Migratory behavior, metabolism, oxidative stress and mercury concentrations
in marine and estuarine European glass eels (Anguilla anguilla)

Valerie Bolliet, Julie Claveau, Marc Jarry, Patrice Gonzalez, Magalie

Baudrimont, Mathilde Monperrus

PII: S0031-9384(16)30584-4
DOI: doi:10.1016/j.physbeh.2016.11.008
Reference: PHB 11542

To appear in: Physiology & Behavior

Received date: 27 July 2016

Revised date: 5 November 2016
Accepted date: 6 November 2016

Please cite this article as: Bolliet Valerie, Claveau Julie, Jarry Marc, Gonzalez Patrice,
Baudrimont Magalie, Monperrus Mathilde, Migratory behavior, metabolism, oxidative
stress and mercury concentrations in marine and estuarine European glass eels (Anguilla
anguilla), Physiology & Behavior (2016), doi:10.1016/j.physbeh.2016.11.008

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 ACCEPTED MANUSCRIPT

Migratory behavior, metabolism, oxidative stress and mercury

concentrations in marine and estuarine European glass eels (Anguilla
anguilla)
Valrie BOLLIETa,b , Julie CLAVEAUa,b, Marc JARRYa,b , Patrice GONZALEZc, Magalie
BAUDRIMONTc, and Mathilde MONPERRUSb, d
a
INRA, UMR 1224 Ecobiop, Aquaple, 64310 Saint Pe sur Nivelle, France
Universit de Pau et des Pays de LAdour, UMR 1224 Ecobiop, UFR Sciences et Techniques cte Basque, Anglet, France

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b

valerie.bolliet@inra.fr /julie.claveau@inra.fr/marc.jarry@univ-pau.fr
c

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Univ. Bordeaux, UMR CNRS 5805 EPOC, team Aquatic Ecotoxicology, Place du Dr Peyneau, 33120 Arcachon, France
patrice.gonzalez@u-bordeaux.fr/ magalie.baudrimont@u-bordeaux.fr
d
Laboratoire de Chimie Analytique Bio-Inorganique et Environnement, Institut Pluridisciplinaire de Recherche sur lEnvironnement et les
Matriaux, UMR 5254 CNRS, Universit de Pau et des Pays de lAdour, Hlioparc Pau Pyrnes, 2 av. P. Angot, 64053 9 Pau cedex 9,

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France, mathilde.monperrus@univ-pau.fr/

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corresponding author:
Valrie Bolliet, UMR 1224 Ecobiop, Aquaple, 64310 Saint Pe sur Nivelle, France
Tel.: +33 05 59 51 59 78, Fax : +33 05 59 54 51 52, E-mail address: valerie.bolliet@inra.fr

Abstract
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The relationships between the migratory behavior, methylmercury (MeHg) concentrations,
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oxidative stress response and detoxification processes were investigated in glass eels collected in
marine (Molliets) and estuarine (Urt) waters (Adour estuary, South West France) at the end of the
fishing season (April). Glass eel migratory behavior was investigated in an experimental flume
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according to their response to dusk. Fish responding to the decrease in light intensity by ascending in
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the water column and moving with or against the flow were considered as having a high propensity to
migrate (migrant). Glass eels still sheltering at the end of the 24 hr catching period were considered as
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having a low propensity to migrate and were called non-migrant. Our results provide some evidence
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that estuarine glass eels were bigger, presented a higher propensity to migrate and a lower oxidative
stress response than marine glass eels. This might reflect a selection process, some marine glass eels
progressively settling or dying before reaching Urt and/or a change in feeding behavior. In April, glass
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eels restart feeding in the Adour estuary which might decrease the oxidative stress possibly related to
starvation, and enhance migration. MeHg concentrations was significantly higher in non-migrant than
in migrant glass eels and it is suggested that non-migrant glass eels might present a higher
vulnerability to stress (at least contamination and/or starvation), although the underlying mechanisms
remain to be elucidated.

