1

Morphology and 18S rDNA gene sequence of Spirostomum minus and Spirostomum 1

teres (Ciliophora: Heterotrichea) from Rio de Janeiro, Brazil.2

Noemi M. Fernandes 1,2 & In�cio D. da Silva Neto 13

1 Laboratório de Protistologia, Departamento de Zoologia, Universidade Federal do Rio de 4Janeiro. 21941-590, Rio de Janeiro, Rio de Janeiro, Brazil.5

2 Corresponding author. E-mail: noemi.fernandes@yahoo.com.br.6

7Abstract8

9Spirostomum species are widely used as model organisms in ecological studies of 10

environmental impact and symbiosis between ciliates and human pathogenic bacteria. However, the 11

taxonomy of this genus is confused as a consequence of the superficiality of morphological 12

descriptions and the use of a few characters for species differentiation. The present study provides 13

details of total infraciliature, nuclear apparatus, morphometric data and 18S rDNA gene sequence of 14

Spirostomum teres Clapar�de & Lachmann, 1858 and S. minus Roux, 1901 isolated respectively 15

from a sewage treatment plant and a freshwater lake in Rio de Janeiro City, Brazil. For 16

morphological descriptions of S. teres and S. minus, living cells were observed using bright-field 17

and differential interference contrast (DIC) microscopy, the total infraciliature and nuclear 18

apparatus were revealed with the protargol method, and ciliature patterns were observed in scanning 19

electron microscopy (SEM). The complete 18S rDNA gene sequences of S. teres and S. minus were20

obtained using the eukaryotic universal primers, and than compared with sequences of other21

Spirostomum species and populations deposited in the GenBank database. S. minus living have 400-22

800 �m in length and 55-115 �m in width; adoral zone of membranelles about 112�m;23

inconspicuous paroral kinetie; somatic ciliature composed of 30-40 kineties; moniliform 24

macronucleus with 9-25 nodes and about 12 micronuclei; single and posterior contractile vacuole; 25

living cells are yellow-brown. S. teres living and fully extended have -250 �m in length and about26

65 μm in width; adoral zone of membranelles about 92 �m; about 30 somatic kineties; compact 27

macronucleus and about 5 micronuclei; presence of macronuclear groove; single and posterior 28

2

contractile vacuole; living cells are colorless. The morphology and 18S rDNA gene sequence 29

confirms the identification of S. minus and S. teres. The utilization of silver impregnation technique30

(protargol) and scanning electron microscopy allowed the observation and description of a greater 31

number of characters in S. minus and S. teres, thus assisting the research that require identification 32

of these species.33

34

Key words: bioindicators; ciliates; heterotrichs; morphology; sewage treatment. 35

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3

Introduction55

Ciliates of the genus Spirostomum Ehrenberg, 1838 are conspicuous organisms that are easily 56

recognizable by their large sizes (500-1000 µm) and elongated body. They are easily confounded57

with small helminths. The name Spirostomum refers to the ability these ciliates have to contract in a 58

spiral mode. This type of contraction is due to the presence of post-ciliar subpellicular fibers, which59

arise on the anterior end and form a spiral in a counterclockwise direction toward the posterior end 60

of the body (ISHIDA et al. 1988).61

The Genus Spirostomum was first mentioned by EHRENBERG (1838), and morphological 62

studies have been subsequently carried out for some species (PACKARD 1948, FINLEY et al. 1964,63

DANIEL & MATTERN 1965, TUFFRAU 1967, KUDO 1971). However, these studies were based64

exclusively on observations of specimens in vivo. REPAK & ISQUITH (1974) emphasize the difficulty 65

in separating and identifying species of Spirostomum as a consequence of the superficiality of the66

morphological descriptions and the use of only a few characters for species differentiation.67

Moreover, most of those characters have a significant intraspecific variability (REPAK & ISQUITH68

1974), making species identification even more difficult. For this reason, the taxonomy of 69

Spirostomum is extremely confusing. To solve this problem, more detailed morphological and 70

morphometric studies that increase the number of characters available for species identification are 71

needed. Descriptions should include observations on infraciliatura patterns and ultrastructural 72

characters.73

The identification of Spirostomum species currently relies only on morphological features, 74

such as cell shape, size, ratio of the total length/ adoral zone of membranelles length, location of the75

contractile vacuole, and configuration of the macronucleus (FOISSNER et al. 1992, BERGER et al.76

