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First published online March 28, 2008
Journal of Experimental Biology 211, 1257-1261 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.015065
Carotenoid intake does not mediate a relationship between reactive oxygen species and bright colouration: experimental test in a lizard
1 School of Biological Sciences, The University of Wollongong, NSW 2522,
Australia
2 Department of Zoology, University of Gothenburg, Box 405, SE 413 90
Gothenburg, Sweden
3 Edward Grey Institute, Department of Zoology, University of Oxford, Oxford OX1
3PS, UK
* Author for correspondence (e-mail: molsson{at}uow.edu.au)
Accepted 27 January 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: reactive oxygen species (ROS), carotenoids, colouration, lizards
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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)
introduced the idea that the expression of male secondary sexual traits may be
constrained by oxidative stress of toxic metabolic by-products, e.g. reactive
oxygen species (ROS), and that tolerance towards these compounds initiated by
the adaptive immune and detoxication systems may be a criterion for female
mate choice. This idea is logically appealing, since male colouration may
function as an indicator to females of male `quality' on these vital aspects
of organismic function (von Schantz et
al., 1999).
More than ten major ROS are potential health hazards to vertebrates
(Peto et al., 1981;
Mayne, 1996;
Whalley, 2001;
Matsuo and Kakato, 2000) and
are countered by two principally different systems: antioxidation and
oxidation damage repair (Matsuo and
Kakato, 2000). The antioxidant defence, which we are primarily
concerned with in the current paper, is made up of three different components:
(i) preventive antioxidant enzymes (e.g. superoxide dismutase), (ii) metal
sequestration (e.g. transferrin), and (iii) dietary antioxidants [e.g.
-tocopherol (vitamin E), carotenoids and ascorbic acid (vitamin C)]
(Matsuo and Kakato, 2000). The
latter category, the most relevant for this study, can be further categorised
with respect to where they primarily occur. Carotenoids and vitamin E (lipid
soluble) are mostly active in biological membranes and lipoproteins, whereas
ascorbic acid (water soluble) is mostly active extracellularly and in the
cytosol (Matsuo and Kakato,
2000). The relative capacity of these systems for antioxidation of
ROS is unstudied in reptiles, but in humans, for example, ascorbic acid is
recognised as the most effective plasma antioxidant
(Alexandrova and Bochev,
2005).
The function of carotenoids as an indicator of male quality in sexual
displays and evolution of mate preferences relies on relatively
straightforward, positive cytological carotenoid effects. The published
literature, however, seems to reveal a more complex picture. Although plasma
and tissue levels of vitamin C and E can be reduced by 35–75% following
immune responses to infectious disease
(Omenn et al., 1996), these
effects may arise in more than one way, of which some but not all may link
antioxidation and secondary sexual trait expression. For example, carotenoids
may have strongly detrimental effects, e.g. significantly increasing the risk
of lung cancer and cardiovascular disease, and the concomitant risk of
mortality in humans (Omenn et al.,
1996). Thus, if carotenoids even have negative health and
viability effects in some circumstances, their link between health and
colouration may be a `red herring' to researchers in sexual selection
signalling [i.e. in this scenario, not accurately reflecting underlying health
status (Hartley and Kennedy,
2004; Isaksson et al.,
2007)].
Previous work has successfully demonstrated positive carotenoid effects on
immune function (Matsuo and Kakato,
2000), reproductive output
(Blount, 2004) and behaviour,
for example flight capacity (Blount and
Matheson, 2006). Few studies have, however, focused on assessing
circulating ROS levels and their effects on colouration [but see McGraw et al.
