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\begin{document}

\title{\textbf{Linking brain size in wild stream-dwelling brown trout with
dietary supply of omega-3 fatty acids}}



\author[1]{Libor Závorka}%
\author[1]{Magnus Lovén Wallerius}%
\author[1]{Martin J. Kainz}%
\author[1]{Johan Höjesjö}%
\affil[1]{Affiliation not available}%


\vspace{-1em}



  
  \date{June 16, 2024}


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\selectlanguage{english}
\begin{abstract}
1. Omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) are key
structural lipids and their dietary intake is essential for brain
development of virtually all vertebrates. The importance of n-3 LC-PUFA
has been demonstrated in clinical and laboratory studies, but little is
known about how differences in availability of n-3 LC-PUFA in natural
prey influence brain development of wild consumers. The availability of
n-3 LC-PUFA in the prey communities is driven by primary producers and
it is therefore distributed heterogeneously, but predictably across
ecosystems, being higher in aquatic than in terrestrial food webs.
Consequently, the numerous consumers foraging on the interface of
aquatic and terrestrial food webs can differ substantially in their
intake of n-3 LC-PUFA, which may lead to in brain development, yet, this
hypothesis still remains to be tested.

2. Here we use the previously demonstrated shift towards higher reliance
on n-3 LC-PUFA deprived terrestrial prey of native brown
trout\emph{Salmo trutta} living in sympatry with invasive brook
trout\emph{Salvelinus fontinalis} to explore this hypothesis.

3. We found that the content of n-3 LC-PUFA in muscle tissues of brown
trout decreased with increasing consumption of n-3 LC-PUFA deprived
terrestrial prey. Brain volume was positively related to content of the
n-3 LC-PUFA, docosahexaenoic acid, in muscle tissues of brown trout.

4. Our study thus suggests that increased reliance on low quality diet
of n-3 LC-PUFA deprived subsidies from terrestrial food web can have a
significant negative impact on brain development of wild trout. These
findings provide the first evidence of an intra-specific link between
n-3 LC-PUFA content in natural prey and brain size of wild vertebrate
consumers.

5. Ongoing global change is predicted to reduce the availability of
dietary n-3 LC-PUFA across food webs. Therefore, our findings emphasise
the need for further research on how wild consumers adapt to the
shortage of dietary n-3 LC-PUFA in order to maintain optimal development
and functioning of their brain, which is crucial for their fitness.%
\end{abstract}%



\sloppy


Abstract content goes here

Libor Z\selectlanguage{ngerman}ávorka\textsuperscript{1}, Magnus Lovén
Wallerius\textsuperscript{2}, Martin J. Kainz\textsuperscript{1*}, Johan
Höjesjö\textsuperscript{2*}

\textsuperscript{1}WasserCluster Lunz -- Inter-university Centre for
Aquatic Ecosystem Research, A-3293, Lunz am See, Austria

\textsuperscript{2}Department of Biological and Environmental Sciences,
University of Gothenburg, Box 463, SE-405 30, Gothenburg, Sweden

\textsuperscript{*}Equal contribution to the manuscript

Key words

essential fatty acids, neurogenesis, encephalization, animal cognition,
ecosystem functioning

\par\null

\textbf{Introduction}

Omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA), in
particular docosahexaenoic acid (DHA; 22:6n-3), are essential for
optimal functioning of metabolism and neural development of most
vertebrate consumers (Pilecky et al. 2021; Twining et al. 2021). DHA is
highly retained by vertebrates (Pilecky et al. 2021) and it has been
shown to be a dominant building block of brain of fishes (Ebm et al.
2021), birds (Speake \& Wood 2005), and mammals (Innis 2007). Laboratory
feeding studies have shown that increased dietary intake of n-3 LC-PUFA
facilitates brain growth and neuron proliferation in rodents (Kawakita
et al. 2006), passerine birds (Hall et al. 2014), and freshwater (Lund
et al. 2012) and marine fishes (Ishizaki et al. 2001). These dietary
induced changes in brain development have also been shown to influence
cognitive skills of vertebrate consumers (Lund et al. 2012;~Sugasini et
al. 2017; Pilecky et al. 2021) and could thus potentially have a direct
impact on their fitness in the wild (Boogert~\emph{et al.}, 2018). Yet,
little is known about how n-3 LC-PUFA content in natural diets
influences the development of vertebrate brain in the wild.~