1997). However, it should be noted that some species of Spirostomum can be easily misidentified 77

(FOISSNER et al. 1992). In the recent decades, the small subunit 18S rDNA gene sequence has been 78

widely used in the phylogenetic study of ciliates, and has been increasingly used as a diagnostic 79

feature for species identification (LYNN & SMALL 2002, GONG et al. 2007, LI et al. 2009). The 80

4

small subunit rRNA gene sequence of European populations of S. minus and S. teres was sequenced81

by SCHMIDT et al. (2007) and have been used in recent phylogenetic studies of the Heterotrichea by 82

several authors.83

Spirostomum species have been used in modern times as model organisms in research on 84

protists, as hosts for human pathogenic bacteria (FOKIN et al. 2003, FOKIN et al. 2005). Some 85

species, for instance S. minus and S. teres, have also been used as a model in environmental impact 86

studies, since they are considered good indicators of water quality (FOISSNER et al. 1992, BERGER et 87

al. 1997, BERGER & FOISSNER 2003) and show sensitivity to certain toxic substances (e.g. heavy 88

metals like nickel, copper, mercury, and zinc; the phenol Na-PCP) (MADONI et al. 1992, MADONI89

2000). Nevertheless, studies on the morphology and taxonomy of Spirostomum species are quite 90

rare.91

In the present work, morphological descriptions and morphometric data of S. teres and S. 92

minus isolated from a sewage treatment plant and a freshwater lake in the city of Rio de Janeiro, 93

Brazil, are provided. Those include descriptions of morphological characters observed on living 94

organisms, and after the use of the protargol impregnation method, which shows details of the95

nuclear apparatus and total infraciliature. Descriptions of ultrastructural characters observed by 96

Scanning Electron Microscopy (SEM) are presented. The small subunit 18S rDNA gene sequence97

of S. minus and S. teres from Rio de Janeiro are also presented and characterized. 98

99

Methodology100

Sampling and morphological investigations. Specimens of Spirostomum teres and S. minus were 101

obtained from samples collected in a sewage treatment plant in Penha, Rio de Janeiro (22 º 52'S, 43 102

º 13'W), and from a freshwater lake located at the Universidade Federal do Rio de Janeiro (22 º 103

51'S, 43 º 13'W), respectively. Samples from both localities were obtained manually using plastic 104

containers, and then maintained in the laboratory for several days at room temperature (about 24 105

°C) as a raw culture for studies as proposed by FOISSNER (1992). Living cells were isolated from 106

5

cultures and observed using bright-field and differential interference contrast microscopy. The 107

infraciliature and nuclear apparatus were revealed with the protargol impregnation method 108

according to DIECKMANN (1995). Specimens were examined at 100× to 1,000× magnification. 109

Somatic and oral ciliature patterns were observed in Scanning Electron Microscopy according to 110

SILVA-NETO et al. (2012). Measurements were carried out with software Image Pro-plus 5.0 by 111

Olympus®. Slides of S. minus and S. teres silver-staining were deposited in collection of 112

Laboratório de Protistologia (Universidade Federal do Rio de Janeiro), with respective registration 113

numbers: IBZ-UFRJ0017-X and IBZ-UFRJ0018-X.114

115

Molecular methods. Genomic DNA extraction, PCR amplification, and 18S rDNA gene 116

sequencing of S. minus and S. teres were performed according to the method described by SCHMIDT 117

et al. (2007). The target segment of the 18S rDNA gene sequences was amplified using the118

eukaryotic universal primers EukA/EukB (MEDLIN et al. 1988), covering the full length of the gene. 119

The sequences obtained were edited on BioEdit (HALL 1999), and compared with the NCBI 120

database using the BLASTN algorithm. The obtained 18S rDNA gene sequences of S. minus and S. 121

teres were deposited at the NCBI/GenBank database with the respective accession number: 122

