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\begin{document}

\title{Young male blackcaps with blood parasite coinfections cope with
oxidative stress favouring anthocyanin-rich food during migratory
fattening}



\author[1]{Lucía Jiménez-Gallardo}%
\author[1]{Jimena López-Arrabé}%
\author[1]{Javier Pérez-Tris}%
\author[1]{Carolina Remacha}%
\affil[1]{Complutense University of Madrid Faculty of Biological Sciences}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Parasites may alter host physiology, which may promote behavioural
adaptations to counteract their effect. Adaptive feeding may help
individuals to cope with infection, especially during physiologically
highly demanding life stages. For instance, migrating birds need to fuel
long-distance flights and repair oxidative damage caused by intense
aerobic exercise, and parasites may influence on how individuals balance
these needs. Infected birds may face increased oxidative challenges,
which could induce them to favour antioxidant defences over other needs,
such as fattening. We tested whether migrating birds can adaptively
choose food according to their needs, favouring dietary antioxidants to
cope with oxidative stress caused by blood parasites during migration.
During autumn migration, we mist-netted young male European blackcaps
(\emph{Sylvia atricapilla}) stopping over in central Spain. We placed
the birds in cages where they were offered fat and anthocyanin-enriched
food alternatives. We measured preference for each food offer. We tested
their infections by haemosporidian parasites with PCR techniques and
their parasitaemia with blood smear inspection. We also measured
physiological variables that account for nutritional and oxidative
status in red blood cells and plasma. We found that birds with multiple
infections favoured anthocyanin-enriched food controlling for an effect
of body mass on food preference (lean blackcaps preferred anthocyanins,
likely because they are urged to repair oxidative damage upon arrival on
stopover with depleted energy reserves). Infected birds had a lower
antioxidant capacity of plasma, and individuals with more oxidative
damage preferred anthocyanin-enriched food. Our results suggest that
parasite infections may increase individuals' antioxidant needs, which
could affect migration performance if the urge to find dietary
antioxidants reduces fuel consumption rate.%
\end{abstract}%



\sloppy


\textbf{Young male blackcaps with blood parasite coinfections cope with
oxidative stress favouring anthocyanin-rich food during migratory
fattening}

\textbf{Abstract}

Parasites may alter host physiology, which may promote behavioural
adaptations to counteract their effect. Adaptive feeding may help
individuals to cope with infection, especially during physiologically
highly demanding life stages. For instance, migrating birds need to fuel
long-distance flights and repair oxidative damage caused by intense
aerobic exercise, and parasites may influence on how individuals balance
these needs. Infected birds may face increased oxidative challenges,
which could induce them to favour antioxidant defences over other needs,
such as fattening. We tested whether migrating birds can adaptively
choose food according to their needs, favouring dietary antioxidants to
cope with oxidative stress caused by blood parasites during migration.
During autumn migration, we mist-netted young male European blackcaps
(\emph{Sylvia atricapilla} ) stopping over in central Spain. We placed
the birds in cages where they were offered fat and anthocyanin-enriched
food alternatives. We measured preference for each food offer. We tested
their infections by haemosporidian parasites with PCR techniques and
their parasitaemia with blood smear inspection. We also measured
physiological variables that account for nutritional and oxidative
status in red blood cells and plasma. We found that birds with multiple
infections favoured anthocyanin-enriched food controlling for an effect
of body mass on food preference (lean blackcaps preferred anthocyanins,
likely because they are urged to repair oxidative damage upon arrival on
stopover with depleted energy reserves). Infected birds had a lower
antioxidant capacity of plasma, and individuals with more oxidative
damage preferred anthocyanin-enriched food. Our results suggest that
parasite infections may increase individuals' antioxidant needs, which
could affect migration performance if the urge to find dietary
antioxidants reduces fuel consumption rate.

\textbf{Keywords:} adaptive food choice, dietary antioxidants,
ecophysiology, haemosporidian parasites, migration stopover,\emph{Sylvia
atricapilla} .

\textbf{Introduction}

Animals face different physiological and energetic challenges during
their life cycle, which entail trade-offs that usually promote
behavioural adaptations (Ricklefs and Wikelski 2002). One such
adaptation is the food choice depending on nutritional requirements
(Catoni et al. 2011). Both dietary needs and the availability of food
sources necessary to meet them vary among life cycle stages, forcing
individuals to adaptively allocate limited resources among different
activities, such as growth (Romero-Haro and Alonso-Alvarez 2014),
reproduction (Seress et al. 2020), daily activity (Beaulieu and Schaefer
2014) or migration (Jenni-Eiermann 2017). Thus, the idea that 'you are
what you eat' may be expanded because 'you need to be different things
at different times'.

The effects of parasites on the physiology and behaviour of their hosts
have been long recognised (Moore 2002; Poulin 2007), and accounting for
parasites may be key in studies of animal performance (Chr\selectlanguage{ngerman}étien et al.
2023). By depleting host resources and eliciting the activation of the
immune system, parasites may influence on nutrient requirements and
resource allocation of their hosts (Cornet et al. 2014; Nwaogu et al.
2020), which may promote dietary shifts as an adaptive response to cope
with parasite infections. All these effects on the physiology of the
host could be enhanced when they are experiencing a demanding situation,
such as migration (Jenni-Eiermann 2017). One important link between
parasite infection and host feeding behaviour may be oxidative stress
(Isaksson et al. 2013; Delhaye et al. 2016; Muriel 2020). Oxidative
stress results from the imbalance between endogenous or exogenous
antioxidants, which protect animals against oxidative damage to DNA,
proteins, or lipids, and reactive oxygen species (ROS) that cause such
damage (Finkel and Holbrook 2000; Monaghan et al. 2009). When animals
can perceive the nutritional content of food and select the most
appropriate food choices for their oxidative state (Schaefer et al.
2008; Schaefer et al. 2014), self-medication arises as a possible way to
maintain oxidative balance (Beaulieu and Schaefer 2013; de Roode et al.
2013).

