Regional Studies in Marine Science

Manuscript Draft

Manuscript Number: RSMA-D-15-00087

Title: The toxic effect of copper on the association between ciliates Euplotes vannus and Euplotes
crassus and their naturally associated bacteria isolated from a polluted and tropical bay.

Article Type: Research Paper

Keywords: Naturally associated bacteria; Heavy metals; Biomass; Sensibility; Coastal pollution

Abstract: The aim of this study was to evaluate the copper sensitivity of Euplotes vannus, Euplotes
crassus, and their naturally associated bacteria sampled from sediments in the northwest and east
regions of Guanabara Bay. The unexposed ciliates and bacteria did not appear to be negatively affected
during assay. In the control group the highest biomass content were reach at 48 h to E. vannus
(6.3x102 μg C cm-3) and E. crassus (1.14x103 μg C cm-3). The maximum biomass was pointed by E.
crassus (8.52x103 µg C cm-3) and their naturally associated bacteria at 24 h (1.90x10-1 µg C cm-3),
both in presence of 0.001 mg Cu L−1. The growth of E. crassus from the east region showed
concentration-dependent manners, and it is more resistant. Naturally associated bacteria showed
better adaptation to increasing concentrations of copper and previous environmental selection. These
results show that new bioremediation methods are needed.
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1 The toxic effect of copper on the association between ciliates Euplotes vannus and Euplotes

2 crassus and their naturally associated bacteria isolated from a polluted and tropical bay

3 J.A.P. Bitencourta*, D.C. Pereiraa, I.D. da Silva Netob, M.A.C. Crapeza.

4
a
5 Universidade Federal Fluminense, Departamento de Biologia Marinha e Meio Ambientes

6 Costeiros, Outeiro São João Batista, s/nº, Centro, Niterói- RJ, Brazil. CEP: 24.020-141.
b
7 Universidade Federal do Rio de Janeiro, Departamento de Zoologia, Ilha do Fundão - Rio de

8 Janeiro, RJ. CEP: 21941-590.

9 * Corresponding author, tel +55(021) 26292261 Fax: +55(021) 26292292, E-mail:

10 jbitencourt@gmail.com.

11

12

1
13

14 Abstract

15 The aim of this study was to evaluate the copper sensitivity of Euplotes vannus,

16 Euplotes crassus, and their naturally associated bacteria sampled from sediments in

17 the northwest and east regions of Guanabara Bay. The unexposed ciliates and bacteria

18 did not appear to be negatively affected during assay. In the control group the highest

19 biomass content were reach at 48 h to E. vannus (6.3x102 μg C cm-3) and E. crassus

20 (1.14x103 μg C cm-3). The maximum biomass was pointed by E. crassus (8.52x103 µg

21 C cm-3) and their naturally associated bacteria at 24 h (1.90x10-1 µg C cm-3), both in

22 presence of 0.001 mg Cu L−1. The growth of E. crassus from the east region showed

23 concentration-dependent manners, and it is more resistant. Naturally associated

24 bacteria showed better adaptation to increasing concentrations of copper and previous

25 environmental selection. These results show that new bioremediation methods are

26 needed.

27 Key words: Ciliates; Naturally associated bacteria; Heavy metals; Biomass; Sensibility;

28 Coastal pollution

29

30 1.Introduction

31 Metals are among the most dangerous and abundant inorganic environmental

32 pollutants, arising from industrial discharge, mining, and other urban activities.

33 Unlike toxic organic compounds, metals cannot be degraded, so they accumulate in

34 the benthic organisms that inhabit sediments (Bertin and Averbeck, 2006; Seebaugh

35 et al., 2005). The toxicity results from alterations in the configurational structure of

36 nucleic acids and proteins and interferes with the oxidative phosphorylation and

37 osmotic balance (Poole and Gadd, 1989).

2
38 Some metals are present in concentrations 10 to 100 times higher in sediments

39 and the interstitial water of both estuarine and coastal marine areas than in the

40 overlying water column (Ruus et al., 2005). Sediment is an important sink and

41 reservoir for copper (Cu) (WHO, 1998) and due to the high complexity of this

42 environmental compartment, a stand-alone chemical characterization often fails to

43 correctly estimate the toxic potential of the sediments. In contrast, a combined

44 chemical–biological approach can overcome this problem and provide valuable

45 information on the bioavailability of pollutants and complex toxic effects of chemical

46 mixtures (Cajaraville et al., 2000).