Keywords: Glass eels, Anguilla anguilla, migration, migratory behavior, oxidative stress
response, metabolism, mercury

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Introduction
The European eel reproduces in the Sargasso Sea and the leptocephalus larvae drift with the
Gulf Stream to join the continental shelf where they metamorphose and are then referred to as
glass eels. Glass eels migrate up estuaries to join rivers for a long period of growth and this

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migration is controlled by several environmental factors such as lunar phase, tides, light,

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temperature and river flow. Glass eels use flood tide transport to migrate up the estuary
(Jellyman, 1979; McCleave & Kleckner, 1982; Gascuel, 1986; Wippelhauser & McCleave,

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1987; Forward & Tankersley, 2001). This transport has a low energy cost (Hickman, 1981)

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and involves moving in the water column during the flood tide to be carried upstream by the
current and to eventually hide in the substratum during the ebb tide. This behaviour is

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triggered at dusk, at least in clear water. However, several studies have suggested that some
glass eels may not migrate to rivers but rather complete their life cycle in coastal or estuarine
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waters (Tsukamoto et al., 1998; Daverat et al., 2006). Different patterns of synchronization to
the tide have also been observed in marine and estuarine glass eels but the reason why some
glass eels stop migration and settle in marine or brackish water is far from clear (Bolliet et al.,
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2007; Bolliet & Labonne, 2008; Bureau du Colombier et al., 2007).

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Several studies have reported that the propensity of glass eels to migrate, or not, to freshwater
may be related to their energy status with migrant individuals having a better body condition
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(Edeline et al., 2006; Bureau Du Colombier et al., 2007; Edeline, 2007). However, this
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relationship has been difficult to observe at the beginning of the fishing season or in marine
glass eels (personal observation). Recently, an experimental study, using isotopic tracers to
contaminate glass eels with methylmercury (MeHg), suggested that non-migrant marine glass
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eels were more sensitive to contamination than migrant glass eels (Claveau et al., 2015b).
This may reflect a higher sensitivity to environmental stress in non-migrant glass eels
although it remains to be confirmed.
Estuaries are often exposed to anthropogenic discharges due to extensive urban development
and can represent a stressful aquatic ecosystem. For example, the Adour estuary exhibits
relatively high sedimentary concentrations of MeHg (Stoichev et al., 2004) that are likely
influenced by anthropogenic point sources of MeHg and the high methylation potential in the
sediment (Stoichev et al., 2004, 2006). However, in a recent study conducted from November
to February, Claveau et al. (2015a) suggested that direct MeHg contamination in the Adour
estuary was low in glass eels, probably because of their starvation during migration. The route
of exposure to MeHg is primarily through the diet (Wiener et al., 2003; Scheuhammer et al.,
2007) and most glass eels feed little or not at all during estuarine migration (Bardonnet &

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Riera 2005). On the other hand, it is also accepted that glass eels restart feeding in the Adour
estuary in April while marine glass eels continue to starve (Charlon & Blanc, 1983). Whether
the different feeding status between marine and estuarine glass eels might affect
contamination and/or migratory behavior has never been investigated.

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The aim of this study was to investigate metabolism, mercury contamination, oxidative stress

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defence and detoxification processes in migrant and non-migrant glass eels collected in
marine and estuarine waters at the end of the fishing season, in April.

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2. Materials and methods
2.1. Sampling site

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Glass eels were caught in April 2013 at night during the flood tide at two stations (Fig.
1): the marine station (Moliets, 43 55N, 1 23W), located 40 km north of the mouth of the
Adour estuary and the estuarine station (Urt, 43 28N, 1 17W), located 21 km from the
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mouth of the Adour estuary, halfway between the estuary mouth and the upstream limit of tidal
influence. Both stations were sampled within the same week. After collection, glass eels were
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brought to the laboratory and kept in an aerated tank. The following morning, a subsample of
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100 glass eels were anesthetized by immersion in a solution of 0.036 ml L-1 clove oil, measured
(mm), weighed (Sartorius CP 153 balance, 1 mg) and the presence of residual food in the
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digestive tract noted. Their pigment stages were determined according to the criteria of Elie et
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al. (1982), who described 8 stages: VA, VB, VIA0, VIA1, VIA2, VIA3, VIA4 and VIB, in
order of increasing pigmentation. The rest of marine and estuarine glass eels were kept in
aerated tanks during one week and progressively acclimatized with freshwater contained in the
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experimental flume used to test their migratory behavior. Then, 900 marine and 550 estuarine
glass eels were placed in the flume to be sorted according to their response to the dusk (see
Claveau et al., 2015b for details).