Avian haemosporidians are the protozoans that cause malaria and similar
diseases of birds (Valki\selectlanguage{polish}ū\selectlanguage{english}nas 2005; Atkinson et al. 2008). These
parasites have great impact on bird populations by decreasing host
reproductive success and survival (Merino et al. 2000; Asghar et al.
2015). Haemosporidian infections induce oxidative stress through
activation of the humoral immune system (Delhaye et al. 2018; Muriel
2020) and breakage of red blood cells (RBC; Pigeault et al. 2015).
Consequently, parasitised individuals must allocate resources to
immunity (Sheldon and Verhulst 1996; Tschirren and Richner 2006), which
may deplete antioxidant defences that otherwise would help to maintain
oxidative balance (Monaghan et al. 2009). Therefore, favouring dietary
antioxidants may help parasitised individuals maintain oxidative status
by reinforcing antioxidant defences, but this feeding behaviour could
compromise the consumption of other important nutrients (Beaulieu and
Schaefer 2014) or increase the oxidative imbalance if antioxidants
become excessive relative to the amount of ROS (Beaulieu and Schaefer
2013). These trade-offs may be most important during life stages when
maintaining oxidative balance is difficult, such as growth (Hall et al.
2010), reproduction (Guindre-Parker and Rubenstein 2018), or migration
(McWilliams et al. 2021), which may exacerbate the influence of
parasites on animal behaviour and performance (Chr\selectlanguage{ngerman}étien et al. 2023).

The impact of haemosporidan parasites on bird oxidative status may be
particularly strong during migration periods. Bird migration involves
intense aerobic exercise, which increases both energy requirements and
oxidative challenges (McWilliams et al. 2021). Migrating birds put on
fat to fuel long-distance flight (Araújo et al. 2019), but unsaturated
fatty acids used as energy stores are prone to oxidation, and endurance
flight effort might increase ROS production that needs to be compensated
with up-regulation of endogenous antioxidant defences and/or increasing
antioxidant-rich food intake during stopovers (Skrip et al. 2015;
Eikenaar et al. 2020; McWilliams et al. 2021). Therefore, parasite
infections are expected to alter migrating bird feeding decisions,
tipping the balance to favour dietary antioxidants although fattening is
a priority (Mancio-Silva et al. 2017; Zuzarte-Luís and Mota 2018).

We conducted a behavioural assay with European blackcaps \emph{Sylvia
atricapilla} to assess if individuals infected by haemosporidian
parasites differ from their uninfected counterparts in their preference
for major dietary antioxidants or fat when faced with this choice during
migration, and whether oxidative status could mediate the relationships
between parasite infection and individual feeding preferences during
this period of high exposure to oxidative challenges. Blackcaps are a
model of research on migratory behaviour and bird-haemosporidian
relationships, and provide an excellent opportunity for this analysis
because they consume both antioxidant-rich and fat-rich fruits on
migration stopovers and can assess the relative content of these
nutrients based on fruit colour (Schaefer et al. 2008; Schaefer et al.
2014). Blackcaps may accrue both energetic and defensive benefits from
the same fruits; for example, wild or cultivated olives may be the
primary source of fat for blackcaps to restore energy reserves on
Mediterranean stopover sites (Rey et al. 1996; Jordano 2013), but also
contain phenolic compounds, \selectlanguage{greek}α-\selectlanguage{english}tocopherol and \selectlanguage{greek}β-\selectlanguage{english}carotene with antioxidant
properties (Visioli et al. 2002; Catoni, Peters, et al. 2008). However,
dietary antioxidants that combine high antioxidant potency and high
availability in bird-consumed fruits may be particularly important to
tackle with parasite-induced oxidative stress, and flavonoids such as
anthocyanins outstand on both regards (Catoni, Peters, et al. 2008).
Anthocyanin-rich diets are known to boost the immune system (Bakuradze
et al. 2019), and blackcaps that select fruits with high anthocyanin
content enhance their humoral immune response (Catoni, Schaefer, et al.
2008). Therefore, we predict that parasitised blackcaps captured during
migration will favour anthocyanin-enriched over fat-enriched food to
control an increased risk of oxidative damage. If preference for
anthocyanins is a defensive behaviour against oxidative stress, we also
predict that infected birds will have poorer oxidative status measured
through physiological indices of oxidative damage or antioxidant
capacity, and that variation in these physiological indices will mediate
individual food preferences. In addition, the physiological impact of
haemosporidian infections may depend on the proportion of RBC that are
infected (intensity of infection) or on the synergistic effects of
multiple infections, where different parasites coexisting in the blood
increase the cost of the disease (Pigeault et al. 2018; Garcia-Longoria
et al. 2022). Therefore, we predict that the more parasites blackcaps
harbour, the greater will be their preference for anthocyanin-enriched
food.

\textbf{Materials and Methods}

\emph{Study Area and Field Methods}

We mist-netted migrating blackcaps in a stopover site located near Campo
Real, central Spain (40\selectlanguage{ngerman}°20'08''N 3°24'27''W, 780 m a.s.l), during
October 2019 and 2020. The area is dominated by olive groves (\emph{Olea
europaea} ) crossed by a stream where fruiting shrubs abound, especially
daneworts (\emph{Sambucus ebulus} ) and brambles (\emph{Rubus
fruticosus} ). Therefore, blackcaps stopping over in this area during
autumn migration can choose between fat-rich olives and anthocyanin-rich
berries typical of their diet during the autumn-winter period (Herrera
1987; Schaefer et al. 2008; Jordano 2013).