47 Due to the global ban on organotin-based compounds, copper is one of the

48 main components of antifouling paints that are applied to submerged structures and

49 used in the construction of some marine pipelines (Sonak et al., 2009). At a

50 community level, copper has generally been demonstrated to have a deleterious effect

51 on the diversity and physiology of some soil microorganisms (Griffiths et al., 1997).

52 Ciliate studies have been carried out to evaluate the toxic effect of metals and toxic

53 compounds on cellular growth and viability (Martín-González et al., 2006). They

54 have been designed to detect ultra-structural alterations due to metal exposure

55 (Martín-González et al., 2006) and the effect on cellular activities (Trielli et al.,

56 2007).

57 Ciliates and bacteria are easily cultivable, a reliable biological model, and a

58 good candidate for use in bioassays (Belkin, 2003; Delmonte-Corrado et al., 2005;

59 Gutiérrez et al., 2003; Mauclaire et al., 2003). However, there are few studies of the

60 interaction between ciliates and bacteria in marine sediments (Beaudoin et al., 2014;

61 Soldo, 1963; Soldo et al., 1982; Vannini et al., 2004), and environmental pollutants

62 such as copper (Belkin, 2003; Gomiero et al., 2013; Gomiero et al., 2012). In

3
63 addition, ciliates perform an important function in the flow of oligo- and macro-

64 elements into higher trophic levels (Hausmann et al., 2003; Porter et al., 1985; Sherr

65 et al., 1988; Sherr et al., 1991) through the microbial loop (Pomeroy et al., 2007;

66 Sherr et al., 1991).

67 Bacteria are members of the base of the marine microbial loop and have a

68 great capacity to metabolize recalcitrant compounds, colonize a wide variety of

69 substrates, and show sophisticated biochemical apparatus that can be used in cross

70 talking and self defense against predators (Friman et al., 2013). The colonization

71 process on ciliate cells is one type of bacterial defense (Esteve and Gaju, 1999). The

72 naturally associated bacteria may produce important compounds, such as fatty acids,

73 for the metabolism of their hosts (Edgcomb et al., 2011; Gast et al., 2009; Soldo,

74 1963; Soldo et al., 1982; Vannini et al., 2007; Vannini et al., 2004). This cooperation

75 process, both in terms of growth potential and the high rate of metabolism, facilitates

76 carbon, energy flux (Fenchel, 1987; Sherr and Sherr, 1994; Sherr and Sherr, 2002),

77 and heavy metal movement through ecosystems (Hassen et al., 1998; Kennish, 2002).

78 In the present study, the ciliated protozoa Euplotes vannus Müller, 1786, and

79 Euplotes crassus Dujardin, 1841, and their naturally associated bacteria, isolated from

80 sediments found in a tropical bay of a developing country with different historical

81 pollution contents, were exposed to increasing concentrations of copper in laboratory

82 conditions. The aim of this study was to evaluate their sensitivity during microbial

83 interaction, assess their resistance and tolerance, and obtain a predictive base of

84 microbial loop activity with copper stress, by biomass production of both

85 microorganisms, during assay.

86 2.Materials and Methods

87 2.1Site Study and Sampling Methods

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 88 Guanabara Bay (Fig. 1), located in Southeast Brazil in the state of Rio de

89 Janeiro between 22°40'-23°00'S, 043°00'–043°18'W, is one of the largest bays along

90 the Brazilian coastline, with an area of approximately 384 km2. It In the past, each of

91 rivers flowing into the bay might create a single estuary, each distinct from the other

92 (Amador, 2013). These estuaries originally made the estuarine system of Guanabara

93 Bay ecologically diverse (Ribeiro and Kjerfve, 2002). However, human and industrial

94 occupation similar to other developing countries caused the bay to lose its natural

95 characteristics and diversity, being reduced to four large and well-demarcated areas

96 with different degrees of environmental pollution, as suggested by data collected by

97 Amador (2012, 2013); Baptista Neto et al. (2013); Carreira et al. (2002); JICA (1994);

98 Kjerfve et al. (1997); Ribeiro and Kjerfve (2002) and proposed by Baptista Neto et al.

99 (2006). Moreover, the bay has a complex bathymetry with a relatively flat central

100 channel that is 400 m wide, stretches more than 5 km into the bay, and is defined by a

101 30 m isobath. The deepest point of the bay (58 m) is located within this channel

102 (Kjerfve et al., 1997).