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Latitude
Moliets
Marine station
FRANCE

4350
SPAIN

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ATLANTIC
OCEAN
4340 Capbreton

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Adour river

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Gave dOloron
River
4330 Adour estuary
Urt Bidouze river

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Bayonne Estuarine station
5 km

130 120 110 Longitude

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Fig. 1. Map of the sampling sites

2.2. Experimental Flume

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Briefly, the flume is an annular structure composed of two parallel sections 10 m long.
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Glass eels were released in each section and subjected to a light/dark cycle (10 hr of light and
13 hr of dark separated by 30 minutes of dawn and dusk) and a constant water current. Traps
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were positioned upstream and downstream in the two sections and an artificial shelter (1 m)
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was placed in the center of each section. Glass eels responding to the decrease in light
intensity at dusk by ascending in the water column and moving with or against the flow were
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trapped downstream or upstream and were considered to have a high propensity to migrate
(migrant). Glass eels still sheltering at the end of the 24 hr catching period were considered as
having a low propensity to migrate and were called non-migrant. Individuals trapped
upstream or downstream but not in response to dusk were called active, which means that
they swam with or against the current but not in relation to migration cues. Length (mm) and
weight (Sartorius CP 153 balance, 1 mg) were measured for Moliets in 77 and 143, migrant
and non-migrant glass eels, respectively and for Urt in 169 and 49, migrant and non-migrant
glass eels, respectively. In this study, the wet weight gave similar results to the condition
factor (data not shown) and was used as a proxy of the body condition.

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2.3. Gene expression analysis

2.3.1. Sampling procedure
After sorting, 10 migrant and 10 non-migrant glass eels from Moliets and Urt were
anesthetized on ice to be measured and weighed. All glass eels were individually placed in

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polyethylene bags under N2 atmosphere, immersed in liquid nitrogen and stored at -80 C

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until analysis.

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2.3.2. RNA extraction and quantitative real-time PCR
The expression levels of 6 genes have been investigated. Two genes were involved in

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mitochondrial metabolism; the small rRNA (12s) and the subunit I of the cytochrome c
oxidase (coxI). Triglyceride lipase (tgl) was studied concerning lipid metabolism. The

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response against oxidative stress was followed using mitochondrial superoxide dismutase
(sod2) and catalase (cat), while the detoxification processes was considered through the
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gluathion S transferase (gstr). Expression of these genes as well as metallothionein
concentrations (MT) were assessed in 10 migrant and 10 non-migrant glass eels for both
Moliets and Urt. Each glass eel was minced to obtain a sample as homogeneous as possible
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and between 20 to 40 mg of wet weight were used to extract total RNA following the
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instructions of the Absolutely RNA Miniprep kit (Agilent). Then, the cDNA was produced
from the total RNA (around 1 g) with the kit AffinityScriptTM Multiple Temperature cDNA
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synthesis kit (Agilent) and stored at -20 C until analysis. Quantitative real-time PCR was
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achieved with a MX3000P (Stratagene). The amplification sequence consisted of a first step
of activation of Taq DNA polymerase at 95 C for 10 mins followed by an amplification of 40
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cycles at 95 C for 30 s, 60 C, 30 s, 72 C, 30 s. Each real-time PCR was performed in a

final volume of 20 L reaction solution containing 1 L cDNA, 2 L of the gene-specific
primer pair at a final concentration of 300 nM for each primer, 7 L of distilled water and 10
L of Brilliant III SYBR Green QPCR Master Mix (Stratagene, Agilent). Reaction
specificity was determined for each reaction from the dissociation curve of the PCR product.
This dissociation curve was obtained by following the SyberGreen fluorescence level during
gradual heating of the PCR products from 60 C to 95 C. Relative quantification of each
gene expression level was normalized according to the -actin gene expression. During the
subsequent qPCR amplifications, the output cycle corresponding to the -actin was examined.
This output was always obtained around the same cycle, demonstrating the relevance of the -
actin as reference gene in our conditions. Relative expression of each gene was expressed
using the 2-CT method as described by Livak and Schmittgen (2001). The Cycle Threshold

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(CT) represents the difference between the CT of a specific gene and the CT of the -actin
gene.
Metallothionein quantification (MT)
On the same glass eels, the concentrations of total MT proteins were determined by

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Hg(II) saturation assay as previously described by Baudrimont et al. (2003). The analysis was

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conducted on 10 glass eels for each condition with the saturation assay being repeated twice
per sample. Trichloroacetic acid was used for the denaturation of non-MT proteins and the

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lyophilized beef haemoglobin (Sigma) prepared in 30 mM Tris-HCl buffer (pH 8.2 at 20 C)

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was added to remove excess mercury not bound to the MT. At the end, the supernatant with
the MT fraction was quantitatively recovered for mercury determination by flameless atomic

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absorption spectrometry (AMA 254, Altec, Prague, Czech Republic). The detection limit of
the experimental instrument was estimated at 0.01 ng Hg binding site g-1 (wet weight). At the
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same time, blanks were performed to validate the method and to deduce the mercury burden
for each sample. During the quantification by flameless atomic absorption spectrometry, a
certified standard (TORT 2, Lobster Hepatopancreas, Canada) was regularly measured as a
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normal sample to check measurements.