We tape-lured blackcaps into mist nets to maximize capture rate. Birds
were sexed and aged according to plumage, distinguishing between young
(individuals born in the previous spring) and older birds (Svensson
1992). We wanted to minimise sample size for ethical reasons, and thus
focused on young male birds to reduce the number of factors in the
statistical design. Males were chosen instead of females because our
sample was sex-biased (63\% males); therefore, our study is relevant to
the young male fraction of the population. Young blackcaps are ideal for
studying the ecological correlates of food preference during migration
because they are facing first parasite infections, which reduces the
behavioural variation associated with a previous infection history
(Remacha et al. 2023). In addition, they are visiting the stopover site
for the first time in their lives and therefore have the same
experience.

A total of 48 young male blackcaps were included in the study (25
captured in 2019 and 23 in 2020). During the mornings, between 9:00 and
15:30 p.m., the birds were kept in individual cotton bags for 30 minutes
to reduce stress and increase motivation to eat after mild fasting.
After that, we weighed birds to the nearest 0.01 g and placed them in
individual cages (cloth mesh, 25 x 25 x 25 cm). We recorded behaviour
using a video camera placed in front of the cage, covering all other
sides and the top of the cage with filter paper to minimise external
stimuli. The cages were fitted with a perch near the side facing the
camera, with two food choices presented on each side nailed to a
semi-vertical twig.

We designed a meaningful nutrient-preference test presenting birds with
two food choices that were equal in all respects except for the dietary
treatment. We used a piece of melon (about 2 cm\textsuperscript{3}) as
the substrate for both food choices, thereby avoiding variation in size,
shape or other fruit attributes known to influence fruit choice by birds
(González\selectlanguage{english}-Varo et al. 2022). Each piece of melon had been soaked
overnight in extra virgin olive oil (Coosur(r)) or cranberry juice
(Granini(r)), hereafter named fat-enriched or anthocyanin-enriched food,
respectively. Blackcaps consume abundant wild and cultivated olives,
whose principal nutritional reward is energy from fats (Herrera 1987;
Jordano 2013). Olive oil is highly energetic and contains a high
proportion of unsaturated fatty acids that help migrant birds to build
fat reserves very efficiently (Pierce et al. 2005). Importantly, olive
oil is also rich in polyunsaturated fatty acids such as omega-3 and
omega-6 (Zarrouk et al. 2019), which enhance the aerobic capacity of
migrating birds but must be obtained from dietary sources (Price and
Guglielmo 2009; Weber 2009). Conversely, cranberry juice provides little
energy rewards but has a high anthocyanin content (Bakuradze et al.
2019). Migratory birds in autumn migration show preference for fruits
with high anthocyanin content, which has been interpreted as a
protective behaviour against oxidative stress associated with endurance
flight (Bolser et al. 2013) Although olives contain other dietary
antioxidants such as phenolic compounds, \selectlanguage{greek}α-\selectlanguage{english}tocopherol and \selectlanguage{greek}β-\selectlanguage{english}carotene
(Visioli et al. 2002; Catoni, Peters, et al. 2008), compared to these
antioxidant compounds anthocyanins are various orders of magnitude more
available in fruits consumed by blackcaps, show much higher antioxidant
potency, and are known to be important antioxidants in the fight against
haemosporidian parasites (Catoni, Peters, et al. 2008; Akinnusi et al.
2023). Fat- and anthocyanin-enriched melon pieces acquired distinctive
golden and purple-reddish tinges, respectively, which we deemed useful
for blackcaps to assess the nutritional rewards of either choice
(Schaefer et al. 2008; Schaefer et al. 2014). The two food choices
swapped cage location between individuals (Supporting information).

Migrating blackcaps arrive at stopover sites lean, start to gain mass
typically on day 2 post-arrival, and put on fat until reaching maximum
body mass at stopover at departure time (Langslow 1976), increasing
approximately 0.2 g day\textsuperscript{-1} in Iberian locations
(Arizaga et al. 2010). In our sample, the heaviest individual (21.4 g)
was 46.6\% heavier than the lightest (14.6 g). Therefore, we used the
residuals of the linear regression of body mass on tarsus length, both
log transformed (residual mass hereafter) as a measure of body condition
of individuals, which was indicative of both their metabolic efficiency
and the time they had spent on stopover. Once the video-recording
finished, we measured the length of the tarsus with 0.01mm precision. At
the end of the manipulation of the birds, we took a blood sample
(\textless{} 1\% body mass) from the jugular vein, used a drop to make a
blood smear, and kept the rest refrigerated in heparinised tubes during
fieldwork. Birds were fitted with a standard aluminium ring to avoid
repetitions and were released unharmed at the capture site.

\emph{Behavioural analyses}

We visualised the video recordings using the VLC media player (3.0.8
Vetinari). We analysed the feeding behaviours observed between time 0,
when the researcher disappeared from the image after leaving the bird in
the cage, and the first time when the researcher reappeared to retrieve
the bird (time end = 1390 s, set by the shortest recording). Because we
minimised the time birds spent captive for ethical reasons, not all
individuals ate during the test, or pecked the fruit one or a few times.
To overcome this limitation, we considered three feeding-oriented
behaviours that could be unequivocally recognised in our video
recordings: pecking the fruit, pecking attempts (when the bird
approached its beak to the fruit without pecking), and showing interest
(when the bird directed its head towards one fruit and kept eye contact
for more than 2 seconds without attempting to peck). Considering the
proportion of occasions in which individuals directed each
feeding-oriented behaviour to one food offer as indicative of their
inclination for that choice, we found that the propensity to peck, to
attempt pecking or to show interest for the same food choice were all
positively correlated (pairwise Pearson correlations between proportions
of each type of behaviour directed to fat-enriched food as the
reference: pecking - pecking attempts r = 0.87, n = 17; pecking -
showing interest r = 0.95, n = 17; pecking attempts - showing interest r
= 0.88, n = 15; all three correlations with \emph{p} \textless{} 0.001),
meaning that all three feeding-oriented behaviours conveyed the same
information on individual food preferences. All videos were blindly
analysed to the infection status, body mass, or physiological condition
of the individuals.