(McGraw et al., 2005b) for an
example of TBARS test demonstration of lipid peroxidation effects on
colouration, and Isaksson et al. (Isaksson
et al., 2007) for links between colouration and total antioxidant
activity]. In the current paper, we tested the prediction, using flow
cytometry, that ingestion of carotenoids at biologically realistic levels and
time frames may reduce the level of ROS in the blood, and have concomitant
effects on male colouration in the polychromatic (red versus yellow)
painted dragon lizard (Ctenophorus pictus) from Australia. This
species was chosen because it is one of the most brilliantly coloured lizards
on the Australian continent (Cogger,
2000), and therefore a suitable model system for assessing
carotenoid effects on ROS, and for illustrating ROS level effects on
colouration.
To analyse ROS, we used flow cytometry in combination with two probes that
freely diffuse into cells, accumulate within mitochondria, and become
fluorescent when oxidised by different ROS: dihydrorhodamine 123 (DHR) is
oxidised by singlet oxygen, superoxide, H2O2 or
peroxynitrite [hereafter unspecified ROS
(Spence, 2005;
Vowells et al., 1995)], while
MitoSOX Red is sensitive to superoxide specifically, and was used to measure
basal superoxide level (bSO).
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). Painted dragons
are sexually dimorphic (females are cryptically greyish-brown), and the
dazzling colours of the adult males are used for signalling to deter
approaching rivals (Healey et al.,
2007; Olsson et al.,
2007). Polymorphism is correlated with differences in behavioural
traits, such as dominance (Healey et al.,
2007). The lizards in our experiments (N=43) were all
caught a week before the onset of the experiments (October, 2005) by noose or
by hand in homogeneous habitat with respect to colour morph spatial
distribution (desert Spinifex and mallee) at Yathong Nature Reserve,
New South Wales (145°35'E; 32°35'S) and were brought back
to holding facilities at the University of Wollongong. Specific sample sizes
are given for each statistical test (see Results), since two samples were
accidentally lost during flow cytometry preparations. The lizards were kept in
cages (330 mmx520 mmx360 mm), on a 12 h:12 h light regime
(light:dark), with a spotlight at one end of the cage to allow
thermoregulation to the preferred body temperature, and fed crickets and meal
worms to satiation at 09:00–10:00 h every second day. Males were weighed
to the nearest 0.01 g and measured snout to vent to the nearest 1.0 mm, and
body condition was calculated as residuals from a mass–snout–vent
length regression. There were no differences between colour morphs or
treatment groups in male mass, snout–vent length or body condition
(P>0.19 for all analyses), nor was there an effect of their
interaction on any of these variables (P>0.11 for all three
analyses). Blood was sampled at 09:00–10:00 h on the day of flow
cytometry in random order of male colour and treatment category (see below for
description). A blood vessel in the corner of the mouth, vena
angularis, was punctured with a syringe (1.20 mmx50 mm BC/SB) and
the blood collected in a capillary tube (for details, see
Olsson, 1994), then
transferred to PBS buffer and put on ice until flow cytometry analysis. Apart
from seven animals that were killed for biochemical analysis, all lizards were
returned to the wild subsequent to the laboratory trials and released unharmed
at the place of capture.
Carotenoid treatment
Our choice of carotenoid supplement was limited by current market
availability and we therefore looked for support of its appropriateness in two
ways. Firstly, we examined what carotenoids were deposited into the skin of
male painted dragon lizards using HPLC (see below). This analysis verified
that two of the main carotenoids in our supplement (lutein and zeaxanthin)
were indeed deposited into the skin (see Results). Secondly, we calculated the
concentration of the carotenoid supplementation (0.1 mg ml–1)
based on liver concentrations in another insectivorous lizard of the same
approximate body size (Czeczuga,
1980), following methodology developed for supplementary studies
in birds (Latscha, 1990). The
carotenoid supplement contained trans-lutein (yellow), trans-capsanthin (red),
beta-carotene and zeaxanthin (8 g kg–1, OroGlo 8 Liquid,
Kemin Industries, Inc., USA), diluted in sunflower oil (with no or only small
traces of carotenoids). Using a pipette, males were fed 100 µl of the
solution or sunflower oil (controls). Each supplementation took less than 10
s. No lizards were confirmed to regurgitate the carotenoid solution. Over 4
weeks, eleven supplementations were administered
(Monday–Wednesday–Friday). This resulted in a total daily intake
of 20 µg day–1 or 220 µg in total. All vitamin
mixtures, which can potentially contain carotenoids, were excluded during the
experiment.