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2.4. Standard metabolism measurement

At the end of the sorting, oxygen consumption was determined in 24 migrant and 23
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non-migrant glass eels collected at Moliets, and in 22 migrant and 23 non-migrant glass eels
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collected at Urt. They were transferred to 16 respirometry chambers of an intermittent flow

respirometer as described by Rgnier et al. (2010). Briefly, oxygen consumption was
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measured individually each day in 16 glass eels (one per chamber). Glass eels were
introduced in the chambers at 4 p.m and oxygen consumption was recorded during 15h
(period of acclimatization). Then, oxygen consumption was recorded during three hours,
referred as measuring sessions. Standard metabolism was expressed in mm3 O2 consumed per
hour and related to wet weight (mm3O2g-1WW). After measurement, the glass eels were
anaesthetized, measured for size and weight. Nine migrant and 11 non-migrant glass eels from
Moliets as well as 14 migrant and 13 non-migrant glass eels from Urt were individually kept
at -20 C for mercury speciation analysis.

2.5. Mercury speciation analysis

Each glass eel was lyophilized, mashed using an agate mortar and then submitted
to microwave extraction according to the procedure previously published by Navarro et al.
(2013). The supernatant was spiked with known amounts of standard solution of MeHg

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and Hg(II) and submitted to propylation. Mercury speciation analysis was performed by
GC-ICPMS using the method with parameters which are detailed by Navarro et al.,
(2013). Both endogenous (naturally assimilated) and exogenous (derived from the spiked
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MeHg) mercury species (i.e. MeHg and Hg(II)) were quantified by the isotopic pattern

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deconvolution methodology (Rodrguez-Gonzlez et al., 2005). Analytical performances

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were evaluated using the Certified Reference Material DOLT- 4 (Dogfish Liver, NRCC,
Ottawa, Canada). Good agreement with certified values (recoveries of 87 % and 101 % for

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MeHg and Hg(II), respectively) confirm the accuracy and precision of the method used.

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Detection limits of 0.04 and 0.11 ng g -1 were found for MeHg and Hg(II), respectively. All
concentrations were expressed in ng Hg g -1 dry weight.

2.6. Statistics
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All statistical analyses were performed using R software (R.2.14.2). Means are given
standard deviation. Chi-square tests were used to compare the behaviors observed in both
sections of the flume. Statistical differences in length and weight between marine and
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estuarine glass eels or between migrant and non-migrant glass eels after sorting were assessed
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using the Student t-test. Statistical differences in gene expression levels, MT, oxygen
consumption and mercury species concentrations between marine and estuarine glass eels or
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between migrant and non-migrant glass eels were assessed using a one-way analysis of
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variance (ANOVA). Relationships between MeHg and eel dry weight were tested by simple
linear regression. A significance level (p < 0.05) was employed for all the statistical analyses.
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3. Results
The control group analyzed just after fishing showed that 71 % of estuarine glass eels
(Urt) presented food residuals in the digestive tract while all marine glass eels (Moliets) were
starving and showed a visible gallbladder. Marine glass eels presented younger developmental
stages than estuarine individuals (Fig.2).
100
90
80 Marine glass eels
Percentage

70 Estuarine glass eels

60
50
40
30
20
10
0
VA VB VIA VA1 VIA2 VIA3 VIA4
0 Glass eel stages

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Fig.2 Distribution in percentages of developmental stages (Elie et al., 1982) in marine (Moliets) and estuarine
(Urt) glass eels.

3.1. Glass eel migratory behavior

After sorting, glass eel behavior in response to dusk was similar between the two sections

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of the flume (Chi square test, X2=3.726, p=0.193) and all individuals were pooled for

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analysis. 40 % of estuarine glass eels were trapped downstream or upstream after dusk and
considered as migrant (Fig. 3; 39,5% ascended in the water column at dusk and moved with

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the current and 0,5 % swam against the current). 19,5% of marine glass eels were trapped

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downstream or upstream after dusk and considered as migrant ( 16% ascended in the water
column at dusk and moved with the current and 3,5% swam against the current). 15 % of

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estuarine glass eels and 40.5 % of marine glass eels stayed in shelters and were considered as
non-migrant i.e. with a low propensity to migrate. Active glass eels were trapped downstream
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or upstream during the light phase (1 and 3% for marine and estuarine glass eels, respectively)
or the dark phase (22 and 36% for marine and estuarine glass eels, respectively) but not in
response to dusk. 17 % of marine glass eels and 6 % of estuarine glass eels were collected at
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the end of the sorting period, either in the water column or at the bottom of the flume sections.
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These individuals probably escaped from shelters during sampling and were not analyzed.
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45
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40
Marine glass eels
35
Estuarine glass eels
30
Percentage

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25

20

15

10

Migrant Non-migrant Active Others

Fig. 3 Percentages of migrant, non-migrant and active glass eels originating from Moliets (marine) and Urt
(estuarine) obtained after sorting. Others correspond to glass eels collected at the end of the sorting period in
the water column or at the bottom of the flume sections (but not in shelters).

