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The high metabolic activity associated with endurance flights and intense fuelling of migrant birds may produce large quantities of reactive oxygen species, which cause oxidative damage. Yet it remains unknown how long-lived birds prepare for oxidative challenges prior to extreme flights. We combined blood measurements of oxidative status and enzyme and fat metabolism in Hudsonian godwits (Limosa haemastica, a long-lived shorebird) before they embarked on non-stop flights longer than 10,000 km during their northbound migrations. We found that godwits increased total antioxidant capacity (TAC) and reduced oxidative damage (TBARS) as the pre-migratory season progressed, despite higher basal metabolic rates before departure. Elevations in plasma β-hydroxybutyrate and uric acid suggest that lipid and protein breakdown supports energetic requirements prior to migration. Significant associations between blood mitochondrial cytochrome-c oxidase and plasma TAC (negative) and TBARS (positive) during winter indicate that greater enzyme activity can result in greater oxidative damage and antioxidant responses. However enzyme activity remained unchanged between winter and premigratory stages, so birds may be unable to adjust metabolic enzyme activity in anticipation of future demands. These results indicate that godwits enhance their oxidative status during migratory preparation, which might represent an adaptation to diminish the physiological costs of long-distance migration.
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SCIENTIFIC REPORTS | (2019) 9:17616 |
Oxidative status and metabolic
prole in a long-lived bird preparing
for extreme endurance migration
Jorge S. Gutiérrez1,2*, Pablo Sabat3,4, Luis E. Castañeda5, Carolina Contreras3,
Lucas Navarrete3, Isaac Peña-Villalobos3 & Juan G. Navedo1,6
The high metabolic activity associated with endurance ights and intense fuelling of migrant birds
may produce large quantities of reactive oxygen species, which cause oxidative damage. Yet it remains
unknown how long-lived birds prepare for oxidative challenges prior to extreme ights. We combined
blood measurements of oxidative status and enzyme and fat metabolism in Hudsonian godwits (Limosa
haemastica, a long-lived shorebird) before they embarked on non-stop ights longer than 10,000 km
during their northbound migrations. We found that godwits increased total antioxidant capacity
(TAC) and reduced oxidative damage (TBARS) as the pre-migratory season progressed, despite higher
basal metabolic rates before departure. Elevations in plasma β-hydroxybutyrate and uric acid suggest
that lipid and protein breakdown supports energetic requirements prior to migration. Signicant
associations between blood mitochondrial cytochrome-c oxidase and plasma TAC (negative) and
TBARS (positive) during winter indicate that greater enzyme activity can result in greater oxidative
damage and antioxidant responses. However enzyme activity remained unchanged between winter
and premigratory stages, so birds may be unable to adjust metabolic enzyme activity in anticipation of
future demands. These results indicate that godwits enhance their oxidative status during migratory
preparation, which might represent an adaptation to diminish the physiological costs of long-distance
Among the most extreme examples of physical performance in the animal kingdom are those of migratory birds,
which make some of the most spectacular long-distance migrations of any organism on the planet1,2. In birds,
the greatest endurance migrations are found among long-distance migratory shorebirds (Charadriiformes), with
several members making annual journeys exceeding 30,000 km, including one or more non-stop trans-oceanic
ights of 8000–12,000 km3. ese powered apping ights of up to 9 days duration, fuelled entirely from stored
nutrients and sustained at very high metabolic rates, extend the limits of what is known of endurance physiology1.
Intense and prolonged physical activity is normally associated with an increased production of reactive oxy-
gen species (ROS), which cause oxidative damage to lipids, DNA and proteins4,5. While there is no one-to-one
correspondence between ROS production and oxygen consumption, ROS production rates are expected to be
substantially higher in animals with inherently high rates of oxygen consumption, particularly birds6,7. erefore,
oxidative stress (i.e. the accumulation of oxidative damage) might occur in migrating individuals as a result of
increased ROS production linked to the high metabolic rates that accompany migration6,8. Unlike other verte-
brates, birds rely primarily on fats (some of which are highly susceptible to attack by ROS) to fuel endurance
migration, and thus face a greater potential for damage from the reactive by-products of their own metabo-
lism8. Accordingly, some studies have shown that migrating birds can experience oxidative damage during
long-duration ights912. For example, Jenni-Eiermann et al.11 found that European robins (Erithacus rubecula)
experience oxidative damage during migratory ights and increase antioxidant capacity. Likewise, Skrip et al.12
found that fat stores positively correlated with circulating antioxidant capacity in blackpoll warblers (Setophaga
1Estación Experimental Quempillén, Facultad de Ciencias, Universidad Austral de Chile, Ancud, Chiloé, Chile.
2Conservation Biology Research Group, Department of Anatomy, Cell Biology and Zoology, Faculty of Sciences,
University of Extremadura, Badajoz, Spain. 3Departamento de Ciencias Ecológicas, Facultad de Ciencias,
Universidad de Chile, Santiago, Chile. 4Center of Applied Ecology and Sustainability, Ponticia Universidad Católica
de Chile, Santiago, Chile. 5Programa de Genética Humana, Facultad de Medicina, Instituto de Ciencias Biomédicas,
Universidad de Chile, Santiago, Chile. 6Bird Ecology Lab, Instituto de Ciencias Marinas y Limnológicas, Universidad
Austral de Chile, Valdivia, Chile. *email:
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striata) and red-eyed vireos (Vireo olivaceus) preparing for fall migration on Block Island, USA, but were uncor-
related in Italian garden warblers (Sylvia borin) aer crossing the Sahara Desert and Mediterranean Sea. Together,
these studies provide evidence that birds on stopover can prepare for, and recover from, oxidative challenges
posed by migratory ights. However, most of the work has been done on passerine species which do not embark
on extreme migration (i.e. non-stop ights >5000 km1). Furthermore, passerines are short-lived birds that usu-
ally compromise oxidative defences when faced with costly activities such as increased breeding eort13. e
‘oxidative stress theory of ageing’5 predicts that long-lived animals will have less cumulative oxidative damage
together with structural characteristics that make them more resistant to oxidative damage itself. Indeed, a recent
comparative study showed that bird species with longer lifespan have higher non-enzymatic antioxidant capacity
and suer less oxidative damage to their lipids14. Long-lived birds, such as shorebirds3, should thus exhibit higher
oxidative defences in response to increased migration workload and thus ROS production.
Besides the oxidative cost caused by prolonged ights, migratory birds are also expected to experience oxida-
tive damage during migratory fuelling1517, as this typically involves increases in metabolism and caloric intake18.
Long-distance migratory shorebirds accumulate large amounts of fat before take-o on migratory ights19,20.
Given the importance of fats to migrant birds, circulating lipid metabolites are oen used to assess individual
energetic state. Overall, some lipid metabolites increase during fat deposition (triglycerides), whereas others
increase during fat catabolism (glycerol and β-hydroxybutyrate)2123; plasma uric acid can also increase due to
the deamination of dietary protein for storage as fat or the catabolism of body protein during fasting2123. Despite
this, the metabolic correlates of long-distance travel in free-living migrants are as yet poorly studied in the context
of oxidative stress12.
Here we integrated blood-based measurements of energy metabolism (lipid metabolites, mitochondrial
enzyme activity) with oxidative status (oxidative damage and antioxidant capacity) and metabolic rate to exam-
ine how Hudsonian godwits (Limosa haemastica; hereaer “godwits”) prepare for oxidative challenges posed by
extreme endurance migration. Individual godwits make non-stop ights of longer than 10,000 km during their
northbound migrations from southern South America to Arctic and sub-Arctic Alaska and Canada24. Moreover,
godwits can live up to 30 years or longer25 and thus provide an excellent model in which to study the link between
oxidative stress and self-maintenance in a migratory context. We compared measurements of godwits sampled at
three stages before embarking on their northbound migration. We hypothesized that godwits readying for depar-
ture would undergo preparation for oxidative challenges posed by extreme endurance migration. Specically, we
predicted (i) that fuelling and pre-departure individuals would increase their antioxidant capacity in anticipation
of ight demands; (ii) that they would show strong correlations between lipid metabolites/fat scores and circulat-
ing non-enzymatic antioxidants; and (iii) that enzyme activity would correlate with metabolic rate and oxidative
Study system and data collection. e study was carried out on a population of Hudsonian godwits
(Fig.1A) that spend the boreal winter on Chiloé Island, southern Chile26. Godwits depart from Chiloé Island
in March and y non-stop to central United States, with most individuals stopping only once before again y-
ing non-stop to their breeding grounds in Alaska24. Individual godwits make non-stop ights of longer than
10,000 km and 7 days during their northbound migrations24 (Fig.1B). Godwits were caught close to the high-tide
roosts (incoming tides) using cannon nets from January to March 2018, spanning three pre-migration stages
(based on body mass, fat scores and departure dates on Chiloé from 2014–2018; J.G. Navedo, unpublished data):
‘wintering’ (early January), ‘fuelling’ (mid-February), and ‘pre-departure’ (mid-March). Time elapsed from
capture to high tide peak averaged 41 min (range 20–76 min). e potential eect of tide-enforced fasting on
metabolite levels22 was probably small since godwits typically extend foraging activity up to 2.5 h aer the low
tide peak26 and their retention time (i.e. the average time for food to pass a bird’s digestive tract) is about 1.5 h27.
Metabolite levels may also change during the time elapsed between capture and blood sampling. Birds were there-
fore blood-sampled as soon as possible aer capture (mean 44 min, range 35–52 min). Approximately 1 ml blood
was taken from the right jugular vein and stored within heparinized Eppendorf tubes maintained at 4 °C for up to
3 h. Next, tubes were centrifuged at 10,000 rpm (relative centrifugal force = 9,250) for 10 min, plasma was sepa-
rated from red blood cells and frozen at 80 °C until analysis. Samples were collected from 54 adults at wintering
(n = 14:6 females and 8 males), fuelling (n = 19:11 females and 8 males), and pre-departure (n = 21:11 females
and 10 males) stages. Time from capture to bleeding (hereaer “bleed time”) was used in analyses to determine
whether there was an eect of time until bleeding on oxidative status and metabolite proles. Upon capture, birds
were also ringed, measured (body mass and standard morphometrics), aged (adult or juvenile), sexed (rst based
on relative bill length and later through molecular sexing), and had their fat depots scored28.