JQ282896 and JQ282897. These two sequences were compared with the 18S rDNA sequence of S. 123

minus (AM398200), S. teres (AM398199) and S. ambiguum (AM398201) obtained by SCHMIDT et 124

al. (2007). The 18S rDNA nucleotide composition of S. minus and S. teres from Rio de Janeiro was 125

obtained using CompSeq software (RICE et al. 2000). The similarity structural matrix and absolute 126

distance matrix between Spirostomum species was generated from BioEdit (HALL 1999) and 127

PAUP* 4.0, respectively. 128

129

Results130

Morphology of Spirostomum minus Roux, 1901 (Figure 1; Table I). Living and fully extended 131

organisms have vermiform, elongated bodies, tapered at the end and a total length ranging from 132

6

400-800 �m (Fig. 1A). Contracted organisms have ellipsoid bodies (Figs 1C, E, F), total length of 133

175-262 �m (mean= 206 �m) and width of 55-115 �m. During the contraction process, specimens 134

of S. minus twist their oral and somatic ciliature in a counterclockwise direction (Fig. 1C-H). The135

adoral zone of membranelles (AZM) have 112�m (64-142 �m) in total length and starts at the136

anterior end of the ciliate and ends in the cytostome, located approximately at the middle of the 137

body (Fig. 1C, G-H). In contracted specimens, the AZM starts on the dorsal surface of the anterior 138

end and makes a counter-clockwise turn around the body up to the cytostome, located on the ventral 139

surface (Fig. 1C, F, H). Inconspicuous paroral kinetie, located to the left of the AZM (Fig. 1C).140

Somatic ciliature composed of 30-40 (mean 35) rows of cilia or kineties (Fig. 1D-E). In contracted141

individuals, the somatic kineties are arranged obliquely with respect to the central axis of the body, 142

following the torsion of the cell (Fig. 1C-F). Nuclear apparatus composed of moniliform 143

macronucleus with 9-25 nodes connected by nuclear bridges, and approximately 12 micronuclei144

near or overlapping the macronucleus (Fig. 1C). Each micronuclei has 1.8 �m in mean diameter. 145

Presence of single contractile vacuole located at the posterior end of the body (Fig. 1A). Living 146

organisms are yellow-brown in color when observed in low magnification (Fig. 1A). This 147

coloration is due to the presence of pale brown cortical granules (approx. 0.5 �m), densely arranged 148

between the kineties (Fig. 1B). Individuals move by crawling slowly on the substrate, and 149

eventually swim freely above it.150

151

Morphology of Spirostomum teres Claparède & Lachmann, 1858 (Figures 2-3; Table II). Fully 152

extended living organisms have rounded posterior end, and tapered anterior end, are 150-250 �m in 153

length and about 65 μm in width (Fig. 2A-C). Contracted cells have ellipsoid body shape, with 154

slightly tapered ends (Figs 2E-F; 3A). During the contraction process, specimens of S. teres twist155

their oral and somatic ciliature in a counterclockwise direction (Figs 2E-G; 3A). The adoral zone of 156

membranelles (AZM) has about 92 �m (56-114 �m) in total length and starts at the anterior end of 157

the ciliate and ends on the cytostome, located at the anterior third of the body (Figs 2E-G; 3A-B). In 158

7

contracted cells, the AZM starts on the dorsal surface of the anterior end and makes a counter-159

clockwise turn around the body, up to cytostome, located on the ventral surface (Figs 2E-G; 3A-B).160

Inconspicuous paroral kineties, located to the left of the AZM (Fig. 2G). Somatic ciliature 161

composed of about 30 kineties (Fig. 3A). In contracted individuals, the somatic kineties are 162

arranged obliquely with respect to the central axis of the body, following the torsion of the cell163

(Figs 2E-F; 3A). Nuclear apparatus, composed of single and compact macronucleus, located 164

approximately at the center of the body (Fig. 2A-F). In some cells, macronuclear grooves that hold 165

micronuclei were observed (Fig. 2H). Presence of about 5 micronuclei (5 µm in diameter)166

dispersed throughout the cytoplasm, or overlapping the macronucleus (Fig. 2E). A single and167

conspicuous contractile vacuole occupying almost the entire posterior third of the body (Fig. 2A-D).168

Live cells are colorless (Fig. 2A-D), and move slowly, crawling up by the substrate. 169