The weight was significantly higher in estuarine than in marine glass eels (Student t-test
p= 0.0353) but no difference was observed between migrant and non-migrant individuals
(Student t-test, p>0.05, Table 1). There was no significant difference for length between

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marine and estuarine glass eels or between migrant and non-migrant glass eels (Student t-test,
p>0.05, Table 1).

Table 1 Biometric measurement (mean standard deviation) in migrant and non-migrant marine (Moliets) and
estuarine (Urt) glass eels.

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Marine glass eels Estuarine glass eels
N Length (mm) Wet weight (mg) N Length (mm) Wet weight (mg)

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Migrant 77 673 24648 169 684 25651

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Non-migrant 143 673 24546 49 684 26457

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3.2. Gene expression levels and metallothionein concentrations
Expression levels of genes 12s, cox and sod2 was significantly higher in marine than in

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estuarine glass eels, as revealed by the ANOVA analysis (Table 2, Site).

Table 2 Gene expression levels, metallothionein concentration (MT, nmol Hg site g-1 wet weight), wet weight (mg)
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(mean standard deviation) in marine and estuarine glass eels (n=10) and results of ANOVA analyses testing
differences between migrant and non-migrant glass eels (behavior) and between marine (Moliets) and estuarine
(Urt) individuals (site). Values for each gene are expressed as relative expression levels compared to -actin
expression level. In bold: significant p values.
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12s cox cat sod2 gstr tgl MT Weight

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Marine glass eels

Mean 4105,576 87,540 0,020 0,022 0,042 0,072 20,1 241,40
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SD 2295,21 105,82 0,02 0,02 0,04 0,08 9,33 39,93
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Estuarine glass eels

Mean 847,212 31,134 0,012 0,011 0,034 0,150 18,2 268,75
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2
SD 594,91 21,83 0,01 0,05 0,05 0,20 9,51 60,54
Behavior
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F value 0,417 0,7670 22,566 0,5954 5,63

51,764 8.30 0.4469
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p value 0,5237 0,3876 <0,0001 0,4458 0,0234 <0,0001 0.00 0.5081
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Site
F value 41,932 15,6566 0,777 7,276 1,246 1,9702 0.57 2.4105
23
p value <0,0001 0,0004 0,3845 0,0109 0,2721 0,1701 0.45 0.1293
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Interaction behavior/site
F value 2,301 7,1449 6,987 1,122 0,6729 0,079 0.64 1.5290
15
p value 0,1404 0,0117 0,0126 0,2971 0,4177 0,7794 0.42 0.2243
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The 12s/cox ratio was half in estuarine than in marine glass eels. Both in Moliets and Urt,
gene expression levels for cat, gstr and tgl were significantly higher in non-migrant than in
migrant glass eels (ANOVA Table 2, Behavior, see Fig. 4). In migrant and non-migrant glass

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eels, gene expression levels for 12s and cox were inversed in marine and estuarine individuals
(Fig. 4). A significant interaction between behavior and site was observed for cox and cat
genes (ANOVA Table 2).
Marine glass eels
12s cox cat sod 2 gstr tgl MT Wet weight

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300
400 0.30

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0.06 0.15
0.06 35 280
8000
0.25
0.05 30 260
300
0.10 0.20
6000

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0.04 0.04 25 240
0.15
200 0.03 220
20
4000

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0.10
0.02 0.05 200
0.02 15
100 0.05 180
2000 0.01 10
0.00 0.00 0.00 160

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M NM M NM M NM M NM M NM M NM M NM M NM

Estuarine glass eels

12s cox cat sod 2 gstr tgl MT Wet weight
100 0.02
2500
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0,035 0.15 0.6 40

