synthesized by some planktonicand benthic cyanobacterial species including of Anabaena spp.(Carmichael et al., 1975; Sivonen et al., 1989), Oscillatoria formosa

(Araoz et al., 2005), Aphanizomenon flos-aque (Rapala et al., 1993),and Phormidium favosum (Gugger et al., 2005). Several studies havereported the occurrence of ATX in freshwater bodies that providedrinking water or are used for public recreation and livestock(Pawlik-Skowronska et al., 2004; Gugger et al., 2005; Al-Sammaket al., 2014). Anatoxin-a concentrations ranging from 0.10 to1750 mg L�1 have been detected in different aquatic ecosystems(Hedman et al., 2008; Al-Sammak et al., 2014). With a LD50 of 200–250 mg kg�1 BW, animals exposed to it suffer from respiratoryarrest, loss of coordination, convulsions and muscular fascicula-tions (Carmichael et al., 1990). Although not conclusive, the deathof a boy was linked to ATX exposure (Behm, 2003). Furthermore,several cases of rapid dog (Gugger et al., 2005; Puschner et al.,2008) and Phoeniconaias minor (Krienitz et al., 2003) deaths havebeen associated with exposure to the toxin in aquatic ecosystems.In response to this, the World Health Organization (WHO)proposed an ATX limit of 3.0 mg L�1 in drinking water (Falconeret al., 1999).

The effect of ATX on phytoplankton species is still poorlyunderstood, but investigations have shown that it induces oxidativestress in aquatic macrophytes (Mitrovic et al., 2004; Ha andPflugmacher, 2013a,b; Ha et al., 2014). Laboratory studies showthat cyanotoxins (e.g. cylindrospermopsins and microcystins) are

released by cyanobacteria to influence the growth and physiologicalresponse of cyanobacteria and other groups of algae duringinterspecific interactions (Rzymski et al., 2014; Bittencourt-Oliveiraet al., 2015). With increasing nutrient enrichment in combinationwith high levels of physical conditions such as light intensity andwater temperature, toxin production by multiple genera ofcyanobacteria is expected to surge (Heisler et al., 2008; Daviset al., 2009). This highlights the need to understand the effect of ATXon phytoplankton growth and physiology in freshwaters, especiallywith species such as Microcystis spp. and Oscillatoria spp. thatco-occur with potential producers (Aphanizomenon flos-aquae,

M.A. Chia et al. / Harmful Algae 49 (2015) 135–146 137

Anabaena flos-aquae, Anabaena circinalis) of this toxin (Pawlik-Skowronska et al., 2004). This situation is further complicated by theability of these species to simultaneously form toxic blooms inhypertrophic lakes/reservoirs and other aquatic ecosystems (Aktanet al., 2009; Solis et al., 2009; Vesna et al., 2010).

Considering that ATX has been demonstrated to affectphotosynthesis and induce oxidative stress in aquatic macrophytes(Mitrovic et al., 2004; Ha and Pflugmacher, 2013a,b; Ha et al.,2014), it can be hypothesized that the biosynthesis of the toxinmay not be meant to prevent grazing alone, but also to deter thegrowth of competing phytoplankton species. Therefore, theobjective of this study was to determine the effect of ATX ongrowth, microcystins (MCs) production and antioxidant responseof Microcystis aeruginosa. The results of the present study willcontribute to our understanding of physiological and ecologicalimplications of the exposure of M. aeruginosa to ATX.

2. Materials and methods

2.1. Cyanobacterial strain and culture conditions

Microcystis aeruginosa BCCUSP232 was chosen based on itsproven toxicity from a previous study (Bittencourt-Oliveira, 2003).It was obtained from the Brazilian Cyanobacteria Collection of theUniversity of Sao Paulo (BCCUSP) and maintained in ASM-1medium at pH 7.4 under controlled environmental conditions(light intensity: 40 mmol m�2 s�1, measured with a LI-COR modelLI-250 quantum meter equipped with a spherical sub-aquatic

Fig. 3. Chlorophyll a content (pg cell�1) of Microcystis aeruginosa as a function of different

with asterisk (*) are significantly different (p < 0.05) from the control.

sensor; photoperiod: 14:10 h, light:dark; and temperature:24 � 1 8C).