170Molecular characterization171

172The 18S rDNA gene sequence and structural similarity confirms the identity of S. minus and S. teres173

characterized in the present study. The 18S rDNA gene sequence of S. minus obtained is composed 174

of 1348 bp of which 27.08% are adenines, 19.21% cytosines, 27.45% guanines and 26.19% are 175

thymines. The GC content is 46.66%, which is within the range of other ciliates. This sequence 176

shows higher similarity (97.3%) with the S. minus sequence obtained by SCHMIDT et al. (2007) 177

(Tab. III) which confirms the identity of the species. 178

The complete sequence of S. teres from Rio de Janeiro, Brazil, is composed of 1558 bp of 179

which 26.32% are adenines, 20.99% cytosines, 27.34% guanines and 25.35% are thymines. The GC 180

content is 48.33%. This sequence is more similar to that of S. teres (98.7%), obtained by SCHMIDT181

et al. (2007) (Tab. III), differing from this it 10 nucleotides. The structural similarity of the 18S 182

rDNA and the absolute distance among Spirostomum populations/species are listed in Table III. 183

184

185

8

Discussion186

Studies on the morphology of Spirostomum species are rare. The most recent and complete work 187

was presented by FOISSNER et al. (1992). In it, aspects of the ecology and morphology of S. 188

ambiguum Ehrenberg, 1838, S. caudatum Delphy, 1939, S. minus Roux, 1901 and S. teres189

Claparède & Lachmann, 1858 are described. The review of REPAK & ISQUITH (1974) and FOISSNER190

et al. (2002) were used as a basis for species identification.191

Among the nine species that currently comprise the genus Spirostomum, five present 192

moniliform macronucleus as in S. minus. These species are distinguished mainly by the shape of the 193

body extremities, and the size of the peristoma (REPAK & ISQUITH 1974): S. inflatum Kahl, 1932 are 194

marine organisms with wider or "inflated" posterior ends, differing from S. minus, which are 195

freshwater organisms and have slightly tapered posterior ends; specimens of S. loxodes Stokes, 196

1885 have tapered anterior ends and peristoma occupying one third of the total length of their 197

bodies (vs 1/2 in S. minus); in S. intermedium Kahl, 1932 the body is smaller than in other species 198

(300-400 µm); The species that most resembles S. minus is S. ambiguum Ehrenberg, 1838 (Tab. 199

IV). Those two are the only species that have yellow pigments. The difference between these two 200

species is the body size. Individuals of S. ambiguum are larger and reach up to 4 mm in length (Tab. 201

IV), have a large peristoma, greater number of macronuclear nodules, somatic kineties and 202

micronuclei in comparison with S. minus (Tab. IV). The S. minus strain described in present study 203

is more similar to the strain described by REPAK & ISQUITH (1974) (Tab. 4), however, has larger 204

number of somatic kineties (20-24 vs. 30-40). According to REPAK & ISQUITH (1974) and FOISSNER205

et al. (1992), the number of somatic kineties is a quite variable characteristic among species of 206

Spirostomum.207

Among Spirostomum species, only S. teres and S. ephrussi Delphy, 1939 have compact 208

macronucleus. However, after the description of S. ephrussi, there were no new observations or new209

reports on this species in the literature. It is possible that S. ephrussi is a junior synonym of S. teres210

(REPAK & ISQUITH 1974). The S. teres strain described in the present study is more similar to the 211

9

population studied by FOISSNER et al. (1992), however, with a greater number of micronuclei (Tab.212

V).213

In comparative studies of Spirostomum species, the phase of the life cycle must be taken into 214

account. PACKARD (1948) performed a morphological study of the nuclear apparatus of S. teres and 215

observed a relationship between age and number of macronuclear nodules. Specimens that have 216

recently gone through a process of division or conjugation have fewer nodules, and micronuclei 217

with smaller diameters, when compared with to mature forms. Internal factors, such as 218

cytoplasmatic pressure, or external, such as chemical composition, can also alter the shape of the 219

macronucleus in S. teres (PACKARD 1948). In the present study, the presence of a macronuclear 220

groove housing one micronuclei was observed in some specimens of S. teres (Fig. 2H). This 221

characteristic was also observed by PACKARD (1948) on populations of S. teres.222