2000 35 350
80 0.5
0,025 30
1500 0.10 0.4
60 300
25
0.01 0.3
1000 0.015 20
250
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40 0.05 0.2
15
500 0.1
20 0.005 10 200
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0 0.00 0.0
0
M NM M NM M NM M NM M NM M NM M NM M NM
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Fig. 4 Gene expression levels, metallothionein concentrations (MT, nmol Hg site g-1 wet weight)) and wet weight
(mg) in migrant (M) and non-migrant (NM) glass eels collected in Moliets (marine) and Urt (estuarine).
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Boxplots show the median, first and third quartiles as well as extreme values and outliers. Values for each gene
are expressed as relative expression levels compared to -actin expression level. Results for ANOVA analyses
are indicated in Table 2.
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Metallothionein concentrations were significantly higher in migrant than in non-migrant glass

eels (ANOVA Table 2, Behavior). There was no significant difference in weight between
marine and estuarine glass eels or between migrant and non-migrant individuals (ANOVA
Table 2, Fig. 4).

3.3. Standard metabolic rates

Oxygen consumption and wet weight of marine and estuarine glass eels are presented in
Figure 5. The ANOVA analyses revealed that oxygen consumptions were significantly
different between marine and estuarine glass eels (0.0450.010 mm3 g-1 WW and 0.063 mm3
g-1 WW, respectively, F=42.840; p<0.0001) but not between migrant and non-migrant glass
eels (F= 1.404; p=0.2390). In this subsample, there was no significant difference in weight

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between marine and estuarine glass eels nor between migrant and non-migrant individuals
(ANOVA, p>0,05).

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02 consumption (mm3g-1WW)

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Wet weight (mg)
Wet weight (mg)

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Marine glass eels Estuarine glass eels

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Fig.5 Wet weight and standard metabolic rate expressed as oxygen consumption in migrant (M) and non-migrant
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(NM) glass eels originating from Moliets (marine) and Urt (estuarine).

3.4. Mercury species concentrations

Concentrations of MeHg and Hg(II) in glass eels were on average 11728 ng Hg g-1dw
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and 1617 ng Hg g-1dw, respectively. The ANOVA analyses revealed no significant

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difference in MeHg and Hg(II) concentrations between marine and estuarine glass eels
(F=0.655, p=0.4245 and F=1.822, p=0.1807, respectively). MeHg concentrations was
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significantly higher in non-migrant than in migrant glass eels (F=4.807, p=0.0311) while no
difference was observed for Hg(II) (F=3.088, p=0.0836). No interactions were observed
between the behavior and the site. There was no significant difference in dry weight related to
the site or the behaviour (F=2.1254, p=0.1487; F=3.7196, p=0.0572, respectively).
When MeHg concentrations were plotted against eel dry weights, a significant inverse
relationship was observed in marine glass eels but not in estuarine ones (F=21.37, p=0.0002
and F=2.235, p=0.1474 respectively, Fig. 6). The range of sizes differed between sites, very
small individuals being present in the marine group but not in the estuarine one. The biggest
individuals were more numerous in the estuary (Fig.6). The smallest glass eels (marine)
presented the highest MeHg concentrations, likely to be because of a concentration effect
(Claveau et al., 2015a). It is also noteworthy that some big estuarine glass eels presented
relatively high levels of MeHg (Fig. 6).

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200

180 Marine glass eels R2 = 0,5174

MeHg (ng Hg g dw )

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160
-1

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140

120

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100

Migrant

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)

80

60
Non-migrant

40

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20 25 30 35 40 45 50 55 60 65
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dry weight (mg) 0
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200
Estuarine glass eels R2 = 0,0453
180
MeHg (ng Hg g dw )

160
-1

140
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120
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100
Migrant
)

80
Non-migrant
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60

40
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20 25 30 35 40 45 50 55 60 65 70

dry weight (mg)

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Fig. 6. Methylmercury (MeHg) concentrations plotted against dry weight in migrant and non-migrant glass eels
collected at Moliets (marine) and Urt (estuarine).

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Discussion
Our study provides evidences that estuarine glass eels collected in April had a higher
propensity to migrate than marine glass eels in our experimental conditions. Cues for

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migration may differ between marine and estuarine individuals, but it seems unlikely. Indeed,

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it is well accepted that glass eels synchronize to the tide and the light-dark cycle to migrate up
estuary in the wild and both marine and estuarine glass eels can synchronize their swimming

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activity to a change in water current direction every 6,2h and to the dusk in experimental

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conditions (Wippelhauser & Mc Cleave, 1987; Bolliet et al., 2007; Bolliet & Labonne, 2008;
Bureau du Colombier et al., 2007, 2009, 2011; Claveau et al., 2015b; personal observation). A