2.2. Experimental design

Pure ATX was obtained from Enzo Life Sciences (New York, USA)as (�/-) anatoxin-a fumarate, which is a racemic preparation of thetoxin. Experiments were conducted using 1000 mL Erlenmeyer flaskshaving 700 mL of ASM-1 growth medium, in culture chamberswith fully regulated conditions as stated above for the stock cultures.All experiments started with an equal initial cell density(1.37 � 105 cells mL�1) of Microcystis aeruginosa, obtained from thesame exponential growth phase stock culture. At exponential growthphase (day 4) of the experimental cultures, the cyanobacterium wasexposed to ATX at 6.5, 12.5, 25 and 50 mg L�1 and incubated for 120 h.The control cultures were not exposed to the toxin. Anatoxin-aconcentrations chosen in this study represent levels commonlyencountered in aquatic ecosystems worldwide (Pawlik-Skowronskaet al., 2004; Al-Sammak et al., 2014). Samples were collected usingsterile techniques at 24, 72 and 120 h during incubation with ATX forcell density, chlorophyll a and total microcystins productionmeasurements, and at 72 and 120 h for reactive oxygen species(H2O2), total proteins and antioxidant enzyme activities determina-tion. All experiments were carried out in triplicate.

Growth was determined using cell density obtained fromcalibration curves, which were made by measuring the absorbance(Biospectro SP-22 spectrophotometer, Biospectro, Curitiba, Brazil)at 750 nm of Microcystis aeruginosa samples of known cell

anatoxin-a concentrations. Error bars are plus/minus standard error for n = 3. Means

M.A. Chia et al. / Harmful Algae 49 (2015) 135–146138

densities. The regression equation obtained from the calibrationcurve is given below:

y ¼ ð3 � 10�8Þx þ 0:0058 (1)

where y is the absorbance, x is the cell density, and r2 = 0.998.For microscopic cell counts to confirm cell density, culture

aliquots were preserved with 10% Lugol’s Iodine solution. Celldensity (cells mL�1) was determined with the Fuchs-Rosenthalcounting chamber under a binocular microscope (Nikon E200,USA). The reliability of the results was increased by counting atleast 400 cells per count to maintain an error within �10%.

2.3. Pigment determination

Microcystis aeruginosa biomass was concentrated by low speedcentrifugation (2200 � g) of 10 mL culture at ambient temperaturefor 5 min. The supernatant was discarded and the pellet (biomass)was re-suspended and macerated in 3 mL acetone. The mixturewas kept in the dark at -20 8C for 3 h for pigment extraction.Afterwards, the extraction solution was centrifuged at 2200 � g for5 min to settle residual biomass, and the supernatant transferredinto 10 mm light path 3.5 mL glass cuvettes. The absorbance of thesupernatant was measured at 663 and 647 nm with a BiospectroSP-22 spectrophotometer. Chlorophyll a concentration wascomputed using the equation provided by Ritchie (2006).

Fig. 4. The effect of anatoxin-a on the production of total microcystins (fg cell�1 quo

2.4. Reactive oxygen species: internal hydrogen peroxide (H2O2)

formation

The method of Jana and Choudhuri (1982) was employed formeasuring internal H2O2 formation by Microcystis aeruginosa withsome modifications. Briefly, M. aeruginosa biomass (cell pellet)obtained by centrifuging 40 mL of culture at 2200 � g and 0 8C, washomogenized in 3 mL of 0.1 M phosphate buffer (pH 6.5) to extractinternal H2O2. Afterwards the homogenate was centrifuged at10,000 � g and 0 8C for 10 min, and the supernatant (extract) usedfor H2O2 determination. To measure H2O2 concentration, 133 mLof 0.1% titanium chloride (in 20% H2SO4) was added to 400 mL ofthe supernatant, and 1 min after incubation, the absorbance of thered-orange color mixture was measured at 410 nm with aBiospectro SP-22 spectrophotometer. The extinction coefficientof 0.28 L mmol�1 cm�1 was used to compute H2O2 concentrationin mmol/mg.