Primary producers drive the availability of dietary n-3 LC-PUFA in prey
communities, which is generally lowest in terrestrial, intermediate in
freshwater, and highest in marine~food webs (Hixson et al. 2015; Brett
et al. 2017; Colombo et al. 2017). Vascular plants dominating primary
production in terrestrial ecosystems~can synthesize only short chain n-3
PUFA alpha-linoleic acid (ALA, 18:3n-3), while multiple aquatic primary
producers, especially microalgae, can~synthesize long-chain n-3
PUFA\emph{~de novo}, mainly eicosapentaenoic acid (EPA; 20:5n-3), but
also DHA (Arts et al. 2009; Brett et al. 2017; Twining et al. 2016). The
dominant determinant of n-3 LC-PUFA synthesis by primary producers
appears to be their phylogeny (Twining et al. 2016;~Marzetz et al.
2017). Therefore, the structure of the primary producer community is
determinant of the n-3 LC-PUFA availability to herbivores (Marzetz et
al. 2017; Guo et al. 2018) and to consumers at higher trophic levels
(Guo et al. 2021).~Observational studies on wild European
perch~\emph{Perca fluviatilis}(Scharnweber et a. 2021) and Eastern
Phoebe~\emph{Sayornis phoebe}~(Twining et al. 2019) have shown that diet
of individuals from the same species can differ substantially in the
content of n-3 LC-PUFA due to intra-specific niche partitioning (Araújo
et al. 2011). Therefore, differences in diet quality in wild vertebrate
consumers can potentially be large enough to cause intra-specific
divergence in brain development, but this hypothesis remains to be
tested.

Dietary ALA and EPA are utilized by many vertebrates for internal
synthesis of DHA (Murray et al. 2014; Twining et al. 2021; Závorka et
al. 2021). However, the synthesis of DHA from short-chain ALA requires
more conversion steps and thus more energy than the synthesis from
long-chain EPA (Pilecky~et al. 2021; Twining et al. 2021). This energy
trade-off can lead to the lower capacity of consumers feeding on diet
deprived of n-3 LC-PUFA to synthetize DHA and retain it in the brain
(Závorka et al. 2021; Pilecky~et al. 2021). In addition to the diet
quality, this energy trade-off can be amplified by intrinsic factors
such as sex of individuals, as females may be able to retain less DHA in
the brain due to the higher investment of n-3 LC-PUFA to gametes
(Maklakov 2008; Hou \& Fuiman 2020). The capacity to synthetize and
retain DHA has also been shown to decrease over ontogeny in freshwater
fishes (Chaguaceda et al. 2020) and mammals (Brenna 2011). A good
example of the energetic trade-off linked to the synthesis of n-3
LC-PUFA from dietary sources of different quality are consumers that
depend on a mix of prey from aquatic and terrestrial resources (Twining
et al.\emph{,}~2019). Aquatic and terrestrial macroinvertebrates are
reciprocal sources of energy and macronutrients (Nakano \& Mukarami
2001; Sullivan et al. 2014), but aquatic macroinvertebrates provide
substantially more long-chain EPA than terrestrial macroinvertebrates,
which contain mainly short-chain ALA (Guo et al. 2018; Brett et al.
2019). Predominantly insectivorous freshwater fishes, such as stream
salmonids, depend on macroinvertebrates from aquatic and terrestrial
subsidies (Nakano \& Mukarami 2001; Syrjänen et al. 2011;
Sánchez\selectlanguage{english}-Hernandez \& Cobo 2018). Therefore, intra-specific dietary
specialization and intrinsic capacity to synthetize and retain n-3
LC-PUFA in a freshwater fishes could provide a first general insight in
how these dietary biomolecules influence brain development of
vertebrates in the wild.