Measurements of plasma and skin colour
Plasma colour
Blood samples (100 µl) were taken once, after the experimental
treatment; 30 µl of this sample was used for assessing plasma colour,
whilst 70 µl was used for flow cytometry analysis (see below). Plasma
colour was qualitatively measured blindly by matching the colour of plasma
from whole blood centrifuged at 5000 r.p.m. for 6 min to Munsell colour charts
(Olsson, 1994). Seven randomly
selected samples were analysed for spectrophotometric (Smartspec Plus, BioRad)
congruence with ocular inspection at wavelengths where peak absorption is
expected for lutein (
=448 nm) and zeaxanthin (=552 nm), and
correlated with their corresponding Munsell scores under the directional
prediction of a positive relationship. This was verified in both cases
(r=0.75, P=0.026, and r=0.74, P=0.030,
respectively, one-tailed test). This analysis was admittedly less sensitive
than for example HPLC but was only performed to qualitatively confirm the
well-established fact that carotenoids are taken up from the diet and
circulated in the bloodstream (e.g. Mayne,
1996). This was also clear from our data (see Results). No samples
contained lysed erythrocytes (which could influence colouration).
Head colour
Skin colour measurements were made twice (at the beginning and end of the
experiment) at the preferred lizard body temperature (
37°C) with a
USB2000 spectrometer system (Ocean Optics Inc., Dunedin, FL, USA), using a
PX-2 pulsed xenon lamp as a light source together with an R200 fibre optic
reflectance probe fitted with a cylindrical plastic sheath to block out
external light. A dark and a reference scan from a WS-2 white standard
(>98% reflectance within wavelengths of 300–800 nm) were obtained
before each individual was measured. The fibre optic probe was held at a
90° angle against the skin, using a fixed stand as a landmark to increase
sampling repeatability. Using OOIBase32 spec software (Ocean Optics Inc.), we
sampled three times in the same location on the head (removing the probe
between each measurement) and used the average measurement for our
analyses.
Colorimetric calculations
From the raw spectral reflectance data, we computed spectral purity
(`chroma';
(Rmax–Rmin)/Raverage)
in the area from 420 nm to 700 nm for head colour, which is an indication of
carotenoid pigments incorporated into the integument
(Johnsen et al., 2003;
Andersson and Prager,
2006).
Carotenoid extraction and HPLC analysis of painted dragon skins
Seven lizards were killed by an overdose of Brietal and the skin was
immediately excised from the body, weighed and then stored overnight in 0.5 ml
acetone. All skin carotenoids dissolved in the acetone phase. The following
day the acetone was filtered (0.2 µm syringe filter, 13 mm GHP Acrodisc)
into a new tube. For saponification, 100 µl ascorbic acid (10%) and 200
µl KOH were added and kept at 70°C for 30 min. The yellow upper phase
was evaporated to dryness under nitrogen gas. The carotenoid residue was
finally dissolved in 20 µl tetrahydrofuran (THF) and 80 µl of the mobile
phase (70:30 acetonitrile:methanol), and immediately analysed by high
performance liquid chromatography (HPLC, see below).
