Journal of Experimental Marine Biology and Ecology 334 (2006) 1 – 9
www.elsevier.com/locate/jembe
Osmotic effect of choline and glycine betaine on the gills
and hepatopancreas of the Chasmagnathus granulata
crab submitted to hyperosmotic stress
Matheus Parmegiani Jahn, Gabriela Maura Cavagni, Danielle Kaiser,
Luiz Carlos Kucharski ⁎
Departamento de Fisiologia, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, CEP: 90050-170,
Porto Alegre, Rio Grande do Sul, Brazil
Received 11 February 2005; received in revised form 28 December 2005; accepted 6 January 2006
Abstract
Choline is the precursor of glycine betaine, a compatible osmolyte that can maintain the osmotic balance of cells in high osmolality
media. This study examined the effect of hyperosmotic stress on the 14C-choline uptake in the hepatopancreas and gills of
Chasmagnathus granulata crabs. Uptake in the hepatopancreas was influenced by different sodium concentrations in the incubation
media. A reduction of uptake was observed in the hepatopancreas, anterior and posterior gills, in the presence of increasing
concentrations of non-radioactive choline. There was a reduction of choline uptake in the anterior and posterior gills of animals
submitted to long-term (72 h) hyperosmotic stress compared to the control group. The hepatopancreas incubated with 14C-choline
during long-term hyperosmotic stress presented choline uptake values approximately two times higher than in the control group. The
glycine betaine synthesis of this group was higher than the control group. These results demonstrate the osmotic effect of glycine
betaine in crabs during hyperosmotic stress, and this effect was only observed in the hepatopancreas, and during the long-term 72-h
stress. This paper shows, for the first time, the effect of choline and glycine betaine in the osmoregulatory process in crustaceans.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Choline; Crab; Glycine betaine; Osmoregulation
1. Introduction
Environmental factors such as drought, salinity and
temperature extremes have been limiting factors for
species survival. Organisms that live in habitats where
these factors are a major issue have developed a few
adaptations to survive in these environments. They
⁎ Corresponding author. Tel.: +55 21 51 3316 3505; fax: +55 21 51
3316 3166.
E-mail address: [email protected] (L.C. Kucharski).
0022-0981/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jembe.2006.01.006
accumulate organic solutes such as polyhydric alcohols,
free amino acids and quaternary ammonium and or
tertiary sulphonium compounds in response to osmotic
stress. The accumulation of these solutes in response to
osmotic stress is a metabolic adaptation found in stresstolerant invertebrates and vertebrates, suggesting convergent evolution for this trait (Rathinasabapathi, 2000).
Betaine is used as a non-disturbing osmolyte by
plants, bacteria, invertebrates and vertebrates to compensate hypertonic stress (Petty and Lucero, 1999). The
choline is taken up by a sodium-dependent transport
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M.P. Jahn et al. / Journal of Experimental Marine Biology and Ecology 334 (2006) 1–9
protein. Okuda et al. (2000) demonstrate that the amino
acid sequence of the choline transporter in cholinergic
neurons of rats has a significant homology with the
members of the sodium dependent glucose transporter
family (26%). The accumulation of osmotically active
compatible solutes, such as glycine betaine, is used by
some organisms to ease growth under stress conditions,
and also to stabilize macromolecules against salt (hyperosmotic stress), increase urea and denaturation by
heat and freezing. In mammals, it is accumulated in the
cells of the kidney medulla, where it regulates hyperosmotic stress and prevents denaturation by the urea
(Randal et al., 1996; Moeckel and Lien, 1997).
Bacteria show tolerance against hyperosmotic stress
by uptake and/or synthesis of the glycine betaine (Van
Der Heide and Poolman, 2000). In Bacillus subtilis two
genes were identified, whose products are used to
convert choline into glycine betaine (Boch et al., 1996).
The internal concentration of betaine in A. halophila may
represent up to 33% of its dry weight and E. halochloris
can show an accumulation of up to 2.5 M of intracellular
betaine concentration (Nyyssola et al., 2000).
In invertebrates, the use of this osmolyte is very
important, mainly during osmotic regulation processes.