Scaled mass index. Godwits are sexually dimorphic in size and their mass is highly variable25. us, we cal-
culated scaled mass index (SMI) for males and females separately following Peig & Green29. is method normal-
izes body mass to a xed value of body size based on the scaling relationship between mass and length measures29.
e xed value of body size was the mean value of wing length for the study population: 220.8 ± 0.5 mm (n = 129)
for females and 210.8 ± 0.4 mm (n = 208) for males (J.G. Navedo unpublished data).
Oxidative status. Lipid peroxidation in red blood cells was measured by the thiobarbituric acid test (thio-
barbituric acid-reactive substances, TBARS), which relies on the ability of polyunsaturated fatty acids of cell
membranes to readily react with ROS by donating a hydrogen atom30. Briey, lipid peroxidation was measured
using a commercial kit (Oxiselect, STA-330 Cell biolabs). e assay evaluates the joint between Malondialdehyde
(MDA, a product of lipid peroxidation) and thiobarbituric acid. Absorbance was monitored in a Thermo
Scientic Multiskan GO UV/VIS spectrophotometer at 25 °C. We determined hydrogen peroxides in plasma
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(H2O2, a major form of ROS) as a measure of the pro-oxidant status4 using a commercial kit (MAK311, Sigma
Aldrich). e assay evaluated colorimetrically the oxidation of Fe+2 to Fe+3 by peroxides present in the sample at
585 nm.
We estimated non-enzymatic total antioxidant capacity (TAC) by measuring the capacity of a plasma sample
to quench a standardized free radical challenge, following Sabat et al.31. Briey, TAC was measured by colori-
metric reaction using a commercial assay kit (Sigma Aldrich, San Diego, CA, #CS0790). e assay is based on
the formation of a ferryl myoglobin radical from metmyoglobin and H2O2 which oxidizes the ABTS (2,2-azi
no-bis(3-ethylbenzthiazoline-6-sulfonic acid)) to form the radical ABTS•+ which in turn produces a chromogen
that can be detected spectrophotometrically at 405 nm (Multiskan GO). is protocol used the Trolox, a Vitamin
E analog to be compared with the antioxidant capacity in a sample. As pointed out by Cohen et al.32 this method
is a functional measure of antioxidant capacity that includes the action of micromolecular components such as
vitamins E and C, and uric acid among others. Because uric acid has antioxidant properties and can reect an
adaptive response to oxidative stress33, we determined plasma uric acid concentrations (see below). Finally, we
calculated oxidative balance as the ratio of TBARS and TAC (×1000) to generate a single variable (TBARS:TAC)
that captured interindividual variation in oxidative balance (with higher values meaning higher oxidative stress9).
Mitochondrial enzyme activity. e enzyme activities of cytochrome c oxidase (COX, E.C. and
citrate synthase (CS, E.C. were measured in mitochondrion of red blood cells as a proxy of metabolic
intensity of other tissues, such as skeletal muscle34. COX is involved in the last reaction of the mitochondrial
respiratory chain that is indicative of the energy capacity of the mitochondrial system, whereas CS catalyses the
reduction of oxygen to water35. Besides, these metabolic enzymes in skeletal muscle are functionally associated
with oxygen consumption (e.g., metabolic rate, see below) at the organismal level in birds31,35,36. COX and CS
activities were measured using the methods described in Sabat et al.31,37. Enzyme activities are reported as spe-
cic activity per gram of protein (µmol min1 mg protein1). Protein concentration in plasma and red blood cell
homogenate was determined aer Bradford38 using bovine serum albumin as standard.
Plasma metabolites. We performed all metabolite assays at the Laboratorio Clínico Veterinario
(Universidad Austral de Chile) using an auto-analyzer (Metrolab 2300, Wiener Lab). Briey, total triglycer-
ides (triglycerides plus free glycerol) was measured by enzymatic colorimetric assay (Triglycerides liquicolor-
mono GPO-PAP kit, HUMAN GmbH) adapted to small sample volumes in 300 μL at-bottom microplates.
Glycerol concentration was determined by a coupled enzyme assay (Sigma-Aldrich MAK117). Concentration
of uric acid was determined using the enzymatic colorimetric test with lipid clearing factor (uric acid liqui-
color PAP kit, HUMAN GmbH). β-hydroxybutyrate was measured using colorimetric enzymatic reaction
Figure 1. (A) Hudsonian godwit overwintering on Chiloé Island (copyright permitted by Juan G. Navedo). (B)
Map showing Chiloé Island and the northbound migration route of the godwit’s local population (adapted from
Senner et al. 2014). Map was created with R version 3.5.1.
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(D-3-hydroxybutyrate kit; Ranbut, Randox Laboratories). Triglyceride levels were calculated by subtracting free
glycerol from total triglyceride levels23,39.
Metabolic rate measurements. A subsample of 18 adult birds was taken into nearby bird facilities at
Quempillén Experimental Station for metabolic rate measurements. Due to logistical limitations we were not able
to measure metabolic rates during the wintering period, so we measured the metabolic rates of fuelling (n = 10)
and pre-departure (n = 8) godwits. Birds were housed in indoor aviaries (5 × 2.5 × 2.5 m) with access to freshwa-
ter and fasted for at least 6 h prior to metabolic trials the same night40. BMR was measured as the overnight oxy-
gen consumption (
O2, ml min1) with a Field Metabolic System (FMS, Sable Systems, Las Vegas, NV). Birds
(four individuals per night) were placed into adjacent metabolic chambers (15 L) in complete darkness and
located in a temperature-controlled room at a constant temperature of 26 °C, i.e. within the thermoneutral zone40.
Incurrent ambient air at a controlled ow rate of 10,000 mL min1 was pumped through needle valves (air ow
manifold MF-8; Sable Systems, Las Vegas, NV), which supplied ve chambers with a constant ow (1400–2000
mL min1). Excurrent air was subsampled at 200 mL min1 and passed through a hygrometer, a CO2 analyser, and
a fuel cell O2 analyser (i.e. the FMS). e O2 analyser was calibrated before each trial using 99.995% pure N2 as the
low reference and ambient air scrubbed of water vapour and CO2 (set to 20.95% O2). e CO2 analyser was cali-
brated daily using pure N2 as the low reference and a certied mixture of 1.01% CO2 as the high reference. e
hygrometer was calibrated weekly according to recommendations of the manufacturer. We used a multiplexer to
monitor each chamber for 20 min before switching to the next chamber. A 10-min baseline of reference airow,
provided by an empty chamber of the same size, was measured aer each round of recordings. All data were
recorded using an analog-to-digital converter (UI-2; Sable Systems, Las Vegas, NV) connected to a laptop com-
puter. We corrected for gas analyser dri and lag time of the respirometry system using ExpeData soware. Main
ow rate was corrected to standard temperature and pressure using eq. 8.6 in Lighton41. We did not scrub water
vapour before gas analysis during measurements, but corrected for this dilution eect during data analysis using
eq. 15.3 in Lighton41.
O2 and CO2 production rate (
CO2) were calculated using eqs. 11.7 and 11.8 in Lighton41,
respectively. We considered BMR as the lowest 5-min average
O2 over the test period. Finally, we calculated the
respiratory quotient (RQ, i.e. the ratio of
CO2 to
O2). RQ varies from 0.7 (pure lipid catabolism) to 1.0 (pure
carbohydrate catabolism); a mix of lipid and carbohydrate catabolism yields intermediate values41. BMR meas-
ured in the absence of CO2 absorbents gives a measure of the non–carbohydrate-fuelled metabolism of the ani-
mal, which can provide interesting data to demonstrate a shift in the catabolic allocation of respiratory
substrates41. e body mass reported for BMR analysis was taken to be the mean of the initial measurement and
the nal measurement.
Statistical analyses. To test for temporal eects on physiological variables, we tted separate general linear
models (GLMs) with each trait measure in turn as the response variable. e full model included all three-way
interactions between the xed eects of pre-migration stage (wintering, fuelling, and pre-departure), sex, mass
(either mass or SMI), and bleed time. For BMR analysis, GLM included stage (fuelling vs. pre-departure), sex,
and mass. Model simplication was conducted using an information theoretical approach42. Because the weights
of the ‘best’ models (lowest AIC) were always <0.9 (AppendixS1), we used model averaging to identify the
most important predictor variables43. Relative importance analysis was carried out with the model-averaging
approach42. In this way, we obtained model-averaged parameter estimates that were directly comparable to each
other. We estimated the parameters from the set of all models for which the sum of Akaike weights reached >0.95.
We considered predictor variables that had model-averaged 95% condence intervals (CIs) that did not cross zero
to be biologically relevant (i.e. signicant). Results were qualitatively similar when using either body mass or SMI
as predictor variable (AppendixS1). For simplicity, here we only present results from analyses using body mass.
We ran all models using the R (version 3.5.1, R Core Team 2016) base function ‘glm’ and the MuMIn package for
model averaging44.
To test for relationships between oxidative and metabolic variables, we ran regression analyses for the whole
study period and for each stage separately. Predictor and response variables were log transformed when necessary
to meet normality assumptions.
Ethics statement. All experimental procedures were carried out under the approval of the Bioethics
Subcommittee of Universidad Austral de Chile (no 260/2016). All methods were carried out in accordance with
these approved guidelines and regulations.