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higher propensity to migrate in estuarine glass eels might rather be explained by a selection
process, non-migrant marine glass eels progressively settling or dying in the estuary before
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reaching Urt and/or by a change in feeding behaviour. Indeed, glass eels feed little or not at all
during migration in the Adour estuary (Bardonnet & Riera, 2005) but they restart feeding at
stages VIA2VIA3 when the temperature reaches 10 C (Charlon & Blanc, 1983). This
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corresponds approximately to April in this estuary (Charlon & Blanc, 1982) and is consistent
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with the present study showing that 71 % of estuarine glass eels presented food in the
digestive tract and stages ranging from VIA2 to VIA4. In contrast, 86 % of marine glass eels
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presented VB stage and all were fasting (no food in the digestive tract and visible gallbladder),
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as previously reported by Charlon & Blanc (1982, 1983). As the propensity to migrate in
glass eels may result, at least in part, from their energetic condition (Bureau du Colombier et
al., 2007; Edeline, 2007), restarting feeding in spring may have contributed to increase mean
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weight and to enhance estuarine glass eels swimming capacity and migratory behavior.

Mercury concentrations in glass eels

Estuarine and marine glass eels showed similar MeHg and Hg(II) concentrations but
when MeHg concentration was plotted against eels dry weight, a significant inverse
relationship was observed in marine but not in estuarine glass eels. In the Adour estuary, a
negative correlation has been previously observed both in marine and estuarine starving glass
eels (Navarro et al., 2013; see Claveau et al., 2015a for discussion). In the present study, the
lack of such correlation in estuarine glass eels might result from the lack of the very small
individuals presenting high MeHg concentrations in the marine group (possibly died or settled
before reaching Urt). But it also seems to result from the biggest individuals, more numerous
at Urt, some of which presenting relatively high levels of MeHg. In aquatic systems, the route

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 ACCEPTED MANUSCRIPT

of pollutant exposure is primarily through the diet (Scheuhammer et al., 2007) and as 71% of
estuarine glass eels restarted feeding, contamination through the diet might explain this
observation.
Methyl mercury concentration levels were higher in non-migrant than in migrant glass

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eels while no difference was observed in the dry weight. This contrasts with previous studies

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showing no difference in MeHg concentrations and weight in migrant and non-migrant glass
eels (Claveau et al., 2015a,b) but is consistent with the higher expression level of the gene

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gstr observed in non-migrant individuals in the present study. Indeed, a previous study

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demonstrated that glass eels exposed to isotopically enriched 201MeHg (50 ng L-1) for 11 days
significantly increased expression level of gstr (Claveau et al., 2015b). In addition, an

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increase in gstr activity has also been reported after mercury contamination in others species
as Paralichthys olivaceus, Brycon amazonicus and Pomatoschistus microps (Monteiro et al.,
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2009; Vieira et al., 2009; Huang et al., 2010).

Oxidative stress defence

Our results provide some evidence that marine glass eels presented a higher oxidative
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stress response than estuarine individuals. Because non-migrant marine glass eels tended to
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have higher expression of genes involved in oxidative stress response than migrant
individuals, a progressive settling or death of the former, before reaching Urt, might partly
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explain this result. Different feeding status might also be involved, because food deprivation
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or restriction has been reported to activate the antioxidant systems in fish and marine glass
eels were starving before being caught (Pascual et al., 2003; Sevcikova et al., 2011; Stoliar et
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al., 2012; Kuhmar Sinha et al., 2015; Morales & Morales, 2004; Bayir et al., 2011;
Antonopoulou et al., 2013). In Sparus auratus, the oxidative stress induced by food
deprivation for 45 days reverted after 1 week of normal feeding (Pascual et al., 2003). In
contrast, in brown trout (Salmo trutta) deprived of food for 49 days and then refed to apparent
satiety over a 21 days period, the main antioxidant enzyme activities remained high after
feeding, indicating that oxidative damage had not yet been counteracted (Bayir et al., 2011).
Also in the rainbow trout and the sturgeon, Furn et al. (2009) reported that the oxidative
stress remained in the hepatic tissue and red blood cells after a 60-day refeeding period. These
contradictory results may reflect specific differences related to the severity of damage induced
by the oxidative stress. Glass eels probably restart feeding one to 4 weeks before catching
(personal observation) but we have no information concerning the duration of their starvation.
The eel is known for its extraordinary resistance to starvation (Homa & Matsui, 1973;

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Olivereau & Olivereau, 1997; Caruso et al., 2010) and the adult European eel possesses the
record for non-hibernating endurance starvation of 1594 days (Boetius Boetius, 1985). In
glass eels, swimming activity synchronized to the water current reversal each 6.2 hr last at
least two months without feeding (unpublished data). This species is considered to have a low

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metabolic rate (Boldsen et al., 2013) suggesting low needs to maintain the metabolism. So, a

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high capacity to adapt metabolism to starvation might prevent glass eels from severe damage
related to ROS and allow a rapid restoration of antioxidant enzyme to control activities when

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they restart feeding.