This is demonstrated in the marine clam Mytilus
californianus, where the betaine transporter present in
the gills is sodium-dependent and the activity of this
transporter decreases with the reduction of the osmolality
(Wright et al., 1992). In the squid Lolliguncula brevis it
was found that the activation of betaine transport in
hypertonic conditions could affect the regulation of
volume and the excitability of motor neurons (Petty and
Lucero, 1999). The oyster Crassostrea virginica presents
a betaine synthesis from exogenous choline. It was found
that mitochondria from oysters adapted to high salinity
do take up choline and synthesize betaine faster than at
low salinities (Pierce et al., 1995). The glycine betaine
synthesis rate is ruled by the rate of choline uptake by
mitochondria from Limulus polyphemus. Choline uptake
by the mitochondria increases when the ion concentration is higher, and stimulates the metabolic pathway,
resulting in an increased glycine betaine production
(Dragolovich and Pierce, 1992, 1994). During the
adaptation period of hyperosmotic stress, the heart
cells of horseshoe crabs accumulated glycine betaine as
an active osmolyte. (Dragolovich and Pierce, 1992)
Although free amino acids are an extremely important
osmolyte in marine organisms, methylamines in general,
and particularly betaine are frequently found at higher
amounts in several euryhaline animals (Pierce et al.,
1995). In crustaceans, most studies have demonstrated
the importance of free amino acids in the osmoregulation
process (Gilles, 1982, 1983, 1997; Gilles and Pequeux,
1985).
The estuarine crab, Chasmagnathus granulata, inhabits salt marshes along the coast of Southern Brazil,
Uruguay and Argentina (Boschi, 1964). This crab is a
good hyper- and hypo-osmoregulator (Bromberg, 1992;
Miranda, 1994, Bromberg et al., 1995; Castilho et al.,
2001), and tolerates long-term exposure to freshwater
and hypersaline medium (40‰) (Nery and Santos, 1993).
A study performed by Deaton (2001) demonstrated
that the concentration of glycine betaine in gills of the
Geukensia demissa clam increases significantly in a period of hyperosmotic stress. Morris and Edwards (1995)
suggest the possibility that gills are not the only major site
of ion absorption during osmotic stress. It would appear
that the Na+/K+-ATPase activity of the hepatopancreas is
much more responsive to external stimulation (osmotic
stress) than the gills, and it becomes the main site of
absorption of these ions for osmotic regulation.
Several studies in crustaceans show the importance
of hepatopancreas in the management of substrates
(Kucharski and Da Silva, 1991; Schein, 1999), nitrogen
production (Koening, 1981; Tan and Choong, 1981;
Schein, 1999), metabolism (Vinagre and Da Silva, 2002)
and PEPCK activity during osmotic shock (Oliveira and
Da Silva, 1997, 2000, Schein et al., 2004).
The crustacean hepatopancreas express a variety of
transporters (Ahearn et al., 1992), but little attention has
been paid to their possible involvement in osmoregulation by euryhaline species.
Although there are many studies concerning choline/
glycine betaine, there is no evidence that glycine betaine
accumulates in crustaceans as an osmotic solute. And
that is the aim of the present study, to determine the effect
of hyperosmotic stress on choline uptake and on the
glycine betaine synthesis, in hepatopancreas, anterior
and posterior gills of the C. granulata crab. Hypothetically, this betaine will then be released by these tissues
into the hemolymph and taken up by other tissues to
increase the cytoplasmatic osmotic concentration and
deal with the higher osmotic concentration of the media.
2. Material and methods
2.1. Animals
Male C. granulata in stage C of the intermolt cycle
(Drach and Tchernigovtzeff, 1967) were collected from
a lagoon (Lagoa Tramandaí) located in the state of Rio
Grande do Sul, Brazil. This is an estuarine environment
where these animals are naturally submitted to a salinity
range of lower than 1‰ up to 35‰. The animals were
M.P. Jahn et al. / Journal of Experimental Marine Biology and Ecology 334 (2006) 1–9
collected according to Brazilian environmental laws
(Portaria no.: 332/90 IBAMA). Animals weighing 15–
17 g were placed in aquaria at a salinity of 20‰, at a
temperature of 25 °C, and natural photoperiod, fed with
meat for two weeks, once a day in the afternoon, and
then were used in the experiments.