Oxidative status. Levels of TBARS were lower at pre-departure (β = 2.88, s.e.m. = 1.110, CI = 5.11,
0.65), compared to wintering and fuelling stages (Fig.2). Stage was the only important predictor for TBARS,
having 71% relative importance. Likewise, H2O2 levels were lowest at pre-departure stage (β = 14.79,
s.e.m. = 6.81, CI = 28.13, 1.46) (Fig.2); stage had 73% relative importance. Conversely, TAC and uric acid
concentrations were substantially higher at pre-departure stage (TAC: β = 1.48, s.e.m. = 0.44, CI = 0.60, 2.35; uric
acid: β = 443.34, s.e.m. = 182.22, CI = 78.92, 807.76; Fig.2), and stage was again the only important predictor
variable for both TAC and uric acid, having 99% and 98% relative importance, respectively. Finally, TBARS:TAC
was lowest at pre-departure (β = 1.62, s.e.m. = 0.49, CI = 2.61, 0.63; Fig.2), indicating that levels oxidative
stress were lowest at this stage. In this case stage was again the only clear signicant predictor variable, having
96% relative importance.
Mitochondrial enzyme activity. ere was no support for temporal (stage) and intrinsic (sex and body
mass) eects on enzyme activity measures (all predictor variables had 95% CIs that crossed zero; Fig.2 and
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AppendixS1). At the wintering stage, however, there were signicant and positive associations between COX
activity and oxidative damage (TBARS and H2O2), as well as a signicantly negative association between COX
activity and TAC (Fig.3). In contrast, CS correlated negatively with TBARS and H2O2, and positively with TAC
during the winter stage (Fig.3). Neither COX nor CS activities were signicantly correlated to oxidative status at
the fuelling and pre-departure stages (always P > 0.05, AppendixS2).
Lipid metabolites and fat scores. Plasma β-hydroxybutyrate concentrations peaked during
pre-departure (β = 0.48, s.e.m. = 0.19, CI = 0.10, 0.85; Fig.4); stage was the only signicant predictor variable
for β-hydroxybutyrate, with a relative importance of 100%. In contrast, glycerol decreased with time, with con-
centration being lower at the fuelling (β = 0.057, s.e.m. = 0.02, CI = 0.10, 0.017) and pre-departure stages
(β = 0.06, s.e.m. = 0.02, CI = 0.11, 0.02) (Fig.4). None of the predictor variables had a strong eect on
triglyceride concentrations (95% CIs crossed zero; Fig.4). However, triglyceride concentrations correlated pos-
itively with fat scores (F1,45 = 4.42, P = 0.041). Fat scores were highest at pre-departure, particularly in males
(GLM: stage(pre-departure): β = 3.83, s.e.m. = 0.58, CI = 2.69, 4.97; stage(pre-departure) × sex(male): β = 1.99,
s.e.m. = 0.80, CI = 0.43, 3.55).
Figure 2. Levels of oxidative stress markers (A–D) and enzyme activities (E,F) in Hudsonian godwits at
dierent pre-migration stages. Data are means ± s.e.m. Dierent letters indicate dierences between stages
based on 95% CIs. Note dierent scales on the y axes.
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Metabolic rate measurements. BMR was higher in pre-departure godwits (β = 1.54, s.e.m. = 0.70,
CI = 0.07, 3.00) than in fuelling godwits; stage had a relative importance of 65%. Overall body mass had a posi-
tive, signicant eect on BMR (β = 0.03, s.e.m. = 0.01, CI = 0.002, 0.050, with a relative importance of 82%). Yet
the interaction ‘body mass × sex’ (β = 0.05, s.e.m. = 0.02, CI = 0.09, 0.02) indicated that body mass had
contrasting eects on BMR in the two sexes: BMR was positively correlated with body mass in females but the
trend was negative in males. When using linear regression analysis between BMR and body mass, irrespective of
sex and stage, values of BMR and body mass were signicantly and positively correlated (F1,17 = 6.59, P = 0.021).
erefore, we used mass-independent BMR (residuals from its regression against body mass) to test for poten-
tial relationships between BMR and enzyme activities. Mass-independent BMR correlated positively with COX
activity (F1,16 = 7.46, P = 0.015; Fig.5), but not with CS activity (F1,11 = 0.81, P = 0.39). Neither whole-animal
nor mass-independent BMR correlated with any markers of oxidative status (always P > 0.05). RQ did not dier
between fuelling (0.74 ± 0.03) and pre-departure (0.76 ± 0.04) godwits (t1,17 = 1.34, P = 0.201), and did not corre-
late signicantly with fat stores, lipid metabolites, and oxidative damage (always P > 0.05).
We showed that Hudsonian godwits prepare for extreme non-stop ights on their ‘wintering’ grounds by simul-
taneously increase antioxidant capacity and reduce oxidative damage, despite the intense fuelling and high met-
abolic rates accompanying the pre-migration period. If considering the context of birds on stopover8, it is no
surprise that godwits increase antioxidants as they build fat stores. Yet one might also expect them to experience
oxidative damage during fuelling15,16. Instead, godwits decreased oxidative damage before take-o, which resulted
in a more positive eect on their oxidative status. is likely represents an adaptation to diminish the oxidative
costs during periods of ight and refuelling17. Moreover, these results support the notion that long-lived species
are more resistant to oxidative damage than shortlived ones14. Godwits are likely to prioritize self-maintenance,
Figure 3. Relationships between (A) TBARS and COX; (B) TBARS and CS; (C) H2O2 and COX; (D) H2O2 and
CS; (E) TAC and COX; and (F) TAC and CS in Hudsonian godwits during the wintering period. Lines represent
linear regressions.
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Figure 4. Levels of plasma metabolites in Hudsonian godwits at dierent pre-migration stages. Data are
means ± s.e.m. Dierent letters indicate dierences between stages based on 95% CIs. Note dierent scales on y
Figure 5. Relationships between mass-independent BMR and COX activity (fuelling and pre-departure data
pooled). Line represents linear regression.
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SCIENTIFIC REPORTS | (2019) 9:17616 |
as shown by their increased antioxidant capacity, and this may underlie the pronounced longevity recorded in this
and other long-distance migratory shorebirds3,25.
e positive trend in antioxidant capacity with time likely reects the abundance of antioxidant-rich food
resources on Chiloé. A recent study indicates that shorebird populations encounter a predictable and abundant
food supply on the intertidal mudats of Chiloé45. Hudsonian godwits mainly feed on polychaetes46,47, which
could act as a source of antioxidants48. Importantly, dietary antioxidants can supplement or replace the action
of endogenous components of the antioxidant system49. us, an increase in the intake of antioxidant-rich food
during the pre-migratory period26 — irrespective of whether birds preferentially select food rich in antioxidants
or antioxidant resources come from a variety of food items available in their natural habitat — is likely to have
reduced oxidative damage in pre-departure godwits. As pointed out by Beaulieu & Schaefer50, migratory birds
may use specic antioxidants in anticipation of impending need. is strategy could be important for godwits as
they may be unable to ameliorate any damage during non-stop ights. Furthermore, the risk of excessive ight
muscle catabolism increases with the duration of non-stop ights11, a situation regularly faced by godwits en
route19,20. us, godwits must heavily invest in antioxidant defences against the consequences of ight muscle
catabolism and resulting oxidative damage.
Additionally, plasma concentrations of three key metabolites varied among stages. e elevation in plasma
β-hydroxybutyrate (a ketone body synthesized from free fatty acids) prior to endurance ight may replace part of
the glucose during short-term fasting22,51. Previous work with Bar-tailed godwits (Limosa lapponica) subjected to
inactive fasting during stopover showed a trend to increase plasma butyrate22, suggesting that it supports general
energetic requirements rather than the specic energetic demands of ight. It is therefore possible that godwits
subjected to short-term, tide-enforced fasting increased plasma β-hydroxybutyrate temporarily. However, the
time elapsed from capture and high tide peak (roosting) was similar across stages, and thus unlikely to have
caused seasonal dierences in levels of β-hydroxybutyrate and other metabolites. An alternative explanation
is that godwits readying for departure had started mobilizing lipids. is is likely as pre-departure godwits had
reached peak fat scores and the numbers of godwits on Chiloé dropped in mid-March (when they were cap-
tured). Elevated levels of β-hydroxybutyrate reduce glucose utilization and play an important role in the sparing
of carbohydrate and protein52. Consequently, the plasma β-hydroxybutyrate levels of pre-departure godwits were
increased compared with wintering and fuelling godwits. is is also consistent with the nding that elevated
β-hydroxybutyrate in semipalmated sandpipers (Calidris pusilla) at stopover areas reects slower weight gain
prior to non-stop ight to the breeding grounds23. In contrast, glycerol concentrations were lower in birds prepar-
ing for migration, compared to those of overwintering ones. Glycerol is produced by lipolysis of triglycerides in
adipose tissue and muscle during periods of negative energy balance and exercise53. Fuelling and pre-departure
godwits were likely in positive energy balance, albeit the higher metabolic rates before ight. us, glycerol levels
may have decreased due to high rates of fat deposition. is is consistent with a study with captive western sand-
pipers (Calidris mauri) showing that plasma glycerol was negatively related to mass gain54.
e signicant increase in uric acid levels at the pre-departure stage likely indicates that birds increased their
intake of dietary protein prior to endurance ight, given that uric acid is the nal product of protein catabo-
lism in birds and correlates closely with protein consumption55. Nonetheless, higher uric acid concentrations
in pre-departure godwits could have also resulted from the breakdown of proteins that originate from body
tissue. is seems to be the case for other birds that change body composition while preparing for a migratory
ight20,56,57, suggesting a common pattern in allocation of lean body mass prior to endurance ight. Notably,
bar-tailed godwits preparing to depart exhibit a reduction in size of digestive tissues, possibly to y with a total
mass as low as possible19,20. Overall, changes in plasma β-hydroxybutyrate and uric acid concentrations sug-
gest that during the premigratory period energy expenditure entails catabolism of lipids and proteins rather
than a shi in fuel substrate. Indeed, the similar RQ in fuelling and pre-departure individuals indicates that
they used predominantly, but not solely, fat as fuel. Unfortunately we did not measured BMR in overwinter-
ing birds, so we do not know whether they increased their BMR throughout the entire pre-migratory period.