103 The drainage basin of Guanabara Bay has an area of 4,080 km2. It comprises

104 55 rivers that carry 4,000,000 t year-1 of solid material from 32 separated sub-

105 watersheds (Amador, 2012; JICA, 1994); however, only six rivers are responsible for

106 85 % of the 100 m3 s-1 total mean annual freshwater input (JICA, 1994). Currently, 11

107 million inhabitants live in this area, the greater Rio de Janeiro metropolitan area,

108 which discharges tons of untreated sewage directly into the bay (Carreira et al., 2002;

109 Ribeiro and Kjerfve, 2002). There are more than 12,000 industries located in the

110 drainage basin, accounting for 25% of the organic pollution released into the bay. The

111 bay also hosts two oil refineries along its shore that process 7% of the national oil

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112 supply, and is the homeport of two naval bases, a shipyard, and a large number of

113 ferries, fishing boats, and yachts (FEEMA, 1990).

114 In this study, sediments were sampled at two distinct locations. Site 1 (Fig. 1)

115 is located in the northwest region of the bay near Governador Island (22°46.589' S,

116 43°11.424' W); this site has fine sand, coarse silt, and an input of 458 g C m-2 year-1

117 of organic matter. Site 2 (Fig. 1) is located at the entrance of the bay at Jurujuba

118 Sound (22°55'32.17"S, 43° 6'28.08"W) and has moderately well-sorted medium sand

119 (Carreira et al., 2002). The copper concentrations in the sediments of these two sites

120 are approximately 53.0 and 66.0 mg kg-1 in the northwest and east, respectively. The

121 organic matter values in these regions are 17.6-19.0% and 0.8-11.0%, respectively.

122 These parameters do not display variability between the dry and wet seasons (Fonseca

123 et al., 2013; Ribeiro and Kjerfve, 2002).

124

125 Fig. 1 Location of sampling sites in the Guanabara Bay, Brazil

126

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127 The sediment samples were obtained using an Eckman sampler for the muddy

128 sediment at Governador Island (22°46.589' S, 43°11.424' W) (site 1) and a Van-Veen

129 grab sampler for the sandy sediment at Jurujuba Sound (22°55'32.17"S, 43°

130 6'28.08"W) (site 2). These samples were stored for 2 h in sealed polythene bags at a

131 container with low temperature (4 ºC) (NJDEP, 2005). This procedure reduces the

132 microbial metabolism and prevents the loss of species or predation during the

133 transport to the laboratory (Fenchel, 1987; NJDEP, 2005).

134 The sediment samples were prepared in Petri dishes according to Dragesco

135 and Dragesco Kernéis (1986) and incubated at 25 ºC in the dark for two weeks. E.

136 vannus and E. crassus were picked from Petri dishes using glass micropipettes and

137 were maintained in the laboratory in plates with a liquid medium containing coarse

138 powdered rice (Dragesco and Dragesco Kernéis, 1986); to reduce contamination of

139 the cultures by other microorganisms, the grains of rice were boiled in sterilized water

140 (Madoni, 2000; Madoni et al., 1994; Madoni et al., 1992; Madoni and Romeo, 2006),

141 and the plate and seawater were sterilized (Krepsky et al., 2007). The identification of

142 the protists was performed using a classical methodology based on structural features

143 (Curds, 1975; Lynn, 2008; Tuffrau, 1960), such as cell morphologic characteristics of

144 the oral and somatic infraciliatures and dorsal argyromes. These features were

145 visualized by the carbonate silver impregnation Protargol method (Silva-Neto, 2000)

146 and a modified Chatton-Lwoff method (Frankel and Heckmann, 1968).