\emph{Infection status}

We assessed the infection status of blackcaps by combining PCR
techniques and microscopy. We extracted total DNA from blood with the
SpeedTools DNA extraction kit, following the manufacturer's protocol,
and diluted the samples to a final concentration of 25 ng/\selectlanguage{greek}µ\selectlanguage{english}l. We first
tested all samples with a multiplex PCR that simultaneously targets DNA
fragments of different sizes of \emph{Haemoproteus} ,
\emph{Plasmodium,}and \emph{Leucocytozoon} parasites (Ciloglu et al.
2019). Reactions were set in total volumes of 10 \selectlanguage{greek}µ\selectlanguage{english}l, including 5 \selectlanguage{greek}µ\selectlanguage{english}l of
2\selectlanguage{ngerman}× Qiagen Multiplex PCR Master Mix (Qiagen, Hilden, Germany), 0.2 \selectlanguage{greek}µ\selectlanguage{english}l of
each of six primers (10 \selectlanguage{greek}µ\selectlanguage{english}M; PMF/R for \emph{Plasmodium,} HMF/R for
\emph{Haemoproteus} and LMF/R for \emph{Leucocytozoon} ), 1.8 \selectlanguage{greek}µ\selectlanguage{english}l of
ddH\textsubscript{2}O, and 2 \selectlanguage{greek}µ\selectlanguage{english}l of DNA template. The temperature profile
included a hot start at 95 \selectlanguage{ngerman}° C for 15 min, followed by 35 cycles at 94 °
C for 30 s, 59 ° C for 90 s, and 72 ° C for 30 s, and an extension at 72
° C for 10 min. The PCR reactions included a negative control
(ddH\textsubscript{2}O) and a triple positive control (DNA positive for
all three genera of parasites). We visualised 4 \selectlanguage{greek}µ\selectlanguage{english}l of the PCR product on
2\% agarose gels stained with 100\selectlanguage{ngerman}× GelRed\textsuperscript{®}.

To identify the parasite lineages infecting each bird, positive samples
for the previous multiplex PCR were amplified with a nested PCR
(Hellgren et al. 2004), which targets 479 bp of the parasite\emph{Cyt-b}
gene broadly used as a DNA barcode for avian haemosporidians (Bensch et
al. 2009). Nested PCR consisted of a preamplification step that targeted
the three genera (with primers HAEMNFi and HAEMNR3) followed by specific
amplifications targeting either \emph{Plasmodium} and
\emph{Haemoproteus} (with primers HAEMF and HAEMR2) or
\emph{Leucocytozoon} (primers HAEMFL and HAEMR2L). We set
pre-amplification reactions at 25 \selectlanguage{greek}µ\selectlanguage{english}l total volume, including 0.5 units
of AmpliTaq DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA),
1 \selectlanguage{greek}µ\selectlanguage{english}l of each 10 \selectlanguage{greek}µ\selectlanguage{english}M primer, 1.25 mM of each dNTP, 1.5 mM of
MgCl\textsubscript{2}, 2.5 \selectlanguage{greek}µ\selectlanguage{english}l of Buffer 10\selectlanguage{ngerman}×, 14.8 \selectlanguage{greek}µ\selectlanguage{english}l of
ddH\textsubscript{2}O, and 2 \selectlanguage{greek}µ\selectlanguage{english}l of DNA template. The thermal profile
included an initial step at 94 \selectlanguage{ngerman}° C for 3 min; 20 cycles of 94 ° C for 30
s, 50 ° C for 30 s, and 72 ° C for 45 s, and an extension of 10 min at
72 ° C. We used 1 \selectlanguage{greek}µ\selectlanguage{english}l of the product for one or both specific
amplification steps depending on the result of multiplex PCR. Specific
reactions involved 35 cycles with the same temperature profile and PCR
conditions as pre-amplification, but including 15.8 \selectlanguage{greek}µ\selectlanguage{english}l of
ddH\textsubscript{2}O. All reactions included negative and triple
positive controls passed from pre-amplification to the specific
amplification step. We sequenced the final PCR products using an Applied
Biosystems 3730xl DNA analyzer, and considered parasite lineages that
differed in one or more base pairs in the MalAvi DNA barcode as distinct
lineages (Bensch et al. 2009).

We stained blood smears with Giemsa (pH 7.2) for 1 h and inspected them
with a light microscope (LEICA DM2500, Leica Microsystems, Wetzlar,
Germany), with a sampling effort of at least 5000 red blood cells (RBC)
observed at 1000\selectlanguage{ngerman}× magnification. We quantified the intensity of the
infection as infected cells in 5000 RBC. Individuals who were PCR
positive but had no visible parasites were assigned an intensity of
infection of 0 infected cells in 5000 RBC. We used any observation of
the presence of more than one parasite lineage in blood (mixed DNA
sequence signal, multigenus amplification in multiplex PCR, or detection
of different parasites by molecular and microscopy methods) as evidence
of multiparasite infection, hereafter multiple infection (Pérez-Tris and
Bensch 2005; Valki\selectlanguage{polish}ū\selectlanguage{english}nas et al. 2006; Ciloglu et al. 2019). Otherwise,
infected individuals were considered to harbour single-parasite
infections.

\emph{Physiological Analyses}

To assess whether parasites could be causing anaemia, on day of capture
we measured haemoglobin concentration in blood with Drabkin reaction
(Spinreact, Girona, Spain), following the manufacturer's instructions.
We set each sample in duplicates on 96-well plates, including a
calibrator and blank (ddH\textsubscript{2}O). We measured absorbance at
540 nm using a Biotek Epoch microplate spectrophotometer (BioTek
Instruments, Inc.). Repeatability of the assay was high
(\emph{r\textsubscript{i}} = 0.97; \emph{n} = 48; \emph{p} \textless{}
0.001), inter-assay CV was 12.61\% and intra-assay CV was 1.03\%.
Immediately after the analysis, we centrifuged the rest of the blood for
10 min at 12000 rpm to separate plasma from RBC. We visually determined
the degree of hemolysis using a three-level scale based on the redness
of plasma (0: clear sample, 1: pale red stain, 2: red sample). Plasma
and red blood cell (RBC) fractions were stored at -80 \selectlanguage{ngerman}° C until further
analysis.