2.5. Antioxidant enzyme activities

Total protein extraction for enzyme activity assays wasperformed with biomass obtained from 40 mL of Microcystis

aeruginosa culture, by homogenizing in 0.1 M phosphate buffer (pH6.5) containing 1% (w/v) polyvinylpyrrolidone (PVP), andcentrifuging the homogenate at 10,000 � g and 0 8C for 10 min.The supernatant was used for total proteins measurement and

ta) by Microcystis aeruginosa. Error bars are plus/minus standard error for n = 3.

M.A. Chia et al. / Harmful Algae 49 (2015) 135–146 139

antioxidant enzyme activity assays. Total proteins concentrationwas determined according to Bradford (1976), with bovine serumalbumin (BSA) as standard. Total protein content per treatmentcondition was used to determine specific activity of the differentantioxidant enzymes.

Catalase (CAT) activity was determined based on its periox-idatic function (Johansson and Borg, 1988). This method usesmethanol as a hydrogen donor for catalase, which results in theformation of formaldehyde that is measured colorimetrically withPurpald. Two hundred and fifty microliters of potassium phos-phate buffer (25 mM, pH 7.0) was incubated with 250 mL ofmethanol and 50 mL of hydrogen peroxide (0.27%, v/v). Thereaction was initiated by the addition of 500 mL enzyme extract atroom temperature (20 8C), with continuous shaking. After 20 min,the reaction was stopped by adding 250 mL of 7.8 M potassiumhydroxide. Then 500 mL of 34.2 mM purpald (4-amino-3-hydra-zino-5-mercapto-1,2,4-triazol) in 480 mM HCl was added, and thereaction mixture incubated again for 10 min at room temperaturewith continuous shaking. The addition of 250 mL 65.2 mMpotassium periodate produced a purple color, whose absorbancewas read at 550 nm with a Biospectro SP-22 spectrophotometer.Enzyme activity was calculated from calibration curves made withformaldehyde (0–120 mM). One unit (U) of CAT was defined as the

Fig. 5. Total proteins (pg cell�1) production by Microcystis aeruginosa after ex

amount of enzyme that caused the formation of 200 mMformaldehyde under the test conditions specified above. CATactivity was expressed in nano katal (nkat)/mg protein; where oneenzyme unit equals to 16.67 nkat.

Peroxidase (POD) activity was assayed according to Reddy et al.(1995). Three milliliters of pyrogallol solution (0.05 M in 0.1 Mphosphate buffer, pH 6.5) and 0.5 mL 1% H2O2 were mixed in acuvette, and the reaction was initiated by the addition of 0.1 mL ofenzyme extract. The change in absorbance per minute wasproportional to the activity of the enzyme. The activity waspresented in nkat/mg protein.

Superoxide (SOD) dismutase activity was performed spectro-photometrically according to the procedures described by Misraand Fridovich (1972). The incubation medium contained in a finalvolume of 3.0 mL, 6.7 mM potassium phosphate buffer (pH 7.8),45 mM methionine, 0.53 mM riboflavin, 84 mM nitro blue tetrazo-lium chloride (NBT). The reaction vials containing the incubationmedium were placed in an aluminum foil-lined chamber andilluminated with two 15 W fluorescent lamps at 25 8C for 10 min.After exposure to light, the absorbance of the incubation mediumwas measured spectrophotometrically at 600 nm. The amount ofhomogenate added to the incubation medium was kept below oneunit of enzyme to ensure sufficient accuracy. One unit of enzyme

posure to anatoxin-a. Error bars are plus/minus standard error for n = 3.

M.A. Chia et al. / Harmful Algae 49 (2015) 135–146140

activity was defined as the amount of enzyme giving a 50%inhibition of the reduction of NBT, according to the equationbelow:

%I ¼ AS � ANC

ANC(2)

Where %I is the percentage inhibition; AS is the absorbance of thesamples, and ANC the absorbance of the negative control (withoutenzyme extract).