However, pre-departure godwits had higher BMR than fuelling godwits, probably reecting changes in the size
or metabolic activity of dierent organs and tissues associated with migratory disposition18,58. Interestingly BMR
increased with mass in females, but not in males. Sexual dimorphism in BMR has been reported for several bird
species59,60. Such sex dierences in the relationships between BMR and body mass may result from between-sex
dierences in metabolically inactive tissues such as fat (males had larger fat scores than females at pre-departure)
and/or dierences in the amount of energy required for energetically demanding tasks, such as migration and
Although the relationship between the rate of oxygen consumption and the generation of ROS is currently
unresolved6163, some studies have found support for a functional relationship between mass-specic metabolic
rates and oxidative status31,64. For example, Sabat et al.31 showed that increased salt intake elicited changes in the
BMR of Rufous-collared sparrows (Zonotrichia capensis), which were in turn coupled with an increase in the
activity of mitochondrial enzymes and changes in oxidative status. Although we did not nd a signicant rela-
tionship between mass-independent BMR and oxidative status, we found that mass-independent BMR correlated
positively with COX activity in mitochondria from red blood cells, suggesting that the rate of oxygen consump-
tion is to some extent driven by tissue-specic metabolic demands. is supports the notion that avian eryth-
rocytes possess functional mitochondria in terms of respiratory activities and ROS production65,66. Moreover,
mitochondrial parameters (such as mitochondrial O2 consumption, the capacity of the electron transport system
and the capacity to produce ATP via oxidative phosphorylation) measured in red blood cells correlated to those
measured in the pectoral muscle in free-ranging king penguins (Aptenodytes patagonica)34. Hence, measures
of mitochondrial function in red blood cells may reect what is happening in other tissues (e.g., pectoral mus-
cle), and thus provide general information at the organismal level34. is warrants further investigation since
blood enzyme activities could represent informative non-invasive markers for monitoring the rate of energy
Content courtesy of Springer Nature, terms of use apply. Rights reserved
SCIENTIFIC REPORTS | (2019) 9:17616 |
expenditure in free-ranging birds, only requiring a small volume (~30 μl) of blood. In addition, we found that
TBARS increased, yet TAC decreased, with COX activity during the wintering period. is nding indicates that
greater enzyme activity can result in greater oxidative damage outside the migration period. Conversely, we found
the opposite pattern for CS. e reason of this contradictory result is puzzling. A recent study67 found CS activity
in blood cells of chicks of yellow-legged gull (Larus michahellis) was negatively correlated with mtDNA damage,
indicating a negative relationship between oxidative damage and mitochondrial activity. Nevertheless, biochem-
ical variables and oxidative damage in gull chicks were also correlated with growth rate. us, we cannot rule out
that the negative association between CS and oxidative damage found in overwintering godwits was caused by
other physiological processes (such as tissue growth and repair) occurring in parallel. It is important to note that
blood enzyme activities did not increased prior to endurance ight. Along this line, adult western sandpipers
ready to migrate did not alter ight muscle catabolic enzymes (including CS activity) from earlier in the winter,
suggesting that modulation of several physiological characteristics occur aer migratory movements began68.
Likewise, the levels of CS in semipalmated sandpipers preparing to migrate non-stop from eastern Canada to
wintering sites on the northwest coast of South America in late summer do not change before their transoceanic
ight, suggesting that changes in body composition can occur without an increase in mitochondrial enzymes69.
Together, these ndings imply that before spring migration, dierent shorebirds may be unable to adjust their
metabolic enzyme activity in anticipation of future demands68.
Our ndings provide an exciting opportunity for future work to test the hypothesis that high levels of ROS
will drive potential long-term costs of oxidative stress in animals responding the challenges of a demanding
migration, which must be scheduled optimally around other activities such as reproduction, moult, and territory
acquisition3. To better understand the oxidative costs and defences, one important challenge in the future will be
to determine how oxidative status may aect timing of migration and thus reproduction. Further studies should
also examine how this physiological parameter is balanced between parents and ospring70. In this context, a
longitudinal approach examining the oxidative status of organisms throughout the dierent stages of their annual
cycle will be valuable. In the face of rapid and extensive human-induced environmental change, understanding
how migratory animals accrue adequate energy stores and regulate the levels of pro-oxidants and antioxidants
have far-reaching implications for evolutionary and population ecology, as well as for animal conservation and
related policy decisions.
In conclusion, our study shows that trans-hemispheric migrant birds can increase circulating antioxidant
capacity and reduce oxidative damage in anticipation of extreme non-stop ights. Our results indicate that god-
wits do not experience greater levels of oxidative stress during intense fuelling. ey also suggest that alleviation
of the accumulation of oxidative damage seems to start early in the pre-migration season, which may be a selec-
tive advantage for individuals that have higher rates of aerobic respiration. While extreme non-stop migration
can represent an oxidative challenge for animals, protective mechanisms like increasing antioxidant defences and
decreasing oxidative damage seem to occur in Hudsonian godwits. is might represent an adaptation to dimin-
ish the physiological costs of endurance migration.
Received: 3 August 2019; Accepted: 8 November 2019;
Published: xx xx xxxx
1. Hedenström, A. Extreme endurance migration: what is the limit to non-stop ight? PLoS Biol. 8, e1000362 (2010).
2. Piersma, T. Why marathon migrants get away with high metabolic ceilings: towards an ecology of physiological restraint. J. Exp. Biol.
214, 295–302 (2011).
3. Conlin, J. ., Senner, N. ., Battley, P. F. & Piersma, T. Extreme migration and the individual quality spectrum. J. Avian Biol. 48,
19–36 (2017).
4. Costantini, D. Oxidative stress and hormesis in evolutionary ecology and physiology. Oxidative Stress and Hormesis in Evolutionary
Ecology and Physiology, (2014).
5. Finel, T. & Holbroo, N. J. Oxidants, oxidative stress and the biology of ageing. Nature 408, 239–247 (2000).
6. Costantini, D. Oxidative stress in ecology and evolution: Lessons from avian studies. Ecol. Lett. 11, 1238–1251 (2008).
7. Buttemer, W. A., Abele, D. & Costantini, D. From bivalves to birds: Oxidative stress and longevity. Funct. Ecol. 24, 971–983 (2010).
8. Srip, M. M. & McWilliams, S. . Oxidative balance in birds: An atoms-to-organisms-to-ecology primer for ornithologists. J. F.
Ornithol. 87, 1–20 (2016).
9. Costantini, D., Cardinale, M. & Carere, C. Oxidative damage and anti-oxidant capacity in two migratory bird species at a stop-over
site. Comp. Biochem. Physiol. - C Toxicol. Pharmacol. 144, 363–371 (2007).
10. Eienaar, C., Hegemann, A., Pacmor, F., leudgen, I. & Isasson, C. Not just fuel: energy stores are correlated with immune
function and oxidative damage in a long-distance migrant. Cu rr. Z ool ., (2019).
11. Jenni-Eiermann, S., Jenni, L., Smith, S. & Costantini, D. Oxidative stress in endurance ight: An unconsidered factor in bird
migration. PLoS One 9, 1–6 (2014).
12. Srip, M. M. et al. Migrating songbirds on stopover prepare for, and recover from, oxidative challenges posed by long-distance ight.
Ecol. Evol. 5, 3198–3209 (2015).
13. Alonso-Alvarez, C. et al. Increased susceptibility to oxidative stress as a proximate cost of reproduction. Ecol. Lett. 7, 363–368
14. Vagasi, C. et al. Longevity and life history coevolve with oxidative stress in birds. Funct. Ecol. 33, 152–161 (2019).
15. Costantini, D., Metcalfe, N. B. & Monaghan, P. Ecological processes in a hormetic framewor. Ecol. Lett. 13, 1435–1447 (2010).
16. Ei enaar, C., Jönsson, J., Fritzsch, A., Wang, H. L. & Isasson, C. Migratory refueling aects non-enzymatic antioxidant capacity, but
does not increase lipid peroxidation. Physiol. Behav. 158, 26–32 (2016).
17. Eienaar, C., ällstig, E., Andersson, M. N., Herrera-Dueñas, A. & Isasson, C. Oxidative challenges of avian migration: A
comparative eld study on a partial migrant. Physiol. Biochem. Zool. 90, 223–229 (2017).
18. Swanson, D. L. Seasonal metabolic variation in birds: functional and mechanistic correlates. Curr. Ornithol. 17, 75–129 (2010).
19. Piersma, T. & Gill, . E. Guts Don’t Fly: Small Digestive Organs in Obese Bar-Tailed Godwits. Auk 115, 196–203 (1998).
20. Landys-Ciannelli, M. M., Piersma, T. & Juema, J. Strategic size changes of internal organs and muscle tissue in the Bar-tailed
Godwit during fat storage on a spring stopover site. Funct. Ecol. 17, 151–159 (2003).
Content courtesy of Springer Nature, terms of use apply. Rights reserved
SCIENTIFIC REPORTS | (2019) 9:17616 |
21. Jenni-Eiermann, S., Jenni, L. & Piersma, T. Plasma metabolites reect seasonally changing metabolic processes in a long-distance
migrant shorebird (Calidris canutus). Zoology 105, 239–246 (2002).
22. Landys, M. M. et al. Metabolic prole of long-distance migratory ight and stopover in a shorebird. Proc. R. Soc. B 272, 295–302, (2005).
23. Lyons, J. E., Collazo, J. A. & Guglielmo, C. G. Plasma metabolites and migration physiology of semipalmated sandpipers: refueling
performance at ve latitudes. Oecologia 155, 417–27 (2008).
24. Senner, N. ., Hochacha, W. M., Fox, J. W. & Afanasyev, V. An exception to the rule: Carry-over eects do not accumulate in a
long-distance migratory bird. PLoS One 9 (2014).