We measured the protein content of the RBC samples to standardize other
measurements (see below) using the Bradford assay (Sigma-Aldrich, St.
Louis, MO), testing them in duplicate on 96-well plates, following the
manufacturer's instructions. In each plate, we added protein standard
(BSA) ranging from 0.1 to 1.4 mg/ml, blank (ddH2O), or RBC. We measured
absorbance at 595 nm using a Synergy HT MultiMode Microplate199 Reader
(BioTek Instruments, Inc.; this spectrophotometer was used in all
subsequent analyzes). The repeatability of the protein measurements was
high (\emph{r\textsubscript{i}} = 0.73; \emph{n} = 48;
\emph{p}\textless{} 0.001), inter-assay CV between the samples was
17.21\% and the CV intra the samples was 5.26\%.

We determined intracellular total glutathione levels in RBC (tGSH) as an
endogenous antioxidant indicator in RBC following Pérez-Rodríguez et al.
(2015). The samples were diluted (1 : 20 w/v) and homogenised in stock
buffer (0.01 M phosphate buffered saline and 0.02 M EDTA) and mixed with
an equal volume of 10\% trichloroacetic acid. We vortexed the mixture
three times during 5 s in 10 min and then centrifuged it at 2000 g for
10 min at 6 ° C, to separate the supernatant. After that, we added NADPH
and DTNB in the first step and GSH reductase in the second step after 15
s. We used 96-well plates including duplicate samples, a blank (buffer),
and a standard curve (serial dilution of GSH from 0.5 to 0.031 mM). Only
one 12-well row from the plate was used at a time. We measured
absorbance at 405 nm after 15 and 45 s, and the change in absorbance was
used to determine the intracellular tGSH concentration by comparing the
output with the standard curve. Levels were standardised to the total
protein content in the sample by dividing tGSH by protein values. The
repeatability of the standardised tGSH calculated in duplicates was high
(\emph{r\textsubscript{i}} = 0.93; \emph{n} = 48; \emph{p} \textless{}
0.001), inter-assay CV was 12.60\% and the intra-assay CV was 8.17\%.

We used malondialdehyde (MDA) to measure lipid peroxidation as an index
of oxidative damage to cell membranes. The integrity of membrane
phospholipids is key for the metabolic performance of pectoral muscle
during endurance flight (Weber 2009), and parasite invasion may cause
peroxidation of phospholipids of RBC membranes due to the combined
effects of parasite metabolism and host immune responses (Percário et
al. 2012). Therefore, we quantified the concentration of MDA both in
plasma (plasma-MDA, as a measure of general lipid peroxidation faced by
the individual) and RBC (RBC-MDA, a measure of oxidative damage to the
RBC membrane), following Romero-Haro and Alonso-Alvarez (2014). We
included a blank and a standard curve per batch diluted from 1,1,3,3
tetraethoxypropane with 40\% ethanol. We added butylated hydroxytoluene,
phosphoric acid, and thiobarbituric acid (TBA) solutions to each sample
(standard or blank) and incubated them for 1 h in a dry bath at 100 ° C.
MDA-TBA adducts were formed, and pure n-butanol was added, vortexed,
centrifuged, and the upper phase was collected to transfer it into an
HPLC vial. We extracted the samples to inject them in duplicate into an
Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA),
and measured them with a fluorescence detector (ref. G1321A, Agilent
Technologies). Repeatability was high for both plasma-MDA
(\emph{r\textsubscript{i}} = 0.87; \emph{n} = 45; \emph{p} \textless{}
0.001; inter-assay CV = 39.46\% and intraassay CV = 15.41\%) and RBC-MDA
(\emph{r\textsubscript{i}} = 0.76, \emph{n} = 48, \emph{p} \textless{}
0.001; inter-assay CV = 18.30\% and intra-assay CV = 14.45\%). We
standardised the levels of RBC-MDA dividing by the total protein
content.

To obtain a biomarker of the general level of circulating antioxidant
defences of the individual, as vitamin C, vitamin E and carotenoids that
are frequently obtained with the diet, we measured the antioxidant
capacity of plasma quantifying total antioxidant status (TAS) following
Miller et al. (1993) with modifications described in López-Arrabé et al.
(2014). We used an \selectlanguage{greek}α-\selectlanguage{english}tocopherol derivative (Trolox) as a calibrator. The
samples were run in duplicates whenever there was enough plasma (44 out
of 46 samples), using 96-well plates with a calibrator and blank. We ran
a row per time to control the reaction delay. We added metmyoglobin (an
equilibrated mixture of myoglobin and potassium ferricyanate) to each
sample, blank, or control. We also added a chromogen (2,2
'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid or ABTS) and
H\textsubscript{2}O\textsubscript{2} to start the reaction. We recorded
absorbance at 660 nm, each 10 s at 37 \selectlanguage{ngerman}° C. The repeatability calculated
in duplicates was high (\emph{r\textsubscript{i}} = 0.95; \emph{n} =
43;\emph{p} \textless{} 0.001), inter-assay CV was 14.19\% and the
intra-assay CV 5.83\%.

Uric acid is the main form of nitrogen excretion due to catabolism of
amino acids in birds. Furthermore, it is also an endogenous antioxidant
whose concentration is frequently positively related to TAS values
(Cohen et al. 2007; Pérez-Rodríguez et al. 2008), potentially
confounding the interpretation of this biomarker (Cohen et al. 2007;
Costantini 2011). Therefore, we measured uric acid in plasma to correct
TAS values and as a measure of nutritional status, using a commercial
kit (Biosystems, Barcelona, Spain). The test details were implemented
according to the manufacturer's instructions, using 96 well plates that
we incubated at 37 ° C for 10 min and measured absorbance at 520 nm. All
duplicates had high repeatability (\emph{r\textsubscript{i}} =
0.99,\emph{n} = 46, \emph{p} \textless{} 0.001), inter-assay CV was
4.07\% and the intra-assay CV was 3.86\%.