147 2.2.Bioassays and Analysis

148 The assay was conducted in triplicate in sterile 24-well polystyrene plates

149 without supplementary nutrients for 0, 24, 48, 72, and 96 h, with and without copper

150 exposure (control group). The plates were incubated at 25 °C in the dark. A final

151 copper concentration of 0, 0.001, 0.009, 0.05, 0.1 and 1.0 mg L-1 was added to

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152 different wells in triplicate. For the copper solution assay, analytical-grade pure

153 copper sulfate (CuSO4.5H2O, Sigma-Aldrich Corp.) was used as a source of free

154 metal ions. A solution of metal salt in the assay was diluted with sterile seawater

155 (Madoni, 2000; Madoni et al., 1992; Madoni and Romeo, 2006). The pH of the tested

156 solutions was 8.0 and did not require any adjustment.

157 For each metal concentration, 30 ciliates were tested. Single ciliates were

158 picked from the culture with a sterile glass micropipette, washed repeatedly in drops

159 of sterile seawater, and individually inoculated into a well. Each well contained 2.0

160 ml of filtered (0.4 µm pore diameter) and sterile heavy metal solution (da Silva et al.,

161 2014; Madoni, 2000; Madoni et al., 1992; Madoni and Romeo, 2006). The same

162 procedure without the copper solution was performed for the control group.

163 The protist and the naturally associated bacterial biomass were quantified as

164 the organic carbon content (µg C.cm-3). Each well content from polystyrene plates

165 was single-filtered through a sterile Millipore membrane (0.22 µm pore diameter) and

166 stained with fluorochrome acridine orange. Intact and healthy cells were enumerated

167 on epifluorescent microscopy at 1000x magnification (Axioskop 1, Zeiss, triple filter

168 Texas Red – DAPI – fluorescein isothiocyanate). When this staining method is used

169 with blue light, the bacterial physiologically healthy cell will emit green light, while

170 healthy protists will emit a green or orange light (Carlucci et al., 1986; Mirrett, 1982).

171 The data obtained was converted to organic carbon following the methodology and

172 equations proposed by Carlucci et al. (1986), Kepner and Pratt (1994) and Mauclaire

173 et al. (2003).

174 In analysis, lethality was estimated by lethal concentration for 50% of the cell

175 population (LD50 - probit) from data of Euplotes spp. survived under 96h of

176 bioassays in accordance with Bliss (1934a, 1934b); Costa et al. (2008); da Silva et al.

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177 (2014). Student T was used to check differences between LD50 results. The Dunnett

178 test with the bootstrap random method (1000x) and a post hoc Tukey test were used to

179 examine the zinc sensitivity of the protists and their naturally associated bacteria.

180 These analyses and LD50 were performed using the SPSS 21 program. All biomass

181 data were transformed using the ranging method before performing the ANOVA

182 parametric test with the bootstrap (1000x), which was used to contrast the differences

183 between the biomass of different Euplotes sp. and their exposure to copper during the

184 assay.

185 3.Results

186 This study compared the effect of increasing concentrations of copper on the

187 growth of resistant marine ciliates and their naturally associated bacteria, isolated

188 from a polluted tropical bay, the Guanabara Bay. Euplotes vannus Müller, 1786 and

189 Euplotes crassus Dujardin, 1841 are crawling (Madoni et al., 1994), bacterivorous,

190 and cosmopolitan protist species (Lynn, 2008). Both survived in laboratory

191 conditions, but we did not previously observe protists in vegetative forms from the

192 sediment samples of site 1, except E. vannus after two weeks of incubation in Petri

193 dishes. At site 2, we only observed E. vannus and E. crassus, but only E. crassus

194 survived in laboratory conditions.

195 Unexposed ciliate E. vannus and E. crassus did not appear to be negatively

196 affected by a period of 96 h (Fig. 2), based on cultures maintained in seawater without

197 copper (control group). In the experiments, both ciliates control group needed 24 h to

198 enhance their biomass to overcome the limit of detection level of the count technique.

199 The E. vannus control group exhibited an increase in biomass content from 8.31x101

200 to 6.3x102 µg C cm-3 between 24 and 72 h and decreased to 2.8x012 µg C cm-3 at 96 h.