2.2. Experimental procedures
The crabs were separated in two groups of hyperosmotic stress: short-term stress, during 1, 2, 6 and
24 h (HPRS) and long-term stress, during 72 h (HPRL).
Animals kept in the acclimatization aquaria (20‰)
were used as control group. The animals submitted to
hyperosmotic stress were placed in an aquarium with
salinity of 35‰ (Luvizotto-Santos et al., 2003; Schein
et al., 2004, 2005). All the groups were killed in the same
day for the experiments. During the stress period, the
animals were kept in aquaria with the same conditions
of temperature, photoperiod, aeration and feeding. The
mortality index during the experiments was zero.
3
400 mM and control group is 300 mM. In these
experiments, the osmolality of PS in hyperosmotic stress
were equilibrated with mannitol, to not disturb the
kinetics of the choline uptake, since there is some sodium
dependency. Other experiments had substituted the sodium in the incubation buffer by other osmolytes to
adjust the osmolality, like sucrose (Pierce et al., 1995)
and mannitol (Petty and Lucero, 1999).
After the removal of the gills and the hepatopancreas,
they were blotted with filter paper and placed in tubes with
500 μl of PS corresponding to each experimental group
(control or hyperosmotic) and 0.4 μCi/ml of [methyl-14C]
choline chloride (55.0 mCi/mmol; Amersham Bioscience). Then the gaseous phase was equilibrated with 5%
CO2 : 95% O2. The tubes were incubated in a Dubnoff
incubator with constant shaking for 120 min at 25 °C.
This protocol was observed in vitro experiments
made with gills and hepatopancreas of C. granulata
(Oliveira and Da Silva, 2000, 2001; Kucharski et al.,
2002).
2.4. Choline uptake
2.3. In vitro experiments
For tissue sampling, the animals were anesthetized by
chilling. The anterior gills (AG) and posterior gills (PG)
and hepatopancreas were removed and kept in cold
physiological solution (PS). The PS used for the control
group incubation was constituted by NaCl 300 mM; KCl
10 mM; MgCl2 10 mM; CaCl2 25 mM; H3BO3 8.8 mM;
HEPES 10 mM and pH 7.8. According to Chittó (2000),
the PS concentration of NaCl in hyperosmotic medium is
To evaluate the effect of different doses of sodium in
the choline uptake in hepatopancreas and anterior and
posterior gills, were utilized tissues from control group
animals and were incubated in a PS plus [methyl-14C]
choline chloride, in which had been added increasing
concentration of sodium chloride (0, 100, 300 and
400 mM), maintaining the control osmolality (300 mM)
measured with Vapro® Vapor Pressure Osmometer
(Wescor).
3
[4]
*#
Uptake (T/M)
2.5
2
[3]
1.5
1
[3]
[3]
0.5
0
0
100
300
400
Sodium concentration (mM)
Fig. 1. The uptake of [methyl-14C] choline by the hepatopancreas of C. granulata with different sodium concentrations in the PS. The uptake is shown
in the tissue / media (T / M) ratio. Each column represents mean ± standard error. Number of samples is shown in brackets. ⁎Significantly different from
control group (P b 0.05, SNK). #Significantly different from 300 mM group (P b 0.05, SNK).
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M.P. Jahn et al. / Journal of Experimental Marine Biology and Ecology 334 (2006) 1–9
1.8
1.6
Uptake (T/M)
1.4
[6]
[9]
1.2
AG
1
PG
0.8
0.6
[3] *
0.4
* [4]
[3] *
* [4]
[4] *
*
[4]
0.2
0
0
1
5
10
Choline concentration (mM)
Fig. 2. The uptake of [methyl-14C] choline by the gills of C. granulata with different doses of non-labeled choline. Anterior gill (AG) and posterior
gill (PG). The uptake is shown in the tissue / media (T / M) ratio. Each column represents mean ± standard error. Number of samples is shown in
brackets. ⁎Significantly different from control group (P b 0.05, SNK).
To analyze the effect of different doses of nonradioactive choline (cold choline) in choline uptake in
hepatopancreas and anterior and posterior gills, these
tissues of animals from control group were incubated in a
PS plus [methyl-14C] choline chloride, in which had
been added increasing concentration of non-radioactive
choline (1, 5 and 10 mM).