In this study, we focus on brown trout~\emph{Salmo trutta}, a salmonid
native to European streams, which is a generalist consumer displaying a
broad range of dietary specialization among populations
(Sanchez-Hernandez 2020) and individuals (Evangelista et al. 2014). We
use a river system where brown trout co-occurs with the invasive brook
trout~\emph{Salvelinus fontinalis,}~as previous studies have shown
that~brown trout increases the consumption of~terrestrial
macroinvertebrates (\emph{i.e.,}~low-quality prey deprived of n-3
LC-PUFA) in sympatry with invasive brook trout (Zavorka et al. 2017;
Cucherousset et al. 2020; but see Horka et al. 2017). Therefore, this
study system allows us to test an entirely natural setting how reduced
dietary intake of n-3 LC-PUFA influence the physiology and brain
development of wild consumers. We explore how dietary reliance on
terrestrial prey affects brain size and brain morphology of brown trout
in sympatry and allopatry with the invasive brook trout, while
accounting for ontogenetic and sex differences among individuals. We
predict that;~\emph{i}) higher reliance on terrestrial prey of brown
trout reduces dietary intake of EPA;~\emph{ii}) the content of EPA and
DHA in brown trout tissues decreases with increasing reliance on
terrestrial prey; and~\emph{iii}) lower DHA content in brown trout
tissues is associated with smaller brain size or changes in brain
morphology (\emph{i.e.,}~region specific brain size reduction).{}

\textbf{Material and Methods}

\emph{Study sites and fish sampling}

This study was conducted in three streams in the upper part of the
catchment of the Viskan River, situated in southwest Sweden
(57deg40\selectlanguage{english}'318\selectlanguage{english}'\selectlanguage{english}'N, 12deg59\selectlanguage{english}'300\selectlanguage{english}'\selectlanguage{english}'E; Appendix S1, Fig. S1). Brook trout was
introduced to Sweden in 1892 (Aas et al. 2018), with the first
electrofishing reports indicating a self-reproducing brook trout
population in our study system dating to 1985 (SERS, 2013). While brook
trout have occurred in European streams for over a century, its impact
on the native species is still significant and it is recognized by many
European countries including Sweden as an invasive alien species
(\emph{e.g.,}~\href{https://www.havochvatten.se/arter-och-livsmiljoer/frammande-arter/sok-frammande-arter/fakta/backroding.html}{}Strand
et al. 2018).~Populations of brook trout are established in the upstream
sections of the sampled streams. Native brown trout occupy the whole
system, and thus each stream contains an allopatric (brown trout only)
and a sympatric (brown and brook trout co-occurring) section. The brown
trout populations in the study stream are landlocked with no reported
lake or seawards migration. Abundance of brown and brook trout in the
studied streams ranges from 0.2 -- 1 individual per meter of the stream
length. At sympatric sites, brook trout is as abundant or more abundant
than brown trout (Zavorka et al. 2017). Other fish species occurring in
the study system are brook lamprey~\emph{Lamperta planeri}, common
minnow~\emph{Phoxinus phoxinus}, and occasionally northern
pike~\emph{Esox lucius}.

Experimental fish were collected by electrofishing (LR-20B; Smith-Root,
Vancouver, WA, USA) carried out over a 500 m stretch at each study site
(Appendix S1, Table S1) between May 11 and June 11, 2019 (Appendix S1,
Table S1). All sampled brown trout were measured (fork
length~\emph{i.e.,}~from the tip of the snout to the end of the central
caudal fin ray) and a small clip of the pelvic fin (\textasciitilde{}0.5
cm\textsuperscript{2}) was stored on ice and later in laboratory kept in
freezer at -80 degC, awaiting stable isotope analysis. We have collected
between 38 to 45 brown trout per sampling site with average fork length
ranging from 93 to 143 mm (Appendix S1). Aquatic macroinvertebrates for
fatty acid and bulk stable isotope analysis were collected using kick
sampling and terrestrial macroinvertebrates were collected by
hand-picking and dragging the kick net over the canopy surrounding the
stream. The macroinvertebrate samples were collected at each sampling
site the same day as the fish and were stored alive in an icebox and
after determination in the laboratory kept frozen at -80 degC until
further processing. Habitat quality was evaluated at each study site on
July 10, 2019 using standardized protocol (Jonkopingsmodellen - Hallden
et al. 2002, Appendix S1, Table S1) and photographs of habitats at each
sampling site were taken (Appendix S1, Fig. S1). Consumption of aquatic
macroinvertebrates by brown trout is known to increase with their
abundance in the stream (Evangelista et al. 2014; Sanchez\selectlanguage{english}-Hernandez \&
Cobo 2018). Therefore, we performed a quantitative assessment of
abundance and biomass of aquatic macroinvertebrates at each sampling
site on July 11 and 12, 2019 during sunny stable weather, with no
precipitation and low water flow. Aquatic macroinvertebrates were
collected by a kick-net (30 x 25 cm, mesh size, 30 seconds of kicking at
three spots within each study site representing all available habitats).
Macroinvertebrates were counted and oven-dried at 60 degC for 48 hours and
total dry biomass of macroinvertebrates at each sampling site was
measured to the nearest 0.001 g (Appendix S1, Table S1).~