201 However, in the presence of 0.001, 0.009, and 0.05 mg Cu L-1, the stationary phase,

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202 low biomass variation occurred up to 48 h of assay. Their biomass values in presence

203 of 0.001 and 0.009 mg Cu L-1 were 6.23x101 and 1.03x101 µg C cm-3 at 0 h, enhanced

204 to 1.87x102 µg C cm-3 at 72 h, and this content was maintained at 96 h. In the

205 presence of 0.1 and 1.0 mg Cu L-1, the biomass values were 1.87x102 µg C cm-3 at 0

206 h. In the presence of 0.1 mg Cu L-1, the biomass value decreased to 1.04x102 µg C

207 cm-3 at 96 h, but with 1.0 mg Cu L-1, the biomass content was below detection level

208 (Fig. 2a).

209

210 Fig. 2 Biomass produced by Euplotes vannus (a) and Euplotes crassus (b) under

211 different copper concentration content. The bars denote standard deviation.

212

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213 The naturally associated bacteria of E. vannus and E. crassus showed a shorter

214 stationary phase period, with 24 h of low biomass production to majority copper

215 tested (Fig. 3). In the naturally associated bacteria of E. vannus, the highest biomass

216 was produced in the control group at 24 h, with 1.63x10-1 µg C cm-3, and decreasing

217 to 4.14x10-1 µg C cm-3 at 96 h. Similarly, with different concentrations of copper, the

218 highest biomass content was produced at 24 h. In the presence of 0.05 and 0.1 mg L-1,

219 these bacterial groups produced 1.14 and 1.32x10-1 µg C cm-3, and that content

220 decreased along assay (96 h) to 3.60 and 2.86x10-2 µg C cm-3, respectively. In the

221 presence of 1.0 mg Cu L-1, the highest biomass content, 6.30x10-2 µg C cm-3, was

222 reached at 72 h and decreased to 1.53x10-2 µg C cm-3, at 96 h (Fig. 3a).

223

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224 Fig. 3 Biomass produced by naturally associated bacteria from Euplotes vannus (a)

225 and Euplotes crassus (b) under different copper concentration content. The bars

226 denote standard deviation.

227

228 For the biomass content of E. crassus, the stationary phase occurred in all

229 treatments, including in the control group. The highest biomass contents were reached

230 at 48 h in most treatments (control, 0.001, 0.05, and 0.1 mg Cu L-1). The E. crassus

231 control group showed a higher biomass content, 1.14x103 µg C cm-3, at 48 h and

232 decreased to 5.61x102 µg C cm-3 at 96 h. In the treatments with 0.001 and 0.009 mg

233 Cu L-1, this ciliate showed 8.52 and 8.11x102 µg C cm-3 at 48 h, and their biomass

234 contents were higher than control at 96 h, measuring 6.65x102 and 8.31x103 µg C cm-
3
235 , respectively. In the presence of 0.1 mg Cu L-1, the highest biomass of E. crassus,

236 7.0x102 µg C cm-3, occurred at 48 h, but with 1.0 mg Cu L-1, this ciliate biomass was

237 below detection level (Fig. 2b).

238 The naturally associated bacteria from E. crassus unexposed to copper

239 decreased the biomass content from 24 to 96 h (4.5 – 1.6x10-2 µg C cm-3), and it was

240 similar to other concentrations of copper. In the presence of 0.1 mg Cu L-1, the

241 biomass content was 6.70x10-2 µg C cm-3 and decreased to 3.00x10-2 µg C cm-3 at 96

242 h. However, this parameter was not much different than the highest copper

243 concentrations, which were higher than other concentration tests in this bioassay,

244 including the control group (p<0.05). In the presence of 1.0 mg Cu L-1, the highest

245 bacterial biomass of naturally associated bacteria, 9.20x10-2 µg C cm-3, was reached

246 at 72 h and decreased to 5.64x10-2 µg C cm-3 at 96 h (Fig. 3b).