The choline uptake in the hepatopancreas and anterior
and posterior gills of animals from control group and
animals submitted to hyperosmotic stress (HPRS and
HPRL) were evaluated by the incubation of the tissue in a
PS plus [methyl-14C] choline chloride.
After the incubation period, the tissues were removed
of the incubation solution, washed two times in cold PS,
blotted with filter paper and placed inside tubes with
distilled water. Choline uptake was determined as described by Machado et al. (1991). Results of the uptake
are expressed as tissue / medium (T / M) ratio, which is,
dpm / ml of tissue per dpm / ml of incubation medium.
2.5. Glycine betaine synthesis
To determinate the synthesis of glycine betaine from
exogenous provided choline, was carried out a thin-layer
chromatography. After the disruption of the tissue by
treatment, 20 μl of internal medium was spotted onto
chromatography sheets (Silica gel G-60 sheet, Darmstadt, Germany). The thin-layer chromatography sheets
1.4
1.2
[4]
Uptake (T/M)
1
0.8
0.6
0.4
*
[4]
0.2
*
[4]
*
[4]
0
0
1
5
10
Choline concentration (mM)
Fig. 3. The uptake of [methyl-14C] choline by the hepatopancreas of C. granulata with different doses of non-labeled choline. The uptake is shown in
the tissue / media (T / M) ratio. Each column represents mean ± standard error. Number of samples is shown in brackets. ⁎Significantly different from
control group (P b 0.01, SNK).
M.P. Jahn et al. / Journal of Experimental Marine Biology and Ecology 334 (2006) 1–9
5
1.8
1.6
Uptake (T/M)
1.4
AG
[6]
PG
[9]
1.2
1
[6]
*
0.8
[6]
*
0.6
0.4
0.2
0
Control
72
Times (hours)
Fig. 4. The uptake of [methyl-14C] choline by the gills of C. granulata submitted to long-term hyperosmotic stress (HPRL). Anterior gill (AG) and
posterior gill (PG). The uptake is shown in the tissue / media (T / M) ratio. Each column represents mean ± standard error. Number of samples is shown
in brackets. ⁎Significantly different when compared to the control group (P b 0.05, SNK).
were placed in a methanol and ammonia 0.88 M (75 : 25)
solution (running solvent) and than subsequently autoradiographed (Boch et al., 1996). The analysis of radioactive spots of glycine betaine were made using Image
Master® VDS (Pharmacia Biotech).
was taken as the level of significance. All tests were
performed with the Jandel Sigma Stat for Windows
version 2.0.
2.6. Statistical analysis
3.1. Uptake of [methyl-14C] choline chloride by the
anterior and posterior gills and hepatopancreas of C.
granulata
To compare the effect of different doses and different
duration of stress among groups was used the one-wayanalysis of variance (ANOVA) followed by Student–
Newman–Keuls (SNK) test, and to compare the glycine
betaine formation among groups was used the Student's
t-test. All data was shown as means ± SEM. P b 0.05
3. Results
Fig. 1 shows the influence of sodium on the choline
uptake in the hepatopancreas. There is a significant rise
in the choline uptake with the higher concentration of
sodium (400 mM), when compared to the concentrations
3
Uptake (T/M)
*
[12]
2.5
2
[9]
1.5
1
0.5
0
Control
72
Times (hours)
Fig. 5. The uptake of [methyl-14C] choline by the hepatopancreas of C. granulata submitted to long-term hyperosmotic stress (HPRL). The uptake is
shown in the tissue / media (T / M) ratio. Each column represents mean ± standard error. Number of samples is shown in brackets. ⁎Significantly higher
when compared to the control group (P b 0.05, t-test).