The assay of glutathione S-transferase (GST) activity wasperformed using the standard model substrate 1-chloro-2,4-dinitrobenzene (CDNB) (Habig et al., 1974). The reaction wasstarted by adding 100 mL of enzyme extract to 2 mL reactionmixture containing 3.6 mM reduced glutathione and 1 mM1-chloro-2,4-dinitrobenzene in 0.1 M potassium phosphate buffer(pH 6.5). Enzyme activity was determined by monitoring thechange in absorbance at 340 nm. The change in absorbance wasdirectly proportional to GST activity.

2.6. Cyanotoxins analyses

For cyanotoxins extraction, frozen culture samples in 1.5 mLmicrocentrifuge tubes were allowed to thaw at room temperature,and rapidly subjected to 3 freeze–thaw cycles to rupture

Fig. 6. The production of internal hydrogen peroxide (pM cell�1) by Microcystis aerugino

n = 3. Means with asterisk (*) are significantly different (p < 0.05) from the control.

cyanobacterial cell wall. Per cycle, the samples were placed inliquid nitrogen for 30 s, and then thawed in a water bath (37 8C) for5 min. Cell rupture was confirmed by microscopic analysis of theextracted samples. Microcystins analysis was carried out 24 and120 h after exposure to ATX, while ATX quantification wasperformed in the beginning and at the end of the experiments,using Beacon ELISA (Enzyme Linked ImmunoSorbent Assay) PlateKits specific to each of the cyanotoxins. Analyses were performedfollowing the manufacturer’s instructions. The color reaction of theELISA test was evaluated at 450 nm using an ASYS model A-5301microtiter plate reader (ASYS Hitech GmbH, Nordstrasse 4,Eugendorf, Austria). Anatoxin-a results showed that only the50 mg L�1 treatment had about 25% of the initial concentration atthe end of the experiment, while in other treatments, ATX was notdetected.

2.7. Data treatment

Data obtained were tested for homogeneity of variance usingthe Mauchly’s sphericity test and normality using the Shapiro-Wilk test. A repeated measure one-way analysis of variance wasused to test for significant differences between the means ofmeasured response parameters, as a function of ATX exposureconcentration and time (Table S1). In cases where sphericity was

sa in response to anatoxin-a exposure. Error bars are plus/minus standard error for

M.A. Chia et al. / Harmful Algae 49 (2015) 135–146 141

violated, the Greenhouse–Geisser correction (Greenhouse andGeisser, 1959) was applied to adjust the degrees freedom andreduce Type I error. For specific growth rate results, single factorANOVA was used to determine significant differences between thetreatments (Table S2). Tukey’s HSD post hoc test was used toseparate significantly different means. A correlation basedprincipal components analysis (PCA) was used to determinepossible relationship between the response variables and ATXexposure concentrations. Analysis of variance was done usingStatistica v.10 (StatSoft, Inc., Tulsa, OK, USA) for windows, whilePCA was done using the Paleontology Statistics (PAST) softwarev.2.7c (Hammer et al., 2001). All analyses were done at 5%significance level.

3. Results

Our experiment with Microcystis aeruginosa showed that celldensity did not significantly (p > 0.05) vary between the treat-ments and the control (Fig. 1). Specific growth rate was lowest at50 mg L�1 ATX and highest at 6.25 mg L�1 ATX treatment. However,the change in specific growth rate between the treatments and thecontrol was not significant (Fig. 2). Within the first 72 h ofexposure to ATX, chlorophyll a content generally declined in aconcentration dependent manner (Fig. 3). The reduction of

Fig. 7. Catalase activity (nkat mg�1 protein) of Microcystis aeruginosa as a function of

n = 3. Means with asterisk (*) are significantly different (p < 0.05) from the control.

chlorophyll a content was only significant (p < 0.05) at 25 mg L�1

1 ATX. However, at 120 h, there was no significant (p > 0.05)difference in chlorophyll a content between the treatments andcontrol.