247 Statistical analysis carried out using an ANOVA test with bootstrap showed

248 significant differences in all biomass variation analysis (n=180; sum of squares=7.0;

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249 df=59; f= 28.76; p≤0.05). We observed significant differences in the influence of time

250 and different copper concentrations on protist biomass variation growth, but we didn’t

251 observe these same characteristics in the production of biomass content from the

252 naturally associated bacteria, in the presence of low Cu concentrations (0.001, 0.009,

253 and 0.05 mg Cu L-1). The biomass value of E. crassus sampled from the eastern

254 region of the Guanabara Bay showed growth showed concentration-dependent

255 manners, and is more resistant to copper, including to high copper concentrations up

256 to 0.1 mg L-1. In the presence of low concentrations of copper, Euplotes isolated from

257 the eastern region produced a larger amount of biomass than E. vannus.

258 These results strongly influenced lethality and sensibility tests (Table 1), as

259 the Dunnett test with bootstrap showed that E. vannus was sensitive to all

260 concentrations of copper (p≤ 0.05), and their population reduced their concentration

261 by 50% (LC50) with 0.30 mg Cu L-1 (Z=36.64; p≤ 0.05). The Dunnett test also

262 showed that E. crassus was more resistant up to 0.05 mg Cu L-1 (p≤ 0.05), it was more

263 sensitive to concentrations higher than 0.1 mg Cu L-1 (p≤ 0.05), and their LC50 was

264 0.34 mg Cu L-1 (Z=70.76; p≤ 0.05). The difference in lethality content between

265 Euplotes spp. was significant (p≤ 0.05; df= 130.15; t=5.3). The naturally associated

266 bacteria from E. vannus and E. crassus were more sensitive to copper concentrations

267 at 1.0 mg. L-1. Conversely, the naturally associated bacteria from E. crassus were

268 positively influenced by the highest concentration. The Tukey and Dunnett tests

269 confirmed these results.

270

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271

272 Table 1: Two sided Dunnett test for ciliates biomass and their naturally associated bacterial biomass values
273
Metal Mean 95% Confidence Interval
Dependent Protist
concentratio Difference Std. Error Sig. Upper
Variable specie Lower Bound
n (I) (I-control) Bound
0.001 -0.30 0.036 * -0.382 -0.195
0.009 -0.33 0.036 * -0.421 -0.234
E. vannus 0.05 -0.36 0.036 * -0.455 -0.267
0.1 -0.33 0.0367 * -0.427 -0.240
Euplotes 1.0 -0.42 0.036 * -0.516 -0.328
biomass 0.001 -0,054 0,038 0,478 -0,153 0,044
0.009 -0,036 0,038 0,806 -0,135 0,063
E. crassus 0.05 -0,170 0,038 * -0,265 -0,068
0.1 -0,081 0,038 0,137 -0,180 0,017
1.0 -0,345 0,038 * -0,444 -0,246
0.001 0,113 0,021 * 0,060 0,167
0.009 0,014 0,021 0,946 -0,040 0,067
E. vannus 0.05 0,010 0,021 0,984 -0,043 0,064
0.1 0,041 0,021 0,182 -0,012 0,095
Bacterial
1.0 0,069 0,021 * 0,016 0,123
biomass
0.001 -0.04 0.026 0.438 -0.105 0.028
0.009 -0.03 0.026 0.528 -0.102 0.032
E. crassus 0.05 -0.037 0.026 0.479 -0.104 0.030
0.1 0.06 0.026 0.077 -0.005 0.129
1.0 -0.16 0.026 * -0.230 -0.096
274 * p≤0.05
275

276 4.Discussion

277 Many developing countries have shown rapid economic growth in recent

278 years, accelerating their population expansion and increasing trends of coastal

279 occupation, and Brazil is no different (Amador, 2013; Baptista Neto et al., 2013; Bell

280 and Pavitt, 1997; Preston, 1979; Rajan and Zingales, 2003). The Brazilian coastline is

281 complex and biologically diverse, which makes the threat from anthropogenic

282 activities all the more dangerous (Wagener, 2005). These may undermine the

283 distribution of nutrients (Wagener, 2005), microbial physiology (Crapez et al., 2003;

284 Fonseca et al., 2009; Sabadini-Santos et al., 2014; Sobrinho da Silva et al., 2008),

285 physical parameters (Nixon, 2010), the distribution of trace elements (Adriano, 2001),

286 and the resistance of the native organisms to the pollution (Nevo et al., 1986).