25. Waler, B. M., Senner, N. ., Elphic, C. S. & lima, J. Hudsonian Godwit (Limosa haemastica). In e Birds of North America
Online (ed. Poole, A.) (Cornell Lab of Ornithology, 2011).
26. Navedo, J. G. et al. Assessing the eects of human activities on the foraging opportunities of migratory shorebirds in Austral high-
latitude bays. PLoS One 14, 1–16 (2019).
27. Navedo, J. G. et al. Unravelling trophic subsidies of agroecosystems for biodiversity conservation: Food consumption and nutrient
recycling by waterbirds in Mediterranean rice elds. Sci. Total Environ. 511, 288–297 (2015).
28. Meissner, W. A classication scheme for scoring subcutaneous fat depots of shorebirds. J. F. Ornithol. 80, 289–296 (2009).
29. Peig, J. & Green, A. J. New perspectives for estimating body condition from mass/length data: e scaled mass index as an alternative
method. Oikos 118, 1883–1891 (2009).
30. Isasson, C., Sturve, J., Almroth, B. C. & Andersson, S. e impact of urban environment on oxidative damage (TBAS) and
antioxidant systems in lungs and liver of great tits, Parus major. Environ. Res. 109, 46–50 (2009).
31. Sabat, P. et al. Coping with salt water habitats: Metabolic and oxidative responses to salt intae in the rufous-collared sparrow. Front.
Physiol. 8 (2017).
32. Cohen, A., lasing, . & iclefs, . Measuring circulating antioxidants in wild birds. Comp. Biochem. Physiol. - B Biochem. Mol.
Biol. 147, 110–121 (2007).
33. Costantini, D. On the measurement of circulating antioxidant capacity and the nightmare of uric acid. Methods Ecol. Evol. 2,
321–325 (2011).
34. Stier, A. et al. How to measure mitochondrial function in birds using red blood cells: A case study in the ing penguin and
perspectives in ecology and evolution. Methods Ecol. Evol. 8, 1172–1182 (2017).
35. Peña-Villalobos, I., Nuñez-Villegas, M., Bozinovic, F. & Sabat, P. Metabolic enzymes in seasonally acclimatized and cold acclimated
rufous-collared sparrow inhabiting a Chilean Mediterranean environment. Curr. Zool. 60, 338–350 (2014).
36. Swanson, D., Zhang, Y. & ing, M. Mechanistic drivers of exibility in summit metabolic rates of small birds. PLoS One 9 (2014).
37. Sabat, P. et al. e interplay between ambient temperature and salt intae aects oxidative status and immune responses in a
ubiquitous Neotropical passerine, the rufous-collared sparrow. Comp. Biochem. Physiol. -Part A Mol. Integr. Physiol. 234, 50–59
38. Bradford, M. A rapid and sensitive assay of protein utilizing the principle of bye dying. Anal. Biochem. 28–264 (1976).
39. Jenni-Eiermann, S. & Jenni, L. Plasma metabolite levels predict individual body-mass changes in a small long-distance migrant, the
Garden warbler. Auk 111, 888–899 (1994).
40. Gutiérrez, J. S., Abad-Gómez, J. M., Sánchez-Guzmán, J. M., Navedo, J. G. & Masero, J. A. Avian BM in marine and non-marine
habitats: A test using shorebirds. PLoS One 7 (2012).
41. Lighton, J. . B. Measuring metabolic rates: a manual for scientists. Measuring metabolic ates: a manual for scientists, https://doi.
org/10.1093/acprof:oso/9780195310610.001.0001 (Oxford University Press, 2008).
42. Burnham, . P. & Anderson, D. . Model selection and multi-model inference: a practical information-theoretic approach. (2nd ed.
Springer, 2002).
43. Grueb er, C. E., Naagawa, S., Laws, . J. & Jamieson, I. G. Multimodel inference in ecology and evolution: Challenges and solutions.
J. Evol. Biol. 24, 699–711 (2011).
44. Barton, . MuMIn: multi-model inference.  pacage version 1.42.1. https://CAN.age=MuMIn (2018).
45. Micael, J. & Navedo, J. G. Macrobenthic communities at high southern latitudes: Food supply for long-distance migratory
shorebirds. Austral Ecol. 43, 955–964 (2018).
46. Martínez-Curci, N. S., Azpiroz, A. B., Isacch, J. P. & Elías, . Dietary relationships among Nearctic and Neotropical migratory
shorebirds in a ey coastal wetland of South America. Emu 115, 326–334 (2015).
47. S enner, N. . & Coddington, . S. C. Habitat use and foraging ecology of Hudsonian Godwits Limosa haemastica in southern South
America. Wader Study Gr. Bull. 118, 105–108 (2011).
48. Moraes, T. B. et al. Antioxidant properties of the mucus secreted by Laeonereis acuta (Polychaeta, Nereididae): A defense against
environmental pro-oxidants? Comp. Biochem. Physiol. - C Toxicol. Pharmacol. 142, 293–300 (2006).
49. Pamplona, . & Costantini, D. Molecular and structural antioxidant defenses against oxidative stress in animals. Am. J. Physiol. -
Regul. Integr. Comp. Physiol. 301, 843–863 (2011).
50. Beaulieu, M. & Schaefer, H. M. ethining the role of dietary antioxidants through the lens of self-medication. Anim. Behav. 86,
17–24 (2013).
51. Jenni-Eiermann, S. & Jenni, L. Metabolic dierences between the postbreeding, moulting and migratory periods in feeding and
fasting passerine birds. Funct. Ecol. 10, 62–72 (1996).
52. obinson, A. M. & Williamson, D. H. Physiological roles of etone bodies as substrates and signals in mammalian tissues. Physiol.
Rev. 60, 143–87 (1980).
53. Stevens, L. Avian Biochemistry and Molecular Biology. Avian Biochemistry and Molecular Biology,
cbo9780511525773 (Cambridge University Press, 2009).
54. Williams, T. D., Guglielmo, C. G. & Martyniu, C. J. Plasma lipid metabolites provide information on mass change over several days
in captive Western sandpipers. Auk 116, 994–1000 (1999).
55. Alan, . . & McWilliams, S. . Oxidative stress, circulating antioxidants, and dietary preferences in songbirds. Comp. Biochem.
Physiol. - B Biochem. Mol. Biol. 164, 185–193 (2013).
56. Jehl, J. . Cyclical Changes in Body Composition in the Annual Cycle and Migration of the Eared Grebe Podiceps nigricollis. J. Av ia n
Biol., (2007).
57. Piersma, T., Gudmundsson, G. A. & Lilliendahl, . apid changes in the size of dierent functional organ and muscle groups during
refueling in a long-distance migrating shorebird. Physiol. Biochem. Zool. 72, 405–15 (1999).
58. vist, A. & Lindstrom, A. Basal metabolic rate in migratory waders: intra-individual, intraspecic, interspecic and seasonal
variation. Funct. Ecol. 15, 465–473 (2001).
59. Mathot, . J., Martin, ., empenaers, B. & Forstmeier, W. Basal metabolic rate can evolve independently of morphological and
behavioural traits. Heredity (Edinb). 111, 175–181 (2013).
60. Maloney, S. . & Dawson, T. J. Sexual dimorphism in basal metabolism and body temperature of a large bird, the Emu. Condor 95,
1034–1037 (1993).
61. Speaman, J. . & Garratt, M. Oxidative stress as a cost of reproduction: Beyond the simplistic trade-o model. BioEssays 36, 93–106
62. Jimenez, A. G. “e same thing that maes you live can ill you in the end”: Exploring the eects of growth rates and longevity on
cellular metabolic rates and oxidative stress in mammals and birds. Integr. Comp. Biol. 58, 544–558 (2018).
Content courtesy of Springer Nature, terms of use apply. Rights reserved
SCIENTIFIC REPORTS | (2019) 9:17616 |
63. Salin, . et al. Individuals with higher metabolic rates have lower levels of reactive oxygen species in vivo. Biol. Lett. 4–7, https://doi.
org/10.1098/rsbl.2015.0538 (2015).
64. Fletcher, Q. E. et al. Oxidative damage increases with reproductive energy expenditure and is reduced by food-supplementation.
Evolution 67, 1527–1536 (2013).
65. Stier, A. et al. Avian erythrocytes have functional mitochondria, opening novel perspectives for birds as animal models in the study
of ageing. Front. Zool. 10, 33 (2013).
66. Niinmaa, M. Vertebrate Red Blood Cells: Adaptations of Function to Respiratory Requirements. (Springer-Verlag, 1990).
67. Velando, A., Noguera, J. C., da Silva, A. & im, S. Y. edox-regulation and life-history trade-os: scavenging mitochondrial OS
improves growth in a wild bird. Sci. Rep. 9, 1–9 (2019).
68. Guglielmo, C. G., Haunerland, N. H., Hochacha, P. W. & Williams, T. D. Seasonal dynamics of ight muscle fatty acid binding
protein and catabolic enzymes in a migratory shorebird. Am. J. Physiol. Integr. Comp. Physiol. 282, 1405–1413 (2002).
69. Driedzic,  ., Crowe, A. N. D. H. L., Hiclin, W. & Sephton, H. Adaptations in pectoralis muscle, heart mass, and energy metabolism
during premigratory fattening in semipalmated sandpipers (Calidris pusilla). Can. J. Zool. 71, 1608 (1993).
70. Blount, J. D., Vitiainen, E. I. ., Stott, I. & Cant, M. A. Oxidative shielding and the cost of reproduction. Biol. Rev. 91, 483–497
We thank members of the Bird Ecology Lab for assistance in the eld and in the lab and member of CECPAN
for essential logistic support. J.S.G. would like to thank Lucy Hawkes, Phil Battley and Jon Blount for helpful
discussions on the oxidative challenges of migration. José A. Masero, Andrea Soriano-Redondo and two
anonymous reviewers provided helpful comments on earlier versions of this manuscript. This study was
supported by the Universidad Austral de Chile (Grant No. SE-2018-01 to J.S.G.), and FONDECYT grants to
J.G.N. (No. 1161224) and P.S. (No. 1160115). J.S.G. was supported by the Government of Extremadura while
writing (Grant No. TA18001).