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M.P. Jahn et al. / Journal of Experimental Marine Biology and Ecology 334 (2006) 1–9
Arbitrary units of intensity
A
1
-
3
2
-
4
12
10
8
6
4
2
0
CTR
HPRS
Group
Arbitrary units of intensity
B
1
-
2
-
4
3
-
5
140
-
6
*
120
100
80
60
40
20
0
CTR
HPRL
Group
Fig. 6. Representative autoradiography of thin-layer chromatography of glycine betaine synthesis from [methyl-14C] choline in the hepatopancreas of
C. granulata. (A) Crabs submitted to short-term hyperosmotic stress (HPRS — 2 h) The samples shown in lanes 1–2 are from control group (CTR)
and in lanes 3–4 are from a short-term hyperosmotic stress group (HPRS). (B) Crabs submitted to long-term hyperosmotic stress (HPRS — 72 h). The
samples shown in lanes 1–3 are from the control group (CTR) and in lanes 4–6 they are from the long-term hyperosmotic stress group (HPRL). Data
are given as arbitrary units of intensity (pixels) mean ± standard error. ⁎Significantly different from control group (P b 0.05, t-test).
of 0 and 300 mM. The same experiment was performed
in the gills, but no influence of sodium on the choline
uptake in this tissue was observed.
The effect of different concentrations of non-labeled
choline on the choline uptake in AG and PG is shown in
Fig. 2, and that in the hepatopancreas is shown in Fig. 3.
A significant reduction was observed (P b 0.05) in the
choline uptake by the anterior and posterior gills
incubated in presence of 1, 5 and 10 mM of non-radioactive choline, compared to the control group (Fig. 2). A
similar reduction was observed (P b 0.01) in hepatopancreas (Fig. 3).
Differences in the uptake of choline in the gills and in
the hepatopancreas in the short-term hyperosmotic stress
(1, 2, 6 and 24 h) were not observed in our experiments.
The effect of long-term hyperosmotic stress (72 h)
on the uptake of choline by the AG and PG is shown in
Fig. 4. It had a significant reduction of the choline uptake
in the AG and PG of the animals submitted to
hyperosmotic stress compared to the control group
(P b 0. 05).
Fig. 5 shows the higher uptake (P b 0.05) of choline in
the hepatopancreas of animals submitted to 72 h of hyperosmotic stress when compared with the control group.
3.2. Synthesis of glycine betaine from [methyl-14C]
choline chloride by the hepatopancreas of C. granulata
The chromatography of glycine betaine synthesis in
the hepatopancreas is seen in Fig. 6. It shows glycine
M.P. Jahn et al. / Journal of Experimental Marine Biology and Ecology 334 (2006) 1–9
betaine formation in the hepatopancreas of animals
submitted to short-term hyperosmotic stress for two
hours (HPRS) (6A) and long-term hyperosmotic stress
for 72 h (HPRL) (6B). There is no difference between
the HPRS group and the control group, however, in
the tissue of animals submitted to HPRL, glycine
betaine synthesis is higher than in the control group
(P b 0.05).
The chromatography of glycine betaine synthesis in
the gills was not shown because no spots were detected
on the autoradiography.
4. Discussion
The investigation of osmotic stress has been the aim of
studies in several organisms such as bacteria, plants,
invertebrates and mammals. In the research performed on
invertebrates studies were found mainly on mollusks
(Pierce et al., 1995, 1997; Perrino and Pierce, 2000a,b;
Deaton, 2001) and crustaceans (Gilles, 1983; Da Silva
and Kucharski, 1992; Morris and Edwards, 1995; Gilles,
1997; Castilho et al., 2001; Schein et al., 2005). The
ability to synthesize betaine from choline is found in many
animal cells. The studies performed on crustaceans have
until now demonstrated the involvement of inorganic and
organic osmolytes, and among organics the most investigated group corresponds to the non-essential amino
acids. However the use of methylamines as osmotic
protection has not been investigated in crustaceans.
The work of Pierce et al. (1995) demonstrates that
mitochondria isolated from oyster gills and incubated with
14
C-choline plus increasing doses of non-labeled choline
presented a linear uptake proportional to the offered doses.
It was also demonstrated that the glycine betaine synthesis
process occurs based on choline, inside the mitochondria,
and it is mediated by the choline dehydrogenase and
betaine aldehyde dehydrogenase enzymes. The experiments carried out in vitro with the hepatopancreas and the
gills demonstrated that the presence of different doses of
non-radioactive choline inhibited choline transport in the
cells (Figs. 2 and 3). These results differ from those
obtained by Pierce et al. (1995) since he used isolated
mitochondria instead of the whole structure utilized in this
experiment. Thus the whole kinetics of choline transport
of the gill cells membrane is probably different from the
mitochondrial membrane. According to Okuda and Haga
(2000) choline uptake occurs through sodium-dependent
cell membrane transporters that show a significant
homology with the members of the family of sodiumdependent glucose transporters, SGLTs. Thus inhibition
of the 14C-choline uptake observed in these experimental
groups may be undergoing a process of competition with
7
the non-labeled choline, as observed by Eichler (2001) in
other transport systems.