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287 Euplotes sp. are cosmopolitan ciliate that were adapted to polluted ecosystems

288 (Lynn, 2008). They can connect bacterial biomass production to high trophic levels

289 (Azam et al., 1983; Pomeroy et al., 2007) and may transport metals by a

290 biomagnification process (Fernandez-Leborans and Novillo, 1996; Madoni and

291 Romeo, 2006; Martín-González et al., 2006). However, there is little information

292 available on heavy metal resistance of ciliated protozoa (Vieira and Volesky, 2000).

293 Copper is a micronutrient, but at low concentrations, it can be toxic and harmful to

294 microzooplankton and microzoobenthos (Fernandez-Leborans and Novillo, 1996;

295 Madoni and Romeo, 2006).

296 The different occurrences of pollution in Guanabara Bay helped to determine

297 the selection of Euplotes vannus and E. crassus, and this was argued by Nevo et al.

298 (1983); Nevo et al. (1986). Harrison et al. (2007) and Sabadini-Santos et al. (2014)

299 showed that bacteria were resistant at site 1, characterized by fine sediment (clay and

300 silt) associated with a high metal content and organic matter, while site 2 included

301 more tolerant bacteria in sandy sediments with a higher copper exposure than site 1.

302 Both sites have a higher copper concentration than is recommended by the Brazilian

303 environmental protection agency (Brasil, 2005), passing the upper limit for the most

304 copper tolerant aquatic organisms (WHO, 1998). This may reflect the behavior of

305 both microorganisms evaluated in bioassays. E. crassus (site 2) is more resistant than

306 E. vannus (site 1) and their naturally associated bacteria is positively influenced in the

307 presence of 1.0 mg Cu L-1, probably by selection on sediments sites and their previous

308 exposure to this metal. However, it should be noted that these processes are much less

309 understood in sediments, especially in marine sediments (Beaudoin et al., 2014).

310 We focus our work on a function of a community that forms the base of the

311 microbial loop. They are multi-species assemblages in which organisms live together

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312 in a contiguous environment and interact with each other, analyzing how biological

313 assemblages are structured, what their functional interactions are, and how

314 community structure changes in space and time (Konopka, 2009). Bacteria are

315 notably known to make associations with other organisms such as metazoans,

316 producing important compounds like vitamins, and assisting in the metabolism of

317 compounds like cellulose. During the assay, the naturally associated bacteria recycle

318 nitrogenous excreta of tested Euplotes sp., keep their feeding process, and bring

319 features to the experiment as a duplicate of Cu exposure of ciliates, the contact by

320 food (biomagnification process). During the experiments, the naturally associated

321 bacteria can metabolize the excreta of the ciliates, particularly nitrogen compounds.

322 However, there are few studies on the association between ciliates and their naturally

323 associated bacteria (Esteve and Gaju, 1999; Soldo, 1963; Soldo et al., 1982; Vannini

324 et al., 2004). Thus, our experiments are a small-scale reproduction of the function of

325 the microbial loop under stress from copper, recreating, in the laboratory, the way

326 both Euplotes suffer in the degraded environment.

327 This metal was free in the medium or closely associated with microorganisms

328 tested because no additional organic matter was in the water (Adriano, 2001), and it

329 was only produced during a 96 h interaction. E. vannus and E. crassus were more

330 significantly influenced by copper in a concentration-dependent manner than biomass

331 produced by bacteria. E. crassus are the most resistant ciliate used in the bioassays

332 because they have been regularly exposed to higher concentrations of copper in

333 Guanabara Bay (Baptista Neto et al., 2006; Sabadini-Santos et al., 2014) and probably

334 less protected by organic matter from sludge outfall. The Dunnett and LD50 tests

335 indicated that previous environmental selection is of great importance. Due to the

336 selective pressure of the metal in the growth environment, microorganisms have

16
337 evolved various mechanisms to resist the heavy metal stress (Harrison et al., 2007).