MCs production by Microcystis aeruginosa was lower in thepresence of ATX than the control (Fig. 4). MCs production at6.25 and 25 mg L�1 ATX was lower than the control after 24 h,while at 120 h after exposure, MCs production at 6.25, 12.5 and50 mg L�1 was lower than the control.

Total protein concentrations decreased with increasing ATXconcentrations (Fig. 5). In general, ATX concentrations from12.5 mg L�1 caused a reduction in total protein concentrationsafter 74 h exposure. At the highest ATX concentration, total proteinconcentrations per cell were as 21.71 and 24.38 pg cell�1, while inthe control, they were 30.23 and 28.98 pg cell�1, after 72 and 120 hof exposure to the toxin, respectively.

The formation of internal H2O2 by Microcystis aeruginosa

increased with increasing ATX concentration (Fig. 6). The highestH2O2 concentrations (123.58–239.92 pM cell�1) were observedafter 72 h, while at 120 h after exposure to ATX, internal H2O2

levels decreased in all treatments. In addition, the levels of H2O2,167.47 and 239.92 pM cell�1, at 25 and 50 mg L�1 ATX, respective-ly, were significantly (p < 0.05) higher than that in the control(57.27 pM cell�1) at 72 h.

different anatoxin-a concentrations. Error bars are plus/minus standard error for

M.A. Chia et al. / Harmful Algae 49 (2015) 135–146142

Antioxidant enzyme results showed that CAT and POD activitieswere increased after exposure to ATX (Figs. 7 and 8). The mostsignificant increase in CAT and POD activities was recorded at25 and 50 mg L�1 ATX concentrations. SOD and GST activitiesincreased with increasing ATX concentrations (Figs. 9 and 10).Generally, concentrations greater than or equal to 6.25 mg L�1 ATXincreased SOD activity after 72 h incubation, while after 120 h,only concentrations greater than or equal to 12.5 mg L�1 ATXincreased its activity (Fig. 9). However, only the highest ATXconcentration resulted in a significant (p < 0.05) SOD activityincrease at 72 and 120 h. For GST, significantly higher activitieswere recorded at ATX concentrations greater than or equal to25.00 mg L�1 ATX (Fig. 10).

The relationship between the response parameters and ATXwas revealed by PCA. The result showed that ATX had significantpositive correlation with SOD, CAT, GST, POD and H2O2 (Fig. 11).Anatoxin-a had a PCA loading of 1.2, while CAT, H2O2, GST, SOD andPOD had PCA loadings generally greater than 1.4, on the sameorthogonal axis of the first principal component. On the otherhand, cell density, chlorophyll a and total proteins content (PCAloadings range from �0.8 to �2.2) had significant negativecorrelation with ATX, on the first principal components. The firsttwo principal components were responsible for 65% of the totalvariation observed.

Fig. 8. Changes in peroxidase activity (nkat mg�1 protein) of Microcystis aeruginosa after

error for n = 3. Means with asterisk (*) are significantly different (p < 0.05) from the co

4. Discussion

4.1. Growth and pigment production

The results of the present study showed that ATX did notsignificantly affect the growth of Microcystis aeruginosa, asreflected in its cell density. However, the opposite was observedfor chlorophyll a, because ATX caused a reduction of itsconcentration per cell. The decline of chlorophyll a concentrationmay affect photosynthesis and possibly carbon dioxide assimila-tion in the cyanobacterium (Smith and Doan, 1999; Larkum et al.,2003; Chia et al., 2015). The decrease in chlorophyll content maybe due to oxidative impairment of structures associated with thechloroplast by H2O2 (Mikula et al., 2012). Oxidative impairmentresults in a decline in quantum yields (Fv/Fm values) in variousalgae, when H2O2 levels are increased within their cells(Dummermuth et al., 2003; Mikula et al., 2012). In addition, itresults in the inhibition of a number of photosynthetic enzymes,such as fructose bisphosphatase, ribulose phosphate kinase andribulose bisphosphate carboxylase/oxygenase (Rubisco), whichcauses a significant reduction in the entire photosynthetic process(Dummermuth et al., 2003). The insignificant difference observedin chlorophyll a concentration at the end of the experimentbetween ATX and non-ATX exposed M. aeruginosa may be related

exposure to different anatoxin-a concentrations. Error bars are plus/minus standard

ntrol.