Pierce et al. (1995), found some dependency of
sodium on the choline uptake in mitochondria isolated
from oyster gills. The fact that choline uptake is performed by a transporter with homology with the family
of SGLTs also explains why the uptake in the hepatopancreas was influenced by different sodium concentrations shown in Fig. 1, demonstrating some sodium
dependency in this transport mechanism.
The gill tissue from animals submitted to longterm hyperosmotic stress presented a reduction of
the 14C-choline uptake. Furthermore, no great formation
of betaine was observed in this tissue. Possibly the gills
do not have the enzymatic capacity to produce betaine
from choline, and consequently, do not take up choline
to perform this osmotic regulation during the long period
of stress. Moreover, the gills should be accumulating
other solutes rather than betaine, such as free amino
acids. A similar study by Deaton (2001) demonstrated
that the concentration of glycine betaine in gills of the
G. demissa clam increased significantly in the first 4 h,
then stabilized, and the total amino acids increased
constantly until the end of the 24-h period of hyperosmotic stress. Morris and Edwards (1995) showed the
possibility that gills are not the prime site for salt balance
in osmoregulation of the Leptograpsus variegatus crab,
suggesting that is the hepatopancreas which is the
primarily responsible for this control.
The results observed in this experiment show that
a significant increase of the uptake 14C-choline occurs in
the hepatopancreas of the animals of the HPRL group
compared with the control group. These results demonstrate the correlation between the increased solution
osmolarity and the choline uptake by hepatopancreas.
Thus we can attribute central activity in substrate
management to the hepatopancreas during the many
different types of metabolic alterations produced by
starvation, anoxia, osmotic stress and hormone levels
(Vinagre and Da Silva, 1992, 2002; Schein, 1999),
functioning as an importer and exporter of substrate
during these situations.
The results obtained demonstrate the difference of
osmotic behavior between the gills and the hepatopancreas of C. granulata submitted to 72 h of hyperosmotic
stress. The difference in choline uptake activity for
osmorregulation in the gills and in the hepatopancreas
can be justified by the different participation of these
tissues in osmotic tolerance. The fact that neither the
hepatopancreas nor the gills showed differences between
experimental groups in the short-term stress demonstrates that choline and glycine betaine may have no
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M.P. Jahn et al. / Journal of Experimental Marine Biology and Ecology 334 (2006) 1–9
action in the osmotic balance during short-time stress.
During the short-time stress the gills and the hepatopancreas should probably use another substrate to perform
this osmotic control, such as ion regulation (Morris and
Edwards, 1995) or free amino acids (Gilles, 1983, 1997).
The osmotic effect of the choline in the hepatopancreas during the 72 h of hyperosmotic stress is clearly
seen when compared with the control group and with the
uptake of gill tissue. Thus we can conclude that the
choline had an osmotic function only in the hepatopancreas during the 72 h of stress.
The greater formation of glycine betaine in the
hepatopancreas during the 72 h of stress once again
shows the importance of choline and glycine betaine in
the osmotic balance during this situation. The tissue is
taking up more choline and converting this compound
into glycine betaine. This betaine will then be released
into the blood and will be the osmolyte accumulated in
the cells to counterbalance the high concentration of the
medium, avoiding some harmful effects of this stress.
This experiment shows for the first time the importance of choline and glycine betaine in osmotic
regulation in crustaceans. New studies will be developed, such as the evaluation of the mitochondrial uptake
of choline and enzymatic activity in the betaine
synthesis, to increase knowledge on the osmotic effect
of choline and glycine betaine in crustaceans.
Acknowledgments
This work was supported by grants from Fundação
de Amparo à Pesquisa do Rio Grande do Sul (FAP
ERGS) and Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), Brazil. We would like
thanks to Gabriel Parmegiani Jahn for the support in the
English version of the manuscript. [SS]
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