Part of the sample (60 µl) was injected with the isocratic mobile phase
into an RP-18 column (ODS-AL, 150 mmx4.0 mm i.d., YMC Europe GmbH,
Schermbeck, Germany), fitted on a ThermoFinnigan (San Jose, CA, USA) HPLC
system with PS4000 ternary pump, AS3000 autosampler and UV6000 diode-array
UV/VIS detector. Column temperature was maintained at 30°C and the
flow-rate at 0.6 ml min–1. Two- (at 450 nm) and
three-dimensional (300–700 nm) chromatograms were obtained and analysed
with ChromQuest 4.0 software (ThermoFinnigan). The major pigment fractions
were identified and quantified by comparison to internal standards and
calibration curves of lutein (β,
-carotene-3,3'-diol) and
zeaxanthin (β,β-carotene-3,3'-diol), kindly provided by Roche
Vitamims Inc. (Basel, Switzerland). All concentrations were calculated as
µg g–1 dry skin.
After the carotenoid extraction, the red-headed morph still had visible red
pigments, whereas yellow-headed skin patches were colourless. To investigate
whether or not the pigments were of carotenoid origin we continued by using a
separation method described by McGraw and Ardia
(McGraw and Ardia, 2003) and
McGraw et al. (McGraw et al.,
2005a). Small pieces of skin from the pigmented areas of the head
were digested by adding acidified pyridine, into which the pigments were
released. Adding an organic solvent to the solution makes it possible to
separate the lipid-soluble carotenoids from other pigments. If carotenoids
were present, the upper phase should be coloured, which we verified for yellow
pigments. Red pigments, however, became deposited in the lower (water) phase,
suggesting that they were not carotenoids [possibly pteridines
(Steffen and McGraw, 2007)],
and, in hindsight, that our treatment possibly did not provide a component for
directly boosting red colour production [since pteridines are synthesised from
basic purine, e.g. guanine (Steffen and
McGraw, 2007)].
Measuring ROS of blood cells by flow cytometry
After administering the final diet treatment, the single sample of
peripheral blood (70 µl) was diluted immediately with 9 volumes of
phosphate buffered saline (PBS; 137 mmol l–1 NaCl, 2.7 mmol
l–1 KCl, 1.5 mmol l–1
KH2PO4, 8 mmol l–1
Na2HPO4, pH 7.4) and stored on ice prior to analyses,
which were completed within 4 h of sampling. Prior to staining, diluted blood
was diluted a further 50-fold with PBS and then centrifuged (300
g for 5 min) to pellet cells; each cell pellet corresponded to
10 µl of whole blood. Cells were resuspended in 100 µl of PBS containing
one of the following: no additions (unstained control), 0.1 mmol
l–1 dihydrorhodamine 123 (DHR; Molecular Probes, Invitrogen,
USA) or 5 µmol l–1 MitoSOX Red (MR; Molecular Probes). DHR
and MR were added from stock solutions in dimethylsulphoxide (DMSO); the final
concentration of DMSO was 0.2% (v/v) or less. Cells were subsequently
incubated at 37°C for 30 min, then washed with PBS by centrifugation as
described above and held on ice until analysed by flow cytometry; 50 000
events were acquired for all samples. Flow cytometry was performed using a
Becton Dickinson LSR II (Becton Dickinson, Sydney, Australia), with excitation
at 488 nm and emitted fluorescence collected using bandpass filters of
515±10 nm (DHR) and 575±13 nm (MR). Data were acquired and
analysed using FACSDiva v4.0.1 and CellQuest Pro v5.1.1 software (Becton
Dickinson), respectively. On the basis of forward angle laser scatter and side
angle laser scatter, a number of blood cell populations were discerned; the
results obtained were similar for all these populations. For each sample, the
arithmetic mean fluorescence for all 50 000 cells acquired was determined
using CellQuest software and used to compare between samples and treatments.
Repeatability of the flow cytometry result for samples from the same
individuals was measured in a separate experiment.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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0.0, P>0.99, d.f.=1). Thus,
carotenoid levels were successfully elevated in carotenoid-treated males.
Test of flow cytometry accuracy
In a separate experiment involving 14 males, we took two blood samples (A
and B) independent of each other and looked for a correlation between samples
A and B. For bSO, the between-sample correlation was r=0.97
(P<0.0001), and for ROS it was r=0.80
(P=0.0006). Thus, our flow cytometry technique was highly
repeatable.