Author contributions
J.S.G. and J.G.N. designed the research and collected the samples. J.S.G. and L.E.C. measured metabolic rates. P.S.
designed biochemical analyses. C.C., L.N. and I.P.-V. performed lab analyses. J.S.G. analysed the data and wrote
the manuscript with contributions and revisions by all authors. All authors approved the nal manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary information is available for this paper at
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... Thus, triglyceride levels were calculated by subtracting free glycerol from total triglyceride level [e.g. 29,48]. Plasma β-hydroxybutyrate was determined by colorimetric assay kit (No. 700190, Cayman Chemical); measurement of βhydroxybutyrate provides a reliable index of the level of ketoacidosis (accumulation ketone bodies, mainly from lipid metabolism during fasting). ...
... The different patterns found in this study do not help clarifying the relationship between this metabolite and fuel deposition rate (but see below). Plasma concentrations of uric acid are related to the protein metabolism and may thus increase during fuelling, following the intake of dietary protein for storage as fat, or during fasting, following the catabolism of body protein [32,29,48,56]. While we found no relationship between uric acid values and SMI in dunlins, previous studies have suggested that potential confounding effects of diet Early, mid and late correspond to three capture events (mid-July, early August and mid-August). ...
... Seaman et al. [27] also found higher levels of glycerol in Western sandpipers (Calidris mauri) during northward compared to southward migration, but failed to provide an explanation to such pattern. On the other hand, Gutiérrez et al. [48] recorded the highest uric acid levels in Blacktailed godwits (Limosa limosa) during pre-departure stages, suggesting that these values resulted from the breakdown of proteins when birds change body composition while preparing for a migratory flight. However, this hypothesis is not supported by the lower haemoglobin values found in late-autumn migrants (see below). ...
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Migratory strategies dictate stopover ecology, particularly concerning decisions of when, where and how long to stop, and what to do at stationary periods. In birds, individuals stop primarily to replenish energy stores, although the functions of stopover events vary among and within species, particularly between pre- and post-breeding seasons. Here, we combined plasma metabolite levels and haematological parameters to compare refuelling rates and physiological state within (early, mid, late) and between (spring, autumn) migratory periods, aiming to identify potentially different migratory strategies in a shorebird, the dunlin Calidris alpina , using a key stopover site in Iberia. Plasma triglycerides and β-hydroxybutyrate concentrations did not differ between seasons, and small differences were found in haematological profiles (higher haemoglobin and hematocrit levels in spring). Similar refuelling rates and physiological status suggests a single migratory strategy in spring and autumn. During both seasons, dunlins arrive at the Tagus estuary with medium-to-high fuel loads, indicating they do not engage in prolonged fuelling. This agrees with a skipping migratory strategy, where birds fly short-to-medium distances while fuelling at moderate rates along a network of sites. Although we may expect late spring migrants to experience stronger pressures to optimally schedule migratory events, we found no significant differences in physiological profiles among early, mid and late migrants. Unexpectedly, such differences were found in autumn: early birds showed the highest triglycerides and haemoglobin levels and lowest β-hydroxybutyrate concentrations. These results denote enhanced refuelling rates and blood oxygen-carrying capacity in early autumn migrants, which is typical of jumpers, i.e., birds travelling with larger fuel loads and performing fewer stops. Our study adds substantially to previous knowledge of stopover ecology in migratory shorebirds in the East Atlantic Flyway. Importantly, it indicates that the Tagus estuary is a high-quality stopover site for intermediate fuelling. Yet, understanding non-fuelling stopping functions is needed to ultimately inform conservation planning.
... How much oxidative damage a bird incurs from a flight depends mainly on the duration of increased ROS production, activation/deactivation of uncoupling proteins, and on the bird's antioxidant capacity [61,12,40,22,30]. In the case of oxidative damage to lipids, another factor comes into play, which is the susceptibility of the substrate (i.e. ...
... Migrating birds may be able to do so by upregulation of the antioxidant defences and, in case of oxidative damage to lipids, by utilizing fuel (FAs) relatively unsusceptible to lipid peroxidation. Although field studies support the idea that during migration, birds upregulate their antioxidant defences [36,57,22,30], there seems to be a ceiling to such upregulation; sometimes clearly not far enough to forego all flight-related oxidative damage [12,36,58]. Furthermore, investment of nutrients and energy into antioxidant defence may come at the expense of investment in other vital physiological functions, such as immune defence [23]. ...
During migratory endurance flights, which are energetically very demanding, migrants have to deal with prolonged elevated generation of reactive oxygen species (ROS). To limit the damaging actions that ROS have on lipids and proteins, migrating birds are known to upregulate their antioxidant defence system. However, there may be additional ways to limit oxidative damage incurred from flying. Migratory endurance flights are fuelled mainly with fatty acids (FAs), and the risk of their peroxidation (resulting in oxidative lipid damage) increases with the number of double bonds in a FA, with polyunsaturated FAs (2 or more double bonds, PUFAs) being most peroxidation-prone. By fuelling their flights with relatively few PUFAs, migratory birds could thus limit oxidative lipid damage. Within migratory birds, there is considerable variation in the length of their flights, with nocturnal migrants making lengthier flight bouts, thus more likely to experience lengthier periods of elevated ROS production, than diurnal migrants. However, whether migrants making lengthier flights incur more oxidative lipid damage is unknown. Neither is it known whether flight length and FA composition are associated. Therefore, we determined plasmatic malondialdehyde level, a marker of oxidative lipid damage, and FA composition of three nocturnal and two diurnal migrant species caught at an autumn stopover site. We found little inter-specific variation in malondialdehyde level, indicating that the amount of oxidative lipid damage was comparable across the species. In contrast, the species strongly differed in their plasmatic FA composition. The nocturnal migrants had significantly lower relative PUFA levels than both diurnal migrants, an effect mainly attributable to linoleic acid, an essential (strictly dietary) FA. Consequently, the susceptibility of plasmatic FAs to lipid peroxidation was significantly lower in the nocturnal than diurnal migrants. Because in birds, energy expenditure during flight decreases with the degree of FA unsaturation, we interpret our observation of lower PUFA levels in nocturnal migrants as support for the idea that utilizing PUFA-poor fuel can help migrating birds to curb oxidative lipid damage.
... For example, uric acid was found to be positively related with allantoin (its' oxidative product) in white-crowned sparrows during intensive exercise (Tsahar et al., 2006). High levels of uric acid were observed in godwits prior to migratory flight, which perhaps was caused from the breakdown of proteins that originate from body tissue (Gutiérrez et al., 2019). Here, when the up-regulated antioxidant capacity was reduced, then the uric acid took over toward the end of the reproduction and most of the variation was explained mainly by the feather-clip treatment regardless of the ambient temperature ( Figure 3A). ...
... Non-enzymatic antioxidant capacity decreased for all females during nestling-rearing period (Figure 3B), uric acid increased, but this was more pronounced for the feather-clipped females, both in warm and cold temperature, than the control ( Figure 3A). These results may suggest the upregulation of the antioxidant defense system could neutralize the adverse effects of oxidative stress (Alan and McWilliams, 2013;Gutiérrez et al., 2019) but the mechanism may differ between the ambient temperatures. The association between reproduction and oxidative stress is rather complex and may also act as a constrain. ...
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Animal life requires hard work but the ability to endure such workload appears to be limited. Heat dissipation limit (HDL) hypothesis proposes that the capacity to dissipate the excess of body heat during hard work may limit sustained energy use. Experimental facilitations of heat loss rate via feather-clipping in free-living birds seem to support HDL hypothesis but testing of HDL through laboratory experiments under controlled conditions is not reported. We employed a two-factorial experimental design to test HDL hypothesis by manipulating the capacity to dissipate heat through exposure of captive zebra finches ( Taeniopygia guttata ) to a cold and warm ambient temperature (14°C and 25°C), and through manipulation of the insulating layer of feathers around the brood patch in females (clipped and unclipped). To simulate foraging costs encountered in the wild we induced foraging effort by employing a feeding system that necessitated hovering to access food, which increased energetic costs of reproduction despite ad libitum conditions in captivity. We quantified the outcome of reproductive performance at the level of both parents, females, and offspring. Thermal limitations due to warm temperature appeared at the beginning of reproduction for both parents with lower egg-laying success, smaller clutch size and lower egg mass, compared to the cold. After hatching, females with an enhanced ability to dissipate heat through feather-clipping revealed higher body mass compared to unclipped females, and these clipped females also raised heavier and bigger nestlings. Higher levels for oxidative stress in plasma of females were detected prior to reproduction in warm conditions than in the cold. However, oxidative stress biomarkers of mothers were neither affected by temperature nor by feather-clipping during the reproductive activities. We document upregulation of antioxidant capacity during reproduction that seems to prevent increased levels of oxidative stress possibly due to the cost of female body condition and offspring growth. Our study on reproduction under laboratory-controlled conditions corroborates evidence in line with the HDL hypothesis. The link between temperature-constrained sustained performance and reproductive output in terms of quality and quantity is of particular interest in light of the current climate change, and illustrates the emerging risks to avian populations.
... p0395 Thus, a more consistent theme arising from a variety of species captured during autumnal migration indicate only moderate elevations of baseline corticosterone, suggesting an allostatic load that falls substantially below stressinduced values Level C (Schwabl et al., 1991;Gwinner et al., 1992;Falsone et al., 2009). At such titers, corticosterone may serve to support metabolic processes supplying energy required for endurance flight in terms of proteinmediated transport of fatty acids into flight muscle and high-oxidative capacity (Lundgren and Kiessling, 1985;McFarlan et al., 2009;Price et al., 2010). ...