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Non-migrant glass eels presented higher levels of cat and gstr expression than migrant
individuals, results being more pronounced in marine than in estuarine individuals. This

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suggests a higher oxidative stress defence and detoxification in non-migrant glass eels. An
increase in mitochondrial function which enhanced ROS production and activated the defence
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system in non-migrant glass eels is also supported by their significantly higher tgl expression
level. Because all marine glass eels starved, the different oxidative stress defence between
migrant and non-migrant individuals cannot result from different feeding status. On the other
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hand, the higher MeHg concentration detected in non-migrant marine glass eels, when
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compared to migrant individuals, may have contributed to this result. Indeed, after exposure
to isotopically enriched 201MeHg (50 ng L-1) non-migrant glass eels significantly increased cat
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expression level but not migrant glass eels (Claveau et al., 2015b). In addition, non-migrant
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contaminated glass eels presented a significantly higher expression level for gstr than migrant
ones (Claveau et al., 2015b). In this study, there were no significant differences in natural or
spiked MeHg concentrations between migrant and non-migrant glass eels but Hg(II) was
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significantly higher in non-migrant glass eels. The authors suggested that non-migrant glass
eels may be more sensitive to environmental stress than migrant individuals.

Starvation and metabolic rate

The standard metabolism was significantly higher in estuarine than in marine glass eels
which is consistent with the lowest 12s/cox ratio observed in the former and suggests that they
have higher energetic needs and mitochondrial activity than marine glass eels. When deprived
of food, fish reduce their energy expenditure which can lead to a decrease in oxygen
consumption depending on the species and the duration of food deprivation (Glass, 1968;
Beamish, 1964; Pusey, 1986; Rios et al., 2002, Yengkokpam et al., 2008). As observed in the
European and the Japanese eel (Homa & Matsui, 1973; Olivereau & Olivereau, 1997; Caruso
et al., 2010), Hoplias malabaricus is able to survive very long periods of food deprivation. In

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this species, oxygen consumption was not affected by 180 days of starvation but decreased
after a period of 240 days (Rios et al., 2002). The authors explained this performance by a
very low metabolic rate when compared to other tropical fish. As observed in three cyprinid
species and the Atlantic salmon (Wieser et al., 1992; OConnors et al., 2000), refeeding after

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starvation rapidly restored the metabolic rate in H. malabaricus (Rios et al., 2002), probably

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due to the restoration of protein synthesis and turnover (Lied et al., 1983; Peragon et al.,
1999). These results suggest that the change in glass eels feeding behavior in the estuary

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during the spring may explain, at least in part, their higher metabolic rate when compared to

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marine individuals who are still fasting.
Surprisingly, despite higher expression levels of cat and gstr in non-migrant than in

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migrant glass eels, the standard metabolic rate did not significantly differed between the two
groups. This might result from the high variability observed in estuarine glass eels, possibly
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resulting from their feeding behaviour.
Conclusion
Our results provided some evidence that estuarine glass eels collected in April presented a
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higher propensity to migrate and a lower oxidative stress defence than marine glass eels. This
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might reflect a selection process, non-migrant marine glass eels progressively settling in the
estuary or dying before reaching Urt and/or a change in feeding behavior. Refeeding in the
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estuary after starvation might have decreased the oxidative stress and enhanced migration. In
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addition, our results support the idea that non-migrant glass eels might present a higher
vulnerability to environmental stresses such as starvation or contamination (Claveau et al.,
2015b) but the underlying mechanisms remain to be elucidated.
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Acknowledgements
Julie Claveaus PhD thesis was funded by The University of Pau & Pays Adour (ED 211).
We wish to thank Pascale Coste, Emmanuel Huchet and Jacques Rives for their technical
assistance. The project was partly supported by funding from the European Union within the
framework of the ORQUE SUDOE project, the Conseil Gnral des Pyrnes Atlantiques
within the framework of the EXPLOR project and by the University of Pau (BQR MIRA) and
the Aquitaine Region GAGILAU program. We also thank Rosie Cox for correcting the
English.

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Highlights
Restarting feeding in glass eels might enhance estuarine migration
Restarting feeding in glass eels might decrease the oxidative stress response
Non-migrant glass eels might present a higher vulnerability to environmental stress

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