~

\emph{Fish dissection}

A subsample of brown trout (Table 1) at each study site was transported
alive to the department of Biology and Environmental Sciences at the
University of Gothenburg for dissection. Before the dissection, fish
were housed in holding tanks (30 L, 300 x 320 x 340 mm), which provided
shelter structures (rocks and plastic plants) and fresh 12 degC water from
a flow-through filtration system (flow rate: 2 L
min\textsuperscript{-1}). Fish were not fed during this period. All fish
were dissected within 20 hours after capture and to randomize the time
effect, individuals from allopatric and sympatric sites were dissected
in alternating order (with even and odd individuals from allopatry and
sympatry respectively). Before dissection, fish were euthanized by an
overdose of 2-phenoxyethanol (0.5 mL L\textsuperscript{-1}) and body
mass and fork length were measured to the nearest 0.1 g and 1.0 mm
respectively. Sex of individuals was determined by inspection of the
gonads. Heads of fish were removed and fixed in 4\% buffered (pH 6.9)
paraformaldehyde solution. Brains were then dissected out as described
in Gonda et al. (2009) by opening the scull along the anteroposterior
axis and removing muscle tissue, nerves and bones until the brain could
be lifted up from the skull and stored in 4\% buffered paraformaldehyde
solution until further procedure. Brains were imaged with a digital
camera (EOS 40D with MP-E 65mm lens; Canon Inc., Tokyo, Japan). Images
of brains were taken using the dorsal, left lateral and ventral views to
calculate the total volume and the volume of cerebellum, optic tectum,
telencephalon, olfactory bulb, and hypothalamus. Measurements were
completed using ImageJ 1.48 (Schneider et al. 2021) and used to
calculate volume with the formulas outlined by Pollen et al. (2007).
Brain morphology was assessed by principal component analysis (Appendix
S2) yielding two dominant principal components. PC1 was positively
related to the volume of olfactory bulb, and hypothalamus and negatively
related to cerebellum and optic tectum, while PC2 was positively related
to the volume of telencephalon.~

Fixing fish brains in paraformaldehyde solution in order to do
morphological measurements prevented us from performing fatty acid
analysis of the brain tissue. Therefore, we have used the muscle tissue
as a proxy of n-3 LC-PUFA content in fish body. Muscles provide a good
proxy for this purpose, because~they represent the majority of fish
biomass, and thus muscle metabolic activity and fatty acids content
reflects individuals as a whole (Norin \& Malte 2012;~Gladyshev et al.
2018). In addition, biochemical composition of muscle tissue of
salmonids has been shown to respond in similar direction to dietary
deprivation of~n-3 LC-PUFA as brain tissue, but with higher
magnitude~(Zavorka et al. 2021). Thus, samples (\textasciitilde{}1 g of
wet mass) of dorsal and ventral muscle tissue samples were taken from
the left side below the dorsal fin, above and below the lateral line
respectively. Bones and skin residuals were mechanically removed from
the tissue samples, before they were stored on dry ice and subsequently
frozen at -80 degC.~\textbf{}

\emph{Bulk stable isotope and fatty acids analysis}

We used bulk tissue analyses of \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C fin clips and
potential prey to estimate diet composition of experimental individuals.
Freeze-dried and homogenized samples were analysed in WasserCluster
Lunz, Austria. Isotope ratios are reported relative to the international
Vienna PeeDee Belemnite carbonate standard. \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C
values were mathematically corrected for lipid content following methods
as described by Post et al. (2007). The baseline~\textsuperscript{13}C
value was further corrected to account for variability in basal
resources across the sampling sites (Olsson~\emph{et al.}~2009; Musseau
et al. 2020) using the following equation:

~\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C\textsubscript{corr}\selectlanguage{greek}\emph{\textsubscript{i ~}=
(δ\textsuperscript{13}C\emph{i}~-~δ\textsuperscript{\emph{13}}C\textsubscript{\emph{inv}}~)/CR\textsubscript{inv}}\selectlanguage{english}

where \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C\textsubscript{corr\emph{i}}~is the
corrected carbon isotopic ratio for individual~\emph{i},
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C\emph{i}~is the carbon isotopic ratio for
individual~\emph{i},
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{\emph{13}}C\textsubscript{\emph{inv}}~is the average
carbon isotope ratio of macroinvertebrates and CR\textsubscript{inv}~is
the carbon range (\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C\textsubscript{\emph{max}}~-
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C\textsubscript{\emph{min}}) of
macroinvertebrates.~\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C\textsubscript{corr\emph{i}}~is
hereafter used as an indicator of reliance on terrestrial prey
(\emph{i.e.,~}increasing value suggests increasing reliance of an
individual on terrestrial prey).~

Fatty acids were extracted and analysed from freeze-dried samples (3--10
mg dry mass) that were homogenized, sonicated and vortexed (4 times) in
a chloroform-methanol (2:1) mixture, following B\selectlanguage{ngerman}öhm et al. (2014). Total
lipid mass ratios were determined via gravimetry. Fatty acids were
derivatized to obtain fatty acid methyl esters (FAME) using toluene and
sulfuric acid-methanol-solution (incubated at 16 h at 50 C). FAME were
identified using a gas chromatograph (Thermo Scientific TRACE GC Ultra)
equipped with a flame ionization detector (FID) and a Supelco SP-2560
column (100 m, 25 mm i.d., 0.2~mm film thickness). Quantification of
fatty acids were performed by comparison with a known concentration of
the internal standard using Excalibur 1.4 (Thermo Electron Corporation).

\emph{Statistical analysis}

All analyses were conducted in R v4.0.2 (http://www.R-project.org/).~The
structure of the final linear models and sample sizes break down by the
levels of categorical explanatory variables are summarized in (Appendix
S3).~Non-significant interactions were removed from the models.
Significance of the final models was evaluated using ANOVA tables using
Type II and III sums of squares for models without and with significant
interaction, respectively. Differences among groups were analysed using
Tukey's HSD post-hoc test.~We have accounted for the ontogenetic effects
by using fork length as a proxy of individuals age. Fork length has been
used as explanatory variable in all models. When models contained other
explanatory variables correlated to the fork length, we used residuals
from a linear model between the log transformed variable of interest and
fork length to prevent collinearity (see~Appendix S3).~{}

We tested the variability in content of ALA and EPA, and of total lipids
in macroinvertebrates across all sampling sites (\emph{i.e.,}~category
with 6 levels -- 3 streams~´~2 populations {[}allopatric, sympatric{]})
and subsidy types (category with 2 levels -- aquatic or terrestrial
macroinvertebrates). Since there was no difference lipid quality of
aquatic and terrestrial subsidies across the six sampling sites (see
results for details), we did not include the stream or sampling site in
the models.~We collected samples of dorsal and ventral muscle tissue
because concentration of adipose cells is usually higher in ventral than
in dorsal muscles and thus these two tissues can differ in their lipid
content (Fauconneau et al. 1995; Ebm et al. 2021). However, the total
lipid content and the relative content of n-3 LC-PUFA were repeatable
across dorsal and ventral muscle samples (total lipids:
R\textsubscript{adj~}= 0.25, 95\% CI {[}0.06, 0.44{]}; ALA:
R\textsubscript{adj~}= 0.59, 95\% CI {[}0.44, 0.72{]}; EPA:
R\textsubscript{adj~}= 0.62, 95\% CI {[}0.48, 0.74{]}; DHA:
R\textsubscript{adj~}= 0.56, 95\% CI {[}0.41, 0.70{]}. Therefore, for
further analysis we have used an average value across tissues for each
individual. Total lipids in all models were reported as mass fractions
(mg/g of the dry mass), while all models and figures including n-3
LC-PUFA reported the relative fatty acid contents (\% of fatty acid
methyl esters; with exception of Fig. 1). This is a common approach
(\emph{e.g.,}~Twining et al. 2019; Ebm et al. 2021) because mass
fractions of fatty acids are often closely correlated to the total lipid
mass fractions, while the relative content of fatty acids is independent
of the total lipids and thus more comparable across the sample types and
studies, particularly when only a part of an organism
(\emph{e.g.,}~muscle tissues) is analysed.