Finally, we measured plasma triglycerides, as an indicator of the
nutritional status of the birds and to standardise MDA measurements if
necessary, using a commercial kit (Biosystems, Barcelona, Spain)
according to the manufacturer's instructions. Triglyceride
concentrations can be an index of lipid absorption a few hours before
sampling and therefore reflect the individual's state of fattening
(Jenni and Jenni-Eiermann 1998). Furthermore, triglyceride levels can be
related to MDA levels, either because MDA is also present in food or
because MDA can also be influenced by the amount of circulating lipids
(Pérez-Rodríguez et al. 2015). We tested 45 out of 48 samples in
duplicate and measured their absorbance at 500 nm. The repeatability of
duplicates was high (\emph{r\textsubscript{i}} = 0.97; \emph{n} =
44;\emph{p} \textless{} 0.001), inter-assay CV was 1.72\% and the
intra-assay CV was 3.98\%.

\emph{Data Analysis}

All analyses were performed using R 4.2.2 (R Core Team 2022). Continuous
variables were checked for normality, transformed when advisable, and
z-standardised for comparison of effect estimates. We checked that
residuals were distributed according to model assumptions with DHARMa R
package (Hartig 2022). When outliers were observed, their influence was
assessed by repeating the analyses with or without outliers. First, we
tested our specific hypothesis, whether there is an effect of infection
on food preference. Then, we analysed the relationships between
infections in each physiological condition variable indicative of
oxidative status. Finally, we examine the effects of physiological
condition as predictors of food choice.

To analyse the effect of infection on food preference, we fitted
generalised linear mixed effects models (GLMM) with binomial error and
logit link function using the lme4 package R (Bates et al. 2015).
Individual food preferences estimated as the proportion of
feeding-oriented behaviours directed to either food choice had variable
reliability depending on the number of behaviours performed by the
individual (Douma and Weedon 2019). Therefore, we use each behaviour as
an observation of the binomial response variable 'preference', with
values 0 (preference for anthocyanin-enriched food) or 1 (preference for
fat-enriched food). Birds that did not show feeding-oriented behaviours
were excluded from the analysis of food choice. We include the identity
of the individuals as a random factor in our models to control the
information available to estimate the preference of the individuals.
Residual mass and infection were included as fixed predictors, along
with year to reduce unexplained variance. We fitted two types of models
to assess the nature of the effect of haemosporidian infections, each
considering a different measure of infection: (1) whether the bird had
no parasites, harboured a single-parasite infection, or had a multiple
infection, and (2) the intensity of infection.

We controlled that haemolysis did not significantly affect physiological
parameters (all differences between haemolysis levels:
\emph{p}\textgreater{} 0.08). Uric acid was controlled in the analysis
of TAS, as both variables were positively correlated (r = 0.86). RBC-MDA
was positively correlated with tGSH (r = 0.58), which we interpreted as
a hormetic response (Costantini 2014) of migrating birds at stopover
regulating antioxidant defences according to oxidative status. To
correctly score the oxidative status of individuals captured at
different stages along this gradient, we performed a Principal Component
Analysis (PCA) with RBC-MDA and tGSH, where PC1 measured hormesis (both
variables with factor loading = 0.89, 79.1 \% of variance explained, RBC
hormesis hereafter), and PC2 measured exposure to lipid peroxidation
relative to enzymatic antioxidant defences of RBC (factor loadings: tGSH
= -0.46 and RBC-MDA = 0.46, 20.9 \% of variance explained, RBC oxidative
stress hereafter).

To assess the relationships between infection, and physiological
condition of individuals, we fitted lineal models (LM) with
physiological parameters as dependent variables. We assessed two types
of models in relation to infection or intensity of infection (as
described above) and included the year and residual mass as covariables.
When an oxidative damage biomarker was analysed as a dependent variable,
we included an antioxidant biomarker as a covariate and vice versa.

Finally, we evaluated the relationships between physiological variables
indicative of oxidative status and bird food preference. We assessed
which physiological variables best predicted food preference using the
Akaike information criterion (AIC) (Burnham and Anderson 2002) with the
MuMIn package (Barton 2023 Mar 15). We used GLMM with preference as a
binomial response and individual identity as a random factor. The
saturated model included the fixed effects of year, three biomarkers of
oxidative status (plasma-MDA, RBC hormesis and RBC oxidative stress), a
biomarker of antioxidant capacity (TAS), and triglycerides and uric acid
to correctly interpret the effects of plasma-MDA and TAS, respectively
(note that uric acid also has antioxidant properties). We calculated
multicollinearity with the variance inflation factor (VIF). We selected
the best model based on AIC values corrected by small sample size
(AICc), and averaged all models with \selectlanguage{greek}Δ\selectlanguage{english}AICc [?] 2 to compute the
significance of effects (Symonds and Moussalli 2011).

\textbf{Results}

Of 48 birds tested, 28 had single infections, 14 had multiple
infections, and 6 were not infected with haemosporidian parasites.
Detailed information on the prevalence of each parasite lineage found in
this sample is available in Supporting information. The infected birds
had a very low intensity of infection (mean = 3.30 parasites in 5000
RBC, sd = 4.33). Twenty-nine blackcaps performed feeding-oriented
behaviours, of which 4 were not infected, 19 had single infections and 6
had multiple infections.