Effects of carotenoids and male colouration on ROS
bSO and ROS, body mass (g) and body condition (residuals from a
mass–snout–vent length regression) were all normally distributed
(Wilks lambda, W, normal: 0.97, P=0.67, ROS, N=34;
0.96, P=0.35, bSO, N=35; 0.94, P=0.06, mass,
N=34; and 0.97, P=0.58, condition, N=34). Body mass
and body condition were not significantly correlated with ROS or bSO
(Pearson's product moment correlations, –0.33<r<0.15,
0.22<P<0.94).
A two-factor ANOVA (carotenoid treatment, colour morph) with interaction term showed no effect on bSO of treatment, colour morph or their interaction (Table 1; mean ± s.e.m. bSO counts, 6.2±0.29, N=18, and 6.0±0.18, N=17, for carotenoid-treated and control males, respectively). For ROS, a similar result was obtained with no effect of treatment, colour morph, or treatment x colour morph (Table 1; mean ± s.e.m. ROS counts, 396±16, N=19, and 405±19, N=15, carotenoid-treated and control males, respectively).
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For the above analyses, Tukey's studentised range tests were performed as
post hoc tests but revealed no difference in means between the two
treatments or the two colour morphs for bSO or ROS (P>0.05 for all
comparisons; the following values are the minimum significant differences,
MSD, at =0.05 followed by the real differences between trait
categories: bSO, treatment MSD=0.73 vs 0.26, colour MSD=0.74
vs 0.12; ROS, treatment MSD=52.1 vs 10.3, colour MSD=52.1
vs 9.0).
Skin carotenoids and colouration
Seven males were analysed using HPLC, resulting in identification of a mean
content of lutein of 3.06 µg g–1 dry skin (±1.66
s.d.; max=448, retention time=4.3 min) and 1.06 µg
g–1 zeaxanthin (±0.94 s.d.;
max=452, retention time=4.6 min).
The effects of carotenoid treatment, natural colouration and their interaction on the change in head chroma through the experiment were analysed in a two-factor ANCOVA with the estimate of chroma at the onset of the experiment as a covariate. None of the factors, or their interaction, significantly influenced head chroma at the end of the treatment, whereas the covariate (colour at onset) did have a significant effect (two-factor ANCOVA, model F4,28=7.86, P=0.0002, R2=0.53; type III statistics, treatment F=0.73, P=0.40, d.f.=1; colour morph F=0.78, P=0.38, d.f.=1; treatment x colour morph F=1.8, P=0.19, d.f.=1; chroma at onset of experiment, F=25.6, P<0.0001, d.f.=1).
We then analysed the change in chroma in response to bSO levels and colour morph with the covariates chroma at onset of experiment and body condition (two-factor ANCOVA, model F5,24=8.85, P<0.0001, R2=0.65; type III statistics, colour morph F=1.43, P=0.40, d.f.=1; bSO F=8.22, P=0.008, d.f.=1; colour morph x bSO F=2.05, P=0.16, d.f.=1; chroma at onset of experiment, F=40.2, P<0.0001, d.f.=1; body condition F=0.22, P=0.64, d.f.=1). Thus, this analysis demonstrated a significant effect of bSO on colouration, as also reflected in a negative correlation coefficient between bSO and head chroma development (r=–0.45, P=0.012, N=31, controlling for body condition in a Pearson's partial correlation analysis, since condition may reflect resource availability necessary for the expression of colouration; Fig. 1).