\textbf{Results}

Reliance on terrestrial prey was higher in sympatric than in allopatric
brown trout (F\textsubscript{1;161} = 52.67, p \textless{} 0.001; Fig.
1). The reliance on terrestrial prey also increased with increasing body
size (F\textsubscript{1;161} = 98.26, p \textless{} 0.001), but it did
not differ between the sexes (F\textsubscript{1;79} = 0.01, p = 0.911;
Fig. 1). Reliance on terrestrial prey increased with increasing
abundance (F\textsubscript{1;161} = 120.01, p \textless{} 0.001) and
biomass (F\textsubscript{1;161} = 63.06, p \textless{} 0.001) of aquatic
macroinvertebrates at the sampling site. This indicates that the
consumption of aquatic and terrestrial prey by brown trout was not
limited by the availability of aquatic prey in the stream, but abundance
and biomass of aquatic macroinvertebrates appeared to be controlled by
the foraging behaviour of brown trout.

Terrestrial macroinvertebrates contained less total lipids (mean±SD =
148±69 mg/dry mass) than aquatic prey macroinvertebrates (mean±SD =
204±86 mg/g dry mass; F\textsubscript{1;51} = 8.93, p = 0.004).
Terrestrial macroinvertebrates had similar content of ALA
(F\textsubscript{1;51} = 1.39, p = 0.245) and significantly lower EPA
(F\textsubscript{1;51} = 30.27, p \textless{} 0.001) than aquatic
macroinvertebrates (Fig 1a). Total lipids (F\textsubscript{5,55} = 1.70,
p = 0.151), and the content of ALA (F\textsubscript{5,55} = 0.40, p =
0.845) and EPA (F\textsubscript{5,55} = 1.00, p = 0.42) of
macroinvertebrates did not differ across the sampling sites. Aquatic
macroinvertebrates were thus a richer dietary source of n-3 LC-PUFA,
particularly of EPA, than terrestrial macroinvertebrates across all
sampling sites (Fig. 1a).

The content of total lipids in brown trout tissues did not differ
between sympatric and allopatric populations (F\textsubscript{1,80} =
0.06, p = 0.803) and was not significantly related to the reliance on
terrestrial prey (F\textsubscript{1,80} = 0.02, p = 0.890), fork length
(F\textsubscript{1,80} = 2.23, p = 0.139), and sex
(F\textsubscript{1,80} = 0.07, p = 0.789) of individuals. The ALA
content in trout tissues was affected by an interaction between the
competition mode and reliance on terrestrial prey
(F\textsubscript{1,79}= 5.71, p = 0.019; Fig. 1b), so ALA increased with
increasing reliance on terrestrial prey in sympatric
(F\textsubscript{1,39} = 8.74, p = 0.005), but not in allopatric
(F\textsubscript{1,38} = 0.73, p = 0.397) brown trout. Sex
(F\textsubscript{1,79} = 0.52, p = 0.472) and body length
(F\textsubscript{1,79} = 3.14, p = 0.080) had no significant effect on
ALA content in trout tissues. The EPA content was higher in allopatric
than in sympatric brown trout (F\textsubscript{1,80}= 14.29, p
\textless{} 0.001; Fig. 1c) and decreased with increasing fork length
(F\textsubscript{1,80} = 146.89, p = 0.001), but was not affected by
individual's sex (F\textsubscript{1,80} = 0.28, p = 0.597) and reliance
on terrestrial prey (F\textsubscript{1,80} = 0.87, p = 0.353). The DHA
content in trout tissues was affected by an interaction between the
competition mode and reliance on terrestrial prey
(F\textsubscript{1,78}= 7.46, p = 0.007; Fig. 1d), so DHA decreased with
increasing reliance on terrestrial prey in sympatric
(F\textsubscript{1,38} = 7.14, p = 0.011), but not in allopatric
(F\textsubscript{1,38} = 1.07, p = 0.307) populations. Sex had no effect
on DHA in trout tissues (F\textsubscript{1,78} = 0.74, p = 0.391), but
DHA content decreased with increasing body size (F\textsubscript{1,78} =
6.33, p = 0.014).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\emph{\textbf{Figure 1} -- (a) Content mg/g of ALA and EPA in aquatic
and terrestrial prey macroinvertebrates. All prey samples contained no
or negligible amount of DHA. Association of the reliance on terrestrial
prey and the relative content of ALA (b), EPA (c), and DHA (d) in trout
tissues. Colours of datapoints, fit lines and box plots displaying
distribution of each variable correspond to AF -- allopatric females
(red), AM -- allopatric males (green), SF - sympatric females (cyan),
and SM -- sympatric males (purple). Variables in figures b-d are
residuals controlled for the fork length of individuals.}