\emph{Food preference and infection}

We found significant effects of year, residual mass, and multiple
infection on food preference (Table 1). Birds that harboured multiple
infections preferred anthocyanin-enriched food (z =
2.75;\emph{p\textsubscript{Tukey}} = 0.02), while individuals infected
by one parasite and non-infected birds showed no detectable preference
(all\emph{p\textsubscript{Tukey}} values \textgreater{} 0.31, Fig. 1).
Fat preference was higher in 2020 and increase as blackcaps were fatter
(Table 1). The intensity of infection did not influence food preference
after controlling for the significant effect of residual mass and year
(Table 1).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Figure 1. Variation in preference for fat-enriched food (as opposed to
preference for anthocyanin-enriched food) between blackcaps with
different levels of multiple status of infection by haemosporidian
parasites. Preference estimates with 95\% confidence intervals represent
the probability (expressed as a percentage) that feeding-oriented
behaviours are directed to fat-enriched food, controlling for residual
body mass and year. The horizontal dashed line set at 50\% indicates the
absence of preference for either food choice. Dots are observations of
single feeding-oriented behaviours, which therefore take values of 0\%
if directed to anthocyanin-enriched food and 100\% if directed to
fat-enriched food, jittered to reduce overlapping. The identity of
individuals expressing these behaviours has been controlled as a random
factor in the model.

Table 1. GLMMs that analyze variation in food preference as a function
of ecological condition (residual mass and parasite infection),
controlling for the fixed effect of year, and including individual
differences as a random factor. Significant results are highlighted in
bold. Different models (in columns) with parasite infection quantified
as the multiple infection status or the intensity of the infection were
tested.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& \textbf{Multiple infection status} & \textbf{Multiple infection
status} & \textbf{Multiple infection status} & \textbf{Intensity of
infection (log)} & \textbf{Intensity of infection (log)} &
\textbf{Intensity of infection (log)}\tabularnewline
\midrule
\endhead
& \selectlanguage{ngerman}\textbf{Estimate ± se} & \emph{LRT} \textbf{(df)} & p &
\selectlanguage{ngerman}\textbf{Estimate ± se} & \emph{LRT} \textbf{(df)} & p\tabularnewline
Parasite infection: & & \textbf{8.02 (2)} & \textbf{0.02} & -0.70 \selectlanguage{ngerman}± 1.11
& 0.38 (1) & 0.54\tabularnewline
Not infected -- single & -1.79 \selectlanguage{ngerman}± 2.31 & & & & &\tabularnewline
Not infected -- multiple & 4.33 \selectlanguage{ngerman}± 2.96 & & & & &\tabularnewline
Single -- multiple & 6.12 \selectlanguage{ngerman}± 2.22 & & & & &\tabularnewline
Residual body mass & \selectlanguage{ngerman}\textbf{1.92 ± 1.03} & \textbf{4.16 (1)} &
\textbf{0.04} & \selectlanguage{ngerman}\textbf{2.08 ± 0.95} & \textbf{5.99 (1)} &
\textbf{0.01}\tabularnewline
Year (2019) & \selectlanguage{ngerman}\textbf{-2.82 ± 0.99} & \textbf{8.64 (1)} & \textbf{0.003}
& \selectlanguage{ngerman}\textbf{-3.10 ± 1.34} & \textbf{7.52 (1)} &
\textbf{0.006}\tabularnewline
\bottomrule
\end{longtable}

\emph{Physiology and infection}

The multiple status of infection of the individuals had a marginal
effect on the antioxidant capacity of plasma (F\textsubscript{2,37} =
2.62; \emph{p} = 0.09), although the plotted data (Fig. 2) suggested
that the infected single and multiple birds had very similar TAS values,
which together were significantly lower than the non-infected bird
values (difference between infected and non-infected
individuals:\selectlanguage{ngerman}\emph{estimate ± SE} = 0.27 ± 0.12; F\textsubscript{1,38} =
5.28;\emph{p} = 0.03). We did not find any significant relationship
between residual mass or infection status and haemoglobin,
triglycerides, uric acid, plasma-MDA, RBC hormesis, or RBC oxidative
stress (Supporting information).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 2. Variation in plasma antioxidant capacity (z-standardised TAS
values) between blackcaps with different levels of multiple status of
infection by haemosporidian parasites. Estimates and 95\% confidence
intervals were computed from a model controlling for uric acid content,
residual body mass, year and plasma MDA. The dots are individual
estimates, jittered horizontally to reduce overlapping.

\emph{Food preference and physiology}

We obtained six models with \selectlanguage{greek}Δ\selectlanguage{english}AIC [?] 2 (Table 2), which were deemed
equally good to explain food preference. The model with lowest AICc
value included RBC oxidative stress, plasma-MDA (log-transformed), uric
acid and year. Given that the best model was not strongly weighted
against competitor models, we used a full model averaging of the six
models to obtain parameter estimates following (Symonds and Moussalli
2011). Plasma-MDA was the only physiological variable with a significant
effect on food preference: individuals with lower log-transformed
plasma-MDA preferred fat-enriched food (full model estimate \selectlanguage{ngerman}± adjusted
SE = -2.08 ± 1.04, \emph{P} = 0.047) controlling for an effect of the
year (estimate ± adjusted SE = -1.99 ± 1.01, \emph{P} = 0.049, all other
effects with \emph{P} \textgreater{} 0.07).