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The corresponding analysis for a change in chroma in response to ROS, however, showed no effect of ROS or other predictors except chroma at the onset of the experiment (two-factor ANOVA, model F5,23=6.48, P=0.0007, R2=0.58; type III statistics, colour morph F=0.07, P=0.79, d.f.=1; ROS F=2.01, P=0.17, d.f.=1; colour morph x ROS F=0.00, P=0.95, d.f.=1; chroma at onset of experiment, F=28.2, P<0.0001, d.f.=1; body condition F=0.20, P=0.66, d.f.=1). The corresponding series of analysis on change in hue from before to after the experiment showed no significant effects of any other variables (P>0.20) except for hue at the onset of the experiment, which was significant in both analyses (P<0.0003 in all cases).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; McGraw,
2005). For example, Blount et al.
(Blount et al., 2003)
demonstrated that carotenoid-treated male zebra finches had better
cell-mediated immune function and were more sexually attractive than controls;
this was later replicated by others
(McGraw and Arida, 2003).
However, in humans, large prospective studies have failed to show an effect of
β-carotene on the health of well-nourished people with a balanced diet
(Hughes, 2001) and subsequent
analysis of carotenoid-induced protection against oxidative stress in birds
have provided similar results
(Hõrak et al., 2006;
Constantinti et al., 2007). Other studies with a very similar treatment
duration to ours (3–4 weeks) suggest that carotenoids may even have
directly harmful effects under some conditions [e.g. in vitamin C-deprived
patients (Omenn et al.,
1996)]. This has contributed to the unclear role of ROS as
moderators of sexually selected traits, and of carotenoids in countering their
systemic effects on health in general and colour in particular
(Hartley and Kennedy, 2004;
Isaksson et al., 2007). The
summed negative effects of ROS on aspects of immunocompetence have previously
been measured (Kurtz et al.,
2006), and one study has assessed the effects of lipid
peroxidation in the context of sexual signalling
(McGraw et al., 2005b).
However, not a single study has to the best of our knowledge measured the
circulating levels of ROS subsequent to antioxidant treatment and concomitant
effects on colouration. Thus, a crucial assumption of ROS-induced ill-health
and colouration, namely that ROS levels decline following carotenoid
administration, remains untested.
Our study demonstrates no overall effect of carotenoid treatment on
circulating ROS levels or bSO. Thus, there is no evidence that dietary
carotenoids reduce circulating levels of free radicals, thereby questioning
the efficiency of carotenoids as antioxidants in vivo. Nevertheless,
since there was still a negative correlation between the change in skin chroma
across the experimental period and bSO across all males, which could indicate
that males already had sufficient carotenoids for colour development, this
suggests that some additional factor(s) to carotenoids links colouration and
superoxide exposure. We do not know the underlying causative mechanism, but it
appears less likely to be a direct effect of superoxide on immune function,
with concomitant pathogenicity, since these males were all in good health and
showed no signs of disease or parasite infestation (whether they would react
differently if unhealthy or not in physiological homeostasis we do not know).
However, one potential explanation is that males varied in superoxide levels
due to events prior to our experiments (and that our carotenoid treatment did
not counter this effect). Such variation could come about through the
production of ROS during innate immune responses, with potential tissue damage
effects and reduced colouration as a result
(Bertrand et al., 2006).
However, for this to be important, it seems likely that superoxide effects
would have to remain high for longer periods of time than is likely in this
study (and in the wild). Alternatively, variation in superoxide among males
could be due to variation in the production of superoxide dismutase (SOD),
i.e. the endogenously produced antioxidant controlling superoxide levels. If
SOD production is costly, and/or traded off against active deposition of
carotenoids in the integument, then this may also contribute to reduced
colouration in males with higher superoxide levels.
In conclusion, consumption of carotenoids has no or limited effect on circulating baseline levels of ROS or superoxide in physiologically unchallenged lizards. Thus, although basal superoxide indeed influences the maintenance of colouration, it remains to be demonstrated that animals in natural populations can access and utilise natural carotenoids to the extent that this depresses circulating levels of ROS, in particular bSO, and that it has concomitant effects on sexually selected traits.
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Acknowledgments |
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