Fig. 9. Superoxide dismutase activity (nkat mg�1 protein) of Microcystis aeruginosa as a function of different anatoxin-a concentrations. Error bars are plus/minus standard

error for n = 3. Means with asterisk (*) are significantly different (p < 0.05) from the control.

M.A. Chia et al. / Harmful Algae 49 (2015) 135–146 143

to the degradation of the toxin and the growth phase of thecyanobacterium. Generally, less than five days are required toquickly and completely degrade ATX, after its release into theenvironment (Matsunaga et al., 1989; Dorr et al., 2010). This issupported by the fact that at the end of the experiment, on day five(120 h), only the highest ATX treatment (50 mg L�1) had a fraction(25%) of the initial exposure concentration remaining in themedium.

4.2. Microcystins production

In the present study, the general trend after exposure ofMicrocystis aeruginosa to ATX was a concentration dependentinhibition of MCs production. Similar results were obtained byRzymski et al. (2014), after exposure of M. aeruginosa tocylindrospermopsin. The authors observed that MC-LR productionby M. aeruginosa decreased as a function of increasing cylindro-permopsin concentrations. Although the role of MCs productionhave been questioned in cyanobacteria and is yet to be fullyunderstood, studies show that aside the allelopathic role of thetoxin, it is involved in the formation and maintenance ofMicrocystis colonies, increase cell size, growth promotion andhigh pigment formation per cell and cell volume (Sedmak andElersek, 2005; Schatz et al., 2007; Gan et al., 2012). Despite thereduction in MC production recorded in our study, the ability of the

cyanobacterium to maintain its growth was not compromised, dueto the lack of statistical difference in cell density recorded betweenthe treatments and the control.

4.3. Reactive oxygen species: internal hydrogen peroxide production

The exposure of photosynthetic organisms to environmentalstress significantly increases the production of reactive oxygenspecies (ROS) in organelles like chloroplasts, mitochondria andperoxisomes (Rhoads et al., 2006; Torres et al., 2008; Qian et al.,2012). Increased H2O2 production increases the risk of oxidativedamage to the cell (Torres et al., 2008), and explains why the initialhigh formation of H2O2 after 72 h correlated with the up-regulation of major ROS combating enzymes assayed in thepresent investigation. At 120 h, the decrease in H2O2 may berelated to the complete degradation of ATX observed in mosttreatments. Therefore, the general reduction in ROS concentrationcan be implicated for the decline in antioxidant enzyme activitiesrecorded at the end of the experiment.

4.4. Total proteins and antioxidant enzyme activities

Our results showed that ATX negatively influenced Microcystis

aeruginosa protein content, as there was a concentrationdependent decrease in its concentration with increasing toxin

Fig. 10. Glutathione S-transferase activity (nkat mg�1 protein) of Microcystis aeruginosa exposed to different anatoxin-a concentrations. Error bars are plus/minus standard

error for n = 3. Means with asterisk (*) are significantly different (p < 0.05) from the control.

M.A. Chia et al. / Harmful Algae 49 (2015) 135–146144

concentration. Organic and inorganic toxic substances are capableof inhibiting protein synthesis in cyanobacteria and othermicroalgae (Carfagna et al., 2013; Chia et al., 2015). This reductionin protein concentration can also be related to a decrease incellular photosynthetic rates, which may result in shortage ofcarbon skeleton, in the form of sugars/monosaccharides and otherorganic molecules from carbon-fixation reactions needed forprotein synthesis (Larkum et al., 2003).