... The lack of correspondence between plasma fatty acid composition and that of fat stores and cell membranes in the same individuals (as described earlier) may explain why oxidative damage did not change with plasma peroxidation index in this latter study. Hudsonian godwits, Limosa haemastica, preparing for a very long-duration migratory flight increased total antioxidant capacity and reduced oxidative damage (TBARS) but did not change metabolic enzyme activity (Gutiérrez et al., 2019). In sum, migratory birds seem to build some component of their antioxidant capacity concomitantly with fat stores and increased oxidative damage may be an inevitable consequence of increasing (or maintaining more) fat stores, especially if they are composed of mostly PUFA. ...
Migratory birds face a host of physiological challenges during their annual peregrinations including (a) maintaining synchrony with the changing environments encountered as they migrate by relying on environmental cues in conjunction with endogenous rhythms to coordinate patterns of movement and stasis; (b) using fats as their primary fuel to satisfy the very high-metabolic costs of continual flapping flight—this requires more oxygen and lipid transporters and energy production, produces more reactive species, produces less metabolic water, and generates more heat. Overcoming these challenges requires flexible (reversble) modifications to physiology including key components of the endocrine, circulatory, respiratory, antioxidant, and thermoregulatory systems, all of which we have described here. A major theme of this chapter is that the life history of a typical migratory bird includes two migration stages—vernal and autumnal—that occur at separate times of the year under differing environmental conditions, that are regulated by unique neuroendocrine mechanisms, and that present somewhat similar yet distinct physiological challenges. Major gaps remain in our understanding of both the network of specific molecules and regulatory relationships that maintain and adjust homeostasis across the life history stages, and especially how the concentrations of key molecules and the relative strengths of certain regulatory relationships change with the context and the conditions of vernal and autumnal migration. More integrative studies (from molecules to genes to physiology to whole organisms) that are also comparative (multiple systems, multiple tissues within the same individual, migration state vs. nonmigration periods, vernal vs. autumnal migration, multiple species that differ in migration strategy) are needed in order to gain a more complete understanding of how the environment influences migration of birds, how birds overcome the multifaceted physiological challenges of migration, and how individuals will cope with the challenges of climate change.
... This protocol using cross-sectional sampling provides the temporal dynamic of the mitochondrial responses to GC secretion and answers the following question: Are oxygen consumption, ATP and ROS productions and the subsequent ratio (ATP/O, ROS/O and ROS/ATP) equivalently impacted by GCs? In addition, the present data bring information about the link between whole organism and cellular metabolisms that is still an open question (Salin et al. 2016;Gutiérrez et al. 2019). ...
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Stress hormones and their impacts on whole organism metabolic rates are usually considered as appropriate proxies for animal energy budget that is the foundation of numerous concepts and models aiming at predicting individual and population responses to environmental stress. However, the dynamics of energy re-allocation under stress make the link between metabolism and corticosterone complex and still unclear. Using ectopic application of corticosterone for 3, 11 and 21 days, we estimated a time effect of stress in a lizard (Zootoca vivipara). We then investigated whole organism metabolism, muscle cellular O2 consumption and liver mitochondrial oxidative phosphorylation processes (O2 consumption and ATP production) and ROS production. The data showed that while skeletal muscle is not impacted, stress regulates the liver mitochondrial functionality in a time-dependent manner with opposing pictures between the different time expositions to corticosterone. While 3 days exposition is characterized by lower ATP synthesis rate and high H2O2 release with no change in the rate of oxygen consumption, the 11 days exposition reduced all three fluxes of about 50%. Oxidative phosphorylation capacities in liver mitochondria of lizard treated with corticosterone for 21 days was similar to the hepatic mitochondrial capacities in lizards that received no corticosterone treatment but with 40% decrease in H2O2 production. This new mitochondrial functioning allows a better capacity to respond to the energetic demands imposed by the environment but do not influence whole organism metabolism. In conclusion, global mitochondrial functioning has to be considered to better understand the proximal causes of the energy budget under stressful periods.
... Lipid peroxidation was assessed by the thiobarbituric acid reactive substances (TBARS) assay using a commercial kit (Oxiselect, STA-330 Cell Biolabs). We calculated an oxidative balance index as the TBARS/TAC ratio to generate a single value that integrates potential variations in the oxidative balance among individuals (Gutiérrez et al., 2019); high index values mean high oxidative stress levels (Costantini et al., 2007). ...
Link for free download: Endogeic earthworms such as Aporrectodea caliginosa play an essential role in the agroecosystems because of their continuous burrowing and feeding (geophagous) activity, which causes a profound impact on soil texture, organic matter decomposition, soil carbon storage, microbial activity, soil biodiversity, and nutrient cycling. Accordingly, endogeic earthworms are being proposed as suitable candidates for the ecotoxicity assessment of polluted soils. However, terrestrial ecotoxicology has little considered the interactive effects from pollutants and environmental variables (temperature, moisture). We acclimatized A. caliginosa for 90 days to two contrasting soil temperatures (10 °C and 20 °C) and moistures (25 % and 35 %, w/v) in 20 mg kg−1 of chlorpyrifos to examine how these two climate change drivers may modulate the pesticide toxicity. We measured the inhibition of cholinesterase (ChE) activities as indicators of organophosphorus exposure, the standard metabolic rate as an integrative physiological biomarker, and the lipid peroxidation (TBARS), and the total antioxidant capacity (TAC) both as indicators of oxidative stress. The main results were: i) chlorpyrifos strongly inhibited ChE activity (>75 % of controls), demonstrating earthworm bioavailability and acute toxicity at the test concentration; 2) a 50 % mortality and loss of body weight (49 %) were found in the earthworms exposed to the most severe environmental conditions (20 °C, 25 %, and pesticide); 3) this latter experimental group displayed a high SMR, which was concomitant with an increase of the oxidative balance index (TBARS/TAC). We postulated that earthworms acclimatized to stressing environmental conditions experienced a higher pesticide-induced metabolic cost and physiological challenges imposed by adverse environmental conditions.
... Evidence from the animal kingdom suggests that endurance flights evoke crucial threats for the antioxidant arsenal of cells. Several markers of oxidative damage, including protein carbonyls (PCs), malondialdehyde (MDA) and proxies of enzymatic antioxidant capacity including glutathione peroxidase (GPx), appear to be affected during bird migration flights [10][11][12][13]. ...
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At high altitude conditions, the low-pressure atmospheric oxygen reduces the generation of energy, thus inducing a decrease in oxygen availability. As a result, endurance flights evoke imbalance in redox signaling, posing a safety risk for the pilots involved. The aim of the present study was to assess changes in the redox status of military pilots during flight simulation conditions according to their flight hours (experts vs. novice). A total of seven expert pilots and an equal number of novice pilots (trainees) were recruited from the Center for Airforce Medicine of the Greek Military Airforce. Glutathione (GSH) levels, catalase activity (CAT), total antioxidant capacity (TAC), lipid peroxidation through the thiobarbituric acid-reactive substances (TBARS), and protein oxidative damage through the assay of protein carbonyls (PCs) levels were assessed at two time points, once prior to and once immediately post a scheduled flight simulation. In the experienced pilots’ arms, GSH was significantly increased post-flight simulation, with TAC being simultaneously reduced. On the other hand, in the trainees’ arms, CAT and TAC were both increased post-flight. No differences were noted with regard to the TBARS and PCs post-simulation. When the two groups were compared, TAC and PCs were significantly lower in the trainees compared to the experienced pilots. The present study provides useful insight into the physiological redox status adaptations to hypobaric hypoxic flight conditions among pilots. In a further detail, an increase in GSH response post-flight simulation is being evoked in more experienced pilots, indicating an adaptation to the extreme flight conditions, as they battle oxidative stress.
... Ducks with CORT implants had lower levels of serum 3-OHB in the presence of elevated glucose, implying that 3-OHB was not acting as an alternative carbon source of energy during the active period of the implant. Multiple studies have reported noticeable rises in plasma 3-OHB in long-distance migrant birds [48][49][50]. ...
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Human-induced environmental changes that act as long-term stressors pose significant impacts on wildlife health. Energy required for maintenance or other functions may be re-routed towards coping with stressors, ultimately resulting in fluctuations in metabolite levels associated with energy metabolism. While metabolomics approaches are used increasingly to study environmental stressors, its use in studying stress in birds is in its infancy. We implanted captive lesser scaup (Aythya affinis) with either a biodegradable corticosterone (CORT) pellet to mimic the effects of a prolonged stressor or a placebo pellet. 1D 1H nuclear magnetic resonance (NMR) spectroscopy was performed on serum samples collected over 20 days after implant surgery. We hypothesized that CORT pellet-induced physiological stress would alter energy metabolism and result in distinct metabolite profiles in ducks compared with placebo (control). Quantitative targeted metabolite analysis revealed that metabolites related to energy metabolism: glucose, formate, lactate, glutamine, 3-hydroxybutyrate, ethanolamine, indole-3- acetate, and threonine differentiated ducks with higher circulatory CORT from controls on day 2. These metabolites function as substrates or intermediates in metabolic pathways related to energy production affected by elevated serum CORT. The use of metabolomics shows promise as a novel tool to identify and characterize physiological responses to stressors in wild birds.