338 Some reports have demonstrated the effect of copper on cell growth or viability for 24

339 h or 48 h. Kim et al. (2011); Madoni et al. (1992); Madoni and Romeo (2006)

340 suggested that the survival rate of ciliate species decreased remarkably after heavy

341 metal exposure (Cu, Cr, Ni, Pb, Zn, and Cd) for 24 h. Kim et al. (2011) showed the

342 toxic effects of heavy metals on the population growth of E. crassus with inhibition of

343 50% growth for 48 h.

344 Some processes help bacteria reduce metal damage through active uptake or

345 biosorption of several toxic metals by the exopolysaccharide (EPS) (Gadd and

346 Griffiths, 1977; Kirchman, 2000; Matz and Kjelleberg, 2005; Morillo Pérez et al.,

347 2008). However ciliates don’t have these resistance mechanisms and can uptake

348 copper from bacteria, which is their basal food (Azam et al., 1983; Pomeroy et al.,

349 2007). These processes can facilitate the bioaccumulation of metals in other

350 organisms of the trophic web (Hassen et al., 1998; Kennish, 2002). Furthermore, the

351 naturally associated bacteria may used their EPS to stay attached (Esteve and Gaju,

352 1999) on both Euplotes and coordinate surface masking on them (Matz and

353 Kjelleberg, 2005) at the beginning of assay, with only sterile seawater, and may

354 detach from Euplotes spp. after 96 h of assay, when the quality of the culture medium

355 becomes ideal for bacterial development, with nitrogen compounds released by

356 ciliates.

357 Ciliates have been identified as significant sources of regenerated nitrogen

358 (Bode et al., 2005; Fenchel, 2008) substrate of some bacteria (Hahn and Höfle, 2001;

359 Kirchman, 2000). The naturally associated bacteria used in assay may be able to grow

360 vigorously using nitrogen compounds released by ciliates as an energy source like

361 ammonia (Hahn and Höfle, 2001; Kirchman, 2000). Their biomass content increases

17
362 and they are resistant to copper, according to Harrison et al. (2007). The bacteria can

363 enhance their biofilm production to immobilize metals like copper (Morillo Pérez et

364 al., 2008) and this process expands the bacterial area (Matz and Kjelleberg, 2005)

365 larger than Euplotes vannus and Euplotes crassus, preventing ciliates grazing, and this

366 may assist in the death of these organisms in a stressful environment with metal.

367 5.Conclusion

368 Guanabara Bay suffers anthropogenic impacts similar to bays in other

369 developing countries. These bays are losing their biological diversity, including their

370 microbiota, due to increasing pollution. Protists were not found in a vegetative form

371 in sediments, probably due to bay pollution. Metals discharged into the bay may

372 select benthonic ciliates and affect their naturally associated bacteria resistance and

373 subsequently biomass production, which were observed during bioassay.

374 Both Euplotes survived and show resistant to copper under laboratory

375 conditions. E. crassus showed more resistance and produced more biomass than E.

376 vannus, probably due to natural selection on the bay. Euplotes spp. may stay in the

377 microbial loop under copper stress in lower concentrations than in actual Guanabara

378 Bay sediments content. These results may exemplify the loss of microbial diversity in

379 this bay and its decreased ability to recycle nutrients that could harm other high

380 trophic levels, like fish and marine mammals.

381 The Euplotes spp. results show that the naturally associated bacteria increased

382 their biomass under both ciliates and copper stress, including with high metal content.

383 They were little influenced by these metals up to 1.0 mg L-1, when naturally

384 associated bacteria from E. crassus biomass was positively influenced. This

385 demonstrates that the basis of the microbial loop is still active under copper stress and

386 can facilitate the biomagnification process.

18
387 Developing countries have a history of rapid development, characterized by

388 unmitigated pollution threatening their ecologically complex environments, which

389 presents a unique challenge to create mechanisms for bioremediation. Guanabara Bay

390 is an example of a polluted tropical bay in a developing country that is losing its

391 biodiversity and ability to self-purify. These results show that new mechanisms for

392 metals bioremediation are necessary, and the base of the microbial loop may be a clue

393 for this future tool.

394 Acknowledgments

395 The authors acknowledge financial support by Coordenação de

396 Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de

397 Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à

398 Pesquisa do Estado do Rio de Janeiro (FEPERJ).

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