Like other photosynthetic organisms, Microcystis aeruginosa isequipped with antioxidant defenses against oxidative stresscaused by toxic substances (Torres et al., 2008; Qian et al., 2012).Among these enzymes, our results showed that CAT and PODactivities were up-regulated as internal H2O2 increased. Theseenzymes sequestrate excess H2O2 produced in stressed M.

aeruginosa and other photosynthetic organisms to produce waterand oxygen (Qian et al., 2012), which explains the increase inactivities recorded in our study. Furthermore, they have a widespectrum of substrate specificity including phenolics, formicacid, formaldehyde, indole, pyrogallol and catechol, whichmakes them principal xenobiotics oxidizing enzymes in plants(Murphy et al., 2000). In agreement with our findings, theexposure of aquatic macrophytes to ATX resulted in an increasein POD and CAT activities (Mitrovic et al., 2004; Ha andPflugmacher, 2013b).

The high SOD activity we observed in response to ATX exposureimplied an increased production of the superoxide radical (O2

�).This is because SOD acts as an antioxidant enzyme in virtually allliving organisms to protect cellular components from thedamaging reactions of O2

� (Wolfe-Simon et al., 2006; Priyaet al., 2007; Miller, 2012). Superoxide dismutase catalyzes thedismutation or partitioning of the toxic O2

� radical into O2 or H2O2,while the produced H2O2 is subsequently converted into water andoxygen by CAT/POD (Lozano et al., 2014). The increased activity ofGST in response to the presence of ATX means it was involved inthe biotransformation and/or detoxification of the toxin, as aprotective measure against its oxidative damage. This biotrans-formation or detoxification is achieved via a conjugation processthat utilizes the reduced form of glutathione (GSH), and iscatalyzed by GST (Salinas and Wong, 1999; Vestena et al.,2011). Previous studies have demonstrated that this enzyme iscapable of bio-transforming and detoxifying cyanobacterial toxinsin phytoplankton species (Bartova et al., 2011) and vascular plants(Ha and Pflugmacher, 2013a,b).

The robust antioxidant response observed in Microcystis aeru-

ginosa after exposure to ATX may explain why it was able towithstand the effect of the toxin, without significantly reducing itscell density or growth rate. Considering that the concentrations ofATX used in this study are similar to those recorded in aquatic

Fig. 11. Principal components analysis biplot showing the relationship between anatoxin-a and different physiological parameters of Microcystis aeruginosa. Variables

grouped together on the same orthogonal axis are positively correlated with each other, while those on opposite axes are negatively correlated.

M.A. Chia et al. / Harmful Algae 49 (2015) 135–146 145

environments, these results provide additional information thatmay partly explain the co-occurrence and sometimes co-dominanceof ATX and MCs producing cyanobacteria, as well as thesimultaneous detection of both cyanotoxins (Pawlik-Skowronskaet al., 2004; Al-Sammak et al., 2014). Thus, as current climaticconditions in some regions of the world favor the excessiveproliferation of potential producers of these cyanotoxins, futureenvironment conditions and human pressures are expected tofurther worsen this situation (IPCC, 2014). The levels of these toxinsin aquatic ecosystems may continue to increase; and thus, presentserious challenges to water quality managers, cause a significantshift in the plankton composition, and negatively affect the generalhealth of aquatic ecosystems.

5. Conclusion

The results of the present investigation showed that ATX did notsignificantly affect the growth of Microcystis aeruginosa. However,chlorophyll a, MCs and total protein content of the cyanobacteriumwere generally inhibited by ATX. The toxin increased internal H2O2

formation, and subsequently increased the activities of importantantioxidant enzymes (CAT, POD, SOD and GST). This may explainwhy M. aeruginosa is able to co-occur with ATX producingcyanobacteria in aquatic ecosystems, and increases the risksimultaneous human and animal exposure to both toxins inaquatic ecosystems.

Acknowledgment

M.A. Chia was supported by post-doctoral fellowships (Proc.FAPESP 2013/11306-3, 2014/26898-6) and M.C Bittencourt-Oliveira by a research grant (Proc. FAPESP 2014/01934-0) fromthe Sao Paulo Research Foundation (FAPESP).[SS]

Appendix A. Supplementary data

Supplementary material related to this article can be found, inthe online version, at doi:10.1016/j.hal.2015.09.006.

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