Many authors state in their descriptions that Spirostomum species lack the paroral membrane 223

(TUFFRAU 1967, REPAK & ISQUITH 1974, ISHIDA et al. 1988). However, FERNANDEZ-LEBORANS224

(1985), using silver-staining methods, observed the presence of a paroral kinetie, consisting of a 225

single row of cilia, located to the right margin of the peristoma in S. teres. Images of S. minus and S. 226

teres protargol staining obtained in the present study show the presence of a paroral kinetie (Figs 227

1C; 2G), corroborating the observations of FERNANDEZ-LEBORANS (1985). This paroral kinetie may 228

correspond to the paroral membrane of other heterotrichs, but evolutionary studies are needed in 229

order to verify their homology.230

Spirostomum species are excellent model organisms and suitable bioindicators for 231

microbiological, ecological, environmental, and ecotoxicological analyses (MADONI et al. 1992,232

BERGER et al. 1997, MADONI 2000, BERGER & FOISSNER 2003). Environmental analyses need233

molecular approaches for fast and accurate species characterization that can complement or even 234

completely substitute traditional morphological methods, which are often resource and time 235

consuming. However, the 18S rDNA gene is known for only three Spirostomum species. This fact 236

10

highlights the need for the molecular characterization of more Spirostomum species, to be used in 237

studies of environmental impact.238

The utilization of silver impregnation technique and scanning electron microscopy allowed 239

the observation and description of a greater number of characters in S. minus and S. teres. The 18S 240

rDNA gene sequences of S. minus and S. teres from Rio de Janeiro confirm the identification of 241

both species. The present study contributes to better understanding of the morphology of these 242

species, which are widely used as models in ecological studies of environmental impact, and in 243

studies of symbiosis between ciliates and human pathogenic bacteria.244

245

Acknowledgements246

This work was supported by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível 247

Superior) and BIOTA-FAPERJ, Proc. E-26/110.022/2011 (Fundação de Amparo à Pesquisa do 248

Estado do Rio de Janeiro). We also thank to Maximiliano Dias for collection of samples from249

sewage treatment plant.250

251

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Legend of figures361362363

Figure 1. Morphology of Spirostomum minus. A: image from phase contrast microscopy of live 364organism showing general shape of the cell, cytostome, and single contractile vacuole in posterior 365end of the body. B: detail of contractile vacuole for visualization of cortical granules. C: S. minus 366after protargol impregnation by Dieckmann (1995) showing nuclear apparatus, somatic and oral 367infraciliature. D: detail of anterior ciliature showing the arrangement of kineties (arrows) obliquely 368disposed in relation to the central axis. E: scanning electron micrographs of morphogenesis with 369oral primordium in formation. G-H: scanning electron micrographs showing detail of the adoral 370zone of membranelles (arrows). AZM: adoral zone of membranelles; PK: paroral kinetie; CV: 371contractile vacuole; Ma: macronucleus; Ct: cytostome; CG: cortical granules; OP: oral primordium.372

373374

Figure 2. Morphology of Spirostomum teres. A-D; image from differential interference contrast 375(DIC) microscopy of live organism showing general shape of the cell, compact macronucleus and 376single contractile vacuole. D: detail of contractile vacuole. E-H: S. teres after protargol 377impregnation. E: ventral view showing adoral zone of membranelles and nuclear apparatus. F: 378dorsal view. G: detail of oral ciliature showing adoral zone of membranelles and paroral kinetie. H: 379macronuclear groove and micronuclei (arrow). AZM: adoral zone of membranelles; PK: paroral 380kinetie; CV: contractile vacuole; Ma: macronucleus; Mi: micronuclei.381

382383

Figure 3. Scanning electron micrographs of Spirostomum teres: A: general view of ventral surface 384showing adoral zone of membranelles and oral infundibulum. B: detail of anterior end showing the 385adoral zone of membranelles. AZM: adoral zone of membranelles; OI: oral infundibulum.386

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398

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Table I. Morphometric data of Spirostomum minus from sewage treatment system by activated sludge in Rio de Janeiro City, Brazil. All measures were performed on specimens protargol impregnated and are given in micrometer (µm). X: mean; M: median; Min: minimum; Max: maximum; SD: standard deviation; SE: standard error; CV: coefficient of variation in %; n: number of specimens investigated; Ma: macronucleus; Mi: micronuclei; AZM: adoral zone of membranelles.