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Ecologically-relevant factors such as exercise and diet quality can directly influence how physiological systems work including those involved in maintaining oxidative balance; however, to our knowledge, no studies to date have focused on how such factors directly affect expression of key components of the endogenous antioxidant system (i.e., transcription factors, select antioxidant genes, and corresponding antioxidant enzymes) in several metabolically active tissues of a migratory songbird. We conducted a 3-factor experiment that tested the following hypotheses: (H1) Daily flying over several weeks increases the expression of transcription factors NRF2 and PPARs as well as endogenous antioxidant genes (i.e., CAT, SOD1, SOD2, GPX1, GPX4), and upregulates endogenous antioxidant enzyme activities (i.e., CAT, SOD, GPx). (H2) Songbirds fed diets composed of more 18:2n-6 PUFA are more susceptible to oxidative damage and thus upregulate their endogenous antioxidant system compared to when fed diets with less PUFA. (H3) Songbirds fed dietary anthocyanins gain additional antioxidant protection and thus upregulate their endogenous antioxidant system less compared to songbirds not fed anthocyanins. Flight training increased the expression of 3 of the 6 antioxidant genes and transcription factors measured in the liver, consistent with H1, but for only one gene (SOD2) in the pectoralis. Dietary fat quality had no effect on antioxidant pathways (H2) whereas dietary anthocyanins increased the expression of select antioxidant enzymes in the pectoralis, but not in the liver (H3). These tissue-specific differences in response to flying and dietary antioxidants are likely explained by functional differences between tissues as well as fundamental differences in their turnover rates. The consumption of dietary antioxidants along with regular flying enables birds during migration to stimulate the expression of genes involved in antioxidant protection likely through increasing the transcriptional activity of NRF2 and PPARs, and thereby demonstrates for the first time that these relevant ecological factors affect the regulation of key antioxidant pathways in wild birds. What remains to be demonstrated is how the extent of these ecological factors (i.e., intensity or duration of flight, amounts of dietary antioxidants) influences the regulation of these antioxidant pathways and thus oxidative balance.
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[This corrects the article DOI: 10.1093/cz/zoz009.][This corrects the article DOI: 10.1093/cz/zoz009.].
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Human presence at intertidal areas could impact coastal biodiversity, including migratory waterbird species and the ecosystem services they provide. Assessing this impact is therefore essential to develop management measures compatible with migratory processes and associated biodiversity. Here, we assess the effects of human presence on the foraging opportunities of Hudsonian godwits (Limosa haemastica, a trans-hemispheric migratory shorebird) during their non-breeding season on Chiloé Island, southern Chile. We compared bird density and time spent foraging in two similar bays with contrasting disturbance levels: human presence (mostly seaweed harvesters accompanied by dogs) was on average 0.9±0.4 people per 10 ha in the disturbed bay, whereas it was negligible (95% days absent) in the non-disturbed bay. Although overall abundances were similar between bays, godwit density was higher in the non-disturbed bay throughout the low tide period. Both days after the start of the non-breeding season and tidal height significantly affected godwit density, with different effects in either bay. Time spent foraging was significantly higher in the non-disturbed bay (86.5±1.1%) than in the disturbed one (81.3±1.4%). As expected, godwit density significantly decreased with the number of people and accompanying dogs in the disturbed bay. Our results indicate that even a low density of people and dogs can significantly reduce the foraging opportunities of shorebirds. These constraints, coupled with additional flushing costs, may negatively affect godwits’ pre-migratory fattening. Hence, as a first step we suggest limiting human presence within bays on Chiloé to 1 person per 10 ha and banning the presence of accompanying dogs in sensitive conservation areas.
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In many animals, catabolic and anabolic periods are temporally separated. Migratory birds alternate energy expenditure during flight with energy accumulation during stopover. The size of the energy stores at stopover affects the decision to resume migration and thus the temporal organization of migration. We now provide data suggesting that it is not only the size of the energy stores per se that may influence migration scheduling, but also the physiological consequences of flying. In two subspecies of the northern wheatear Oenanthe oenanthe, a long-distance migrant, estimated energy stores at a stopover during autumn migration were positively related with both constitutive innate and acquired immune function, and negatively related with oxidative damage to lipids. In other words, migrants’ physiological condition was associated with their energetic condition. Although time spent at stopover before sampling may have contributed to this relationship, our results suggest that migrants have to trade-off the depletion of energy stores during flight with incurring physiological costs. This will affect migrants’ decisions when to start and when to terminate a migratory flight. The physiological costs associated with the depletion of energy stores may also help explaining why migrants often arrive at and depart from stopover sites with more energy stores than expected. We propose that studies on the role of energy stores as drivers of the temporal organization of (avian) migration need to consider physiological condition, such as immunological and oxidative states.
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It has been proposed that animals usually restrain their growth because fast growth leads to an increased production of mitochondrial reactive oxygen species (mtROS), which can damage mitochondrial DNA and promote mitochondrial dysfunction. Here, we explicitly test whether this occurs in a wild bird by supplementing chicks with a mitochondria-targeted ROS scavenger, mitoubiquinone (mitoQ), and examining growth rates and mtDNA damage. In the yellow-legged gull Larus michahellis, mitoQ supplementation increased the early growth rate of chicks but did not reduce mtDNA damage. The level of mtDNA damage was negatively correlated with chick mass, but this relationship was not affected by the mitoQ treatment. We also found that chick growth was positively correlated with both mtDNA copy number and the mitochondrial enzymatic activity of citrate synthase, suggesting a link between mitochondrial content and growth. Additionally, we found that MitoQ supplementation increased mitochondrial content (in males), altered the relationship between mtDNA copy number and damage, and downregulated some transcriptional pathways related to cell rejuvenation, suggesting that scavenging mtROS during development enhanced growth rates but at the expense of cellular turnover. Our study confirms the central role of mitochondria modulating life-history trade-offs during development by other mechanisms than mtROS-inflicted damage.
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Summary 1. The mechanisms that underpin the evolution of ageing and life histories remain elusive. Oxidative stress, which results in accumulated cellular damages, is one of the mechanisms suggested to play a role. 2. In this paper we set out to test the ‘oxidative stress theory of ageing’ and the ‘oxidative stress hypothesis of life histories’ using a comprehensive phylogenetic comparison based on an unprecedented dataset of oxidative physiology in 88 free-living bird species. 3. We show for the first time that bird species with longer lifespan have higher non-enzymatic antioxidant capacity and suffer less oxidative damage to their lipids. We also found that bird species featuring a faster pace-of-life either feature lower non-enzymatic antioxidant capacity or are exposed to higher levels of oxidative damage, while adult annual mortality does not relate to oxidative state. 4. These results reinforce the role of oxidative stress in the evolution of lifespan and also corroborate the role of oxidative state in the evolution of life histories among free-living birds.
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All aerobic organisms are subjected to metabolic by-products known as reactive species (RS).RS can wreak havoc on macromolecules by structurally altering proteins and inducing mutations in DNA, among other deleterious effects. . To combat accumulating damage, organisms have an antioxidant system to sequester RS before they cause cellular damage. The balance between RS production, antioxidant defences, and accumulated cellular damage is termed oxidative stress. Physiological ecologists, gerontologists and metabolic biochemists have turned their attention to whether oxidative stress is the principal, generalized mechanism that mediates and limits longevity, growth rates and other life-history trade-offs in animals, as may be the case in mammals and birds. At the crux of this theory lies the regulation and activities of the mitochondria with respect to the organism and its metabolic rate. At the whole-animal level, evolutionary theory suggests that developmental trajectories and growth rates can shape the onset and rate of aging. Mitochondrial function is important for aging since it is the main source of energy in cells, and the main source of RS. Altering oxidative stress levels, either increases in oxidative damage or reduction in antioxidants, has proven to also decrease growth rates, which implies that oxidative stress is a cost of, as well as a constraint on, growth. Yet, in nature, many animals exhibit fast growth rates that lead to higher loads of oxidative stress, which are often linked to shorter lifespans. In this paper, I summarize the latest findings on whole-animal life history trade-offs, such as growth rates and longevity, and how these can be affected by mitochondrial cellular metabolism, and oxidative stress.
Physiological traits associated with maintenance, growth, and reproduction demand a large amount of energy and thus directly influence an animal's energy budget, which is also regulated by environmental conditions. In this study, we evaluated the interplay between ambient temperature and salinity of drinking water on energy budgets and physiological responses in adult Rufous-collared sparrow (Zonotrichia capensis), an omnivorous passerine that is ubiquitous in Chile and inhabits a wide range of environments. We acclimated birds to 30 days at two ambient temperatures (27 °C and 17 °C) and drinking water salinity (200 mM NaCl and fresh water) conditions. We evaluated: 1) the aerobic scope and the activities of mitochondrial metabolic enzymes, 2) osmoregulatory parameters, 3) the skin-swelling immune response to an antigen, 4) oxidative status, and 5) the length of telomeres of red blood cells. Our results confirm that Z. capensis tolerates the chronic consumption of moderate levels of salt, maintaining body mass but increasing their basal metabolic rates consistent with expected osmoregulatory costs. Additionally, the factorial aerobic scope was higher in birds acclimated to fresh (tap) water at both 17° and 27 °C. Drinking water salinity and low ambient temperatures negatively impacted inflammatory response, and we observed an increase in lipid peroxidation and high levels of circulating antioxidants at low temperatures. Finally, telomere length was not affected by osmo- and thermoregulatory stress. Our results did not support the existence of an interplay between environmental temperature and drinking water salinity on most physiological and biochemical traits in Z. capensis, but the negative effect of these two factors on the inflammatory immune response suggests the existence of an energetic trade-off between biological functions that act in parallel to control immune function.
Intertidal soft‐bottom assemblages located at high latitudes provide a critical food source for long‐distance migratory animals which link biodiversity across distant areas. On the southern Pacific coasts of South America, however, comprehensive information about macrobenthic assemblages at these habitats is lacking. Here we provide an inter‐annual estimation of food supply at the southern limit of the Pacific temperate zone within a Site of Hemispheric Importance for the conservation of Arctic breeding shorebird populations during the nonbreeding season. Macrobenthic communities on Isla Grande of Chiloé are dominated by Polychaetes, Bivalves and Malacostraca. Average biomass (4.4–9.6 g ash‐free dry weight m⁻²) falls within the values reported for temperate intertidal areas located at around 40°S latitude. While total available biomass within each bay was similar during the three sampling years, the annual contribution of each class varied. The major contributor to zoobenthic biomass (Polychaetes) was indeed one of the main preys for the most abundant shorebird species at Chiloé. Arctic breeding migratory shorebird populations seem thus to encounter a predictable and abundant food supply at high southern latitudes on Isla Grande of Chiloé.