Table 2. Selection of the best models to explain food preference as a
function of physiological parameters involved in oxidative status. The
models are arranged according to the Akaike Information Criterion
corrected by sample size (AICc), where the best model (number 1) is the
one with lowest AICc. Models with \selectlanguage{greek}Δ\selectlanguage{english}AICc [?] 2 are shown, indicating their
AICc value, the increase in AICc compared to the best model, the model
weight and the number of parameters. The same information is shown for
the null model (ranked 95) at the end of the table.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Model & AICc & \selectlanguage{greek}Δ\selectlanguage{english}AICc & Weight & k\tabularnewline
\midrule
\endhead
1.RBC stress + plasma MDA (log) + uric acid + year & 210,671 & 0,000 &
0,067 & 6\tabularnewline
2. plasma MDA (log) + uric acid + year & 211,118 & 0,447 & 0,054 &
5\tabularnewline
3.RBC stress + plasma MDA (log) + uric acid + TAS + year & 211,916 &
1,245 & 0,036 & 7\tabularnewline
4.RBC stress + plasma MDA (log) + year & 212,008 & 1,337 & 0,034 &
5\tabularnewline
5. RBC hormesis + RBC stress + plasma MDA (log) + uric acid + year &
212,392 & 1,721 & 0,028 & 7\tabularnewline
6.RBC stress + plasma MDA (log) + TAS + year & 212,584 & 1,913 & 0,026 &
6\tabularnewline
95. Null model & 217,546 & 6,875 & 0,002 & 2\tabularnewline
\bottomrule
\end{longtable}

\textbf{Discussion}

Our behavioural test with young male blackcaps captured on migration
showed that birds preferred anthocyanin- or fat-enriched food
alternatives depending on their physiological condition. The results
supported a scenario in which feeding decisions helped birds to cope
with the oxidative challenges they face as they recover from and prepare
to the intense aerobic exercise during endurance flight. In this
scenario, haemosporidian parasites had an impact on host physiology by
exacerbating oxidative challenges, which influenced on individual food
preferences supporting the idea that increasing the intake of dietary
antioxidants may be a self-medication mechanism to cope with elevated
risk of oxidative stress due to infection (Mancio-Silva et al. 2017;
Zuzarte-Lu\selectlanguage{ngerman}ís and Mota 2018). This interpretation was supported by our
observation of infected birds having lower total antioxidant capacity,
and individuals with higher levels of oxidative damage to lipids having
more preference for anthocyanin-enriched food.

Co-infected blackcaps preferred anthocyanin-enriched food. Consumption
of antioxidants such as anthocyanins could be a means of self-medicating
if it reduced oxidative damage caused by the parasite or by activation
of the immune system during the infection (Beaulieu and Schaefer 2013;
de Roode et al. 2013; Muriel 2020). This effect of parasitism was only
significant in multiple infections, which could increase parasite
virulence or the cost of the host's immune response (Pigeault et al.
2018; Garcia-Longoria et al. 2022). In this context, the consumption of
antioxidants could be detrimental to the energy intake essential for
migration performance (Beaulieu and Schaefer 2014), or it could
compromise the active search of alternative essential nutrients, such as
polyunsaturated fatty acids that may enhance physiological performance
during migration (Weber 2009).

We found a correlation between infection and the oxidative balance,
since infected birds showed lower values of plasma antioxidant capacity.
Antioxidant depletion associated with immune activation (Costantini and
Møller 2009) or production of free radicals as a result of parasite
exploitation (Percário et al. 2012) may induce an increase in ROS in
infected birds (Delhaye et al. 2016). Similar results of infections have
been found in other stressful situations such as during reproduction
(Badás et al. 2015), or in poor habitat (Messina et al. 2022). However,
we did not detect any correlation between infection status and other
biomarkers of oxidative status. These correlations could have been
concealed by a hormetic response, which is supported by a positive
correlation between tGSH and RBC-MDA. Faced with predictable oxidative
stress, migrating birds could up-regulate antioxidant defences before
suffering too much oxidative damage (Costantini 2010; McWilliams et al.
2021), thus buffering the relationships between oxidative parameters and
the sources of stress.

We did not find any relationship between parasitemia and biomarkers of
oxidative stress, although other studies have found it in other species
such as great tits \emph{Parus major} (Isaksson et al. 2013; Delhaye et
al. 2016). However, blackcaps showed very low parasitemia in our study.
This result indicates that blackcaps during autumn migration have mostly
chronic infections with few parasites in the bloodstream, yet such
low-intensity infections may have an impact on oxidative status during
migration, which could contribute to the mortality cost of
haemosporidian infections observed in other migratory bird species
(Asghar et al. 2015).

We found a positive correlation between relative body mass and
preference for fat-enriched food, which we attribute to the fact that
body mass of migrating blackcaps captured on stopover is most dependent
on the time they have spent at the stopover site, as birds gain body
mass from arrival to departure (Langslow 1976; Arizaga et al. 2010).
Thus, leaner birds may prefer anthocyanin-enriched food if dietary
antioxidants help them cope with oxidative damage associated to the
aerobic effort of the preceding flight stage (Costantini 2008; Eikenaar
et al. 2020). Conversely, fatter birds closer to departure may focus on
putting on fats to fuel the next flight stage, thereby preferring
fat-enriched food. Preference for olive oil could be further increased
close to departure if birds benefit from acquiring so-called ''natural
doping'' polyunsaturated fatty acids present in olive oil as these may
increase metabolic efficiency during endurance flight (Weber 2009). Our
interpretation of the relationship between body mass and food
preferences observed in blackcaps is supported by the fact that
blackcaps lose weight on arrival at stopover sites (Langslow 1976),
which may be indicative of a period of tissue repair during which birds
do not put on any fat. Furthermore, birds with less oxidative damage in
plasma (as measured by MDA) selected fats rather than anthocyanins,
supporting the idea that individuals with a good oxidative status could
focus on rebuilding fat stores or prioritise the intake of alternative
essential nutrients that may give them a physiological advantage during
endurance flight (Weber 2009; Costantini 2010; Beaulieu and Schaefer
2014).

Understanding the intricate relationships between body condition,
oxidative status and dietary preferences during migration has long been
at the centre of research on the physiological performance of migrating
birds (Weber 2009). Our results support the idea that haemosporidian
infections influence on these relationships by exacerbating the
oxidative challenges faced by birds during this critical life cycle
stage, thereby providing a physiological mechanism to explain the
negative impact of chronic avian malaria infections on survival observed
in migrating birds (Asghar et al. 2015), and emphasising the importance
of accounting for parasite infections in studies of animal performance
(Chrétien et al. 2023).

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