Table II. Morphometric data of Spirostomum teres from a freshwater lake in Rio de Janeiro City, Brazil. All measures were performed on specimens protargol impregnated and are given in micrometer (µm). X: mean; M: median; Min: minimum; Max: maximum; SD: standard deviation; SE: standard error; CV: coefficient of variation in %; n: number ofspecimens investigated; Ma: macronucleus; Mi: micronuclei; AZM: adoral zone of membranelles.

Caracteres X M Min Max SD SE CV n

Body length 165 167 93 196 25.4 4.6 0.1 30

Body width 66 61 38 96 15.5 2.8 0.2 30

Length of AZM 92 93 56 114 15.3 2.8 0.1 30

Total length of Ma 38 38 20 52 6.2 1.1 0.1 30

Width of Ma 12 13 7 16 2.6 0.4 0.2 30

Diameter of Mi 2 2 1 2.9 0.4 0 0.2 30

Number of Mi 5 5 1 11 2.9 0.5 0.5 30

Number of macronuclear nodes 1 1 1 1 0.0 0.0 0.0 30

Number of somatic kineties 30 40 22 37 5.9 0.1 0.1 30

Characters X M Min Max SD SE CV n

Body length 206 200 175 262 23.5 4.7 0.1 25

Body width 82 82 55 115 15.1 3 0.1 25

Length of AZM 112 111 64 142 19.3 3.8 0.1 25

Total length of Ma 232 228 125 362 60.9 12.1 0.2 25

Width of Ma 8 8 5 12 1.8 0.3 0.2 25

Diameter of Mi 1.8 1.9 1.3 3 0.3 0 0.1 25

Number of Mi 12 8 3 30 3.8 2.7 0.3 25

Number of macronuclear nodes 17 17 9 25 4.4 0.8 0.2 25

Number of somatic kineties 35 34 30 40 1.1 0.6 0.1 25

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Table III. The structural similarities (%) of the 18S rDNA gene sequences of S. minus and S. teresfrom Rio de Janeiro, Brazil (RJ), and others species/populations of Spirostomum species. sequenced. Absolute distance (number of nucleotidic differences) is shown in parenthesis.

Table IV. Morphological comparisons between similar species and populations of Spirostomum minus.

Table V. Morphological comparisons between similar species and populations of Spirostomum teres.

Species S. ephrussi S. teres S. teres S. teres

Total lenght 450 µm 150-400 µm 150-600 µm 150-250 µm

Lenght of peristoma 3/5 of body 1/2 of body 1/2 of body 1/2 of body

Color - colorless colorless colorless

Macronuclear shape compact ellipsoid ellipsoid ellipsoid

Number of macronuclear nodes 1 1 1 1

Number of micronuclei - - 1-2 1-11

Number of kineties - 14-24 25-30 22-37

Reference Repak & Isquith 1974 Repak & Isquith 1974 Foissner et al. 2002 Present study

Species S. minus S. minus RJ S. ambiguum S. teres

S. minus -

S. minus RJ 0.973 (31) -

S. ambiguum 0.982 (23) 0.970 (35) -

S. teres 0.978 (28) 0.972 (32) 0.975 (32) -

S. teres RJ 0.973 (28) 0.964 (36) 0.971 (31) 0.987 (10)

Species S. ambiguum S. ambiguum S. minus S. minus S. minus

Total lenght 1000-4000 µm 1000-4000 µm 500-800 µm 300-800 µm 400-800 µm

Lenght of peristoma 2/3 of body - 1/2 of body - 1/2 of body

Color yellow yellow yellow yellow yellow

Macronuclear shape moniliform moniliform moniliform moniliform moniliform

Number of macronuclear nodes 12-50 10-50 24 8-50 9-25

Number of micronuclei 12-100 - 4-20 - 3-30

Number of kineties 46 70-90 20-24 20-24 30-40

Reference Repak & Isquith1974

Foissner et al. 2002

Repak & Isquith1974

Foissner et al.2002

Present study

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Figure 1

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Figure 2

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Figure 3