Total brain volume increased with increasing DHA content in trout
tissues (F\textsubscript{1,80} = 11.57, p = 0.001, Fig. 2a), but DHA
content had no effect on brain morphology (PC 1: F\textsubscript{1,80} =
0.92, p = 0.341, Fig. 2b; PC2: F\textsubscript{1,80} = 3.89, p = 0.052,
Fig. 2c). Total lipids content in trout tissues had no significant
effect on total brain volume (F\textsubscript{1,81} = 2.92, p = 0.092)
and morphology (PC 1: F\textsubscript{1,81} = 1.27, p = 0.263; PC2:
F\textsubscript{1,81} = 3.94, p = 0.051). The brain volume increased
with increasing fork length of individuals (F\textsubscript{1,80} =
657.45, p \textless{} 0.001), but brain morphology was independent of
individual fork length (PC 1: F\textsubscript{1,81} = 0.07, p = 0.786;
PC2: F\textsubscript{1,80} = 0.01, p = 0.943). Brain volume and brain
morphology did not differ between allopatric and sympatric populations
(brain volume: F\textsubscript{1,80} = 0.51, p = 0.476; PC 1:
F\textsubscript{1,80} = 0.99, p = 0.322; PC2: F\textsubscript{1,80} =
0.00, p = 0.990) or between the sexes (brain volume:
F\textsubscript{1,80} = 1.54, p = 0.219; PC 1: F\textsubscript{1,81} =
0.57, p = 0.454; PC2: F\textsubscript{1,80} = 0.05, p = 0.943).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig2/Fig2}
\caption{{\textbf{Figure 2} \emph{-- Association between DHA in trout tissues and
the brain volume (a), brain morphology PC1 {[}PC1 was positively related
to the volume of olfactory bulb, and hypothalamus and negatively related
to cerebellum and optic tectum{]} (b) and brain morphology PC2 {[}PC2
was positively related to the volume of telencephalon{]} (c). Colours of
datapoints, fit lines and box plots displaying distribution of each
variable correspond to AF -- allopatric females (red), AM -- allopatric
males (green), SF - sympatric females (cyan), and SM -- sympatric males
(purple). All variables are residuals controlled for the fork length of
individuals.}
{\label{239012}}%
}}
\end{center}
\end{figure}

~\textbf{Discussion}

Dietary reliance of brown trout on low-quality terrestrial prey,
deprived of n-3 LC-PUFA, increased in sympatry with invasive brook
trout. Higher reliance on terrestrial prey of sympatric brown trout
resulted in lower content of EPA in their muscle tissues compare to the
allopatric conspecifics. In the sympatric populations, the content of
the short-chain ALA increased and the content of the long-chain DHA
decreased in muscle tissues with increasing reliance on the low-quality
terrestrial prey, but the relative content of ALA and DHA in allopatric
brown trout was not related to the diet quality. Sex had no effect on
diet quality and muscle content of n-3 LC-PUFA, but larger (and
presumably older,~\emph{i.e.,}~Bowker 1995; \selectlanguage{ngerman}Öhlund et al. 2008)
individuals relied more on terrestrial prey and consequently had lower
relative content of n-3 LC-PUFA in muscles. We found that the relative
content of DHA in muscle tissues was positively related to the
encephalization of brown trout (\emph{i.e.}, higher relative brain size
after controlling for the body size). These findings demonstrate that
intra-specific differences in diet quality in wild vertebrates, foraging
at interface of aquatic and terrestrial food webs, are linked to their
brain size. In addition, our findings indicate that external stressors,
such as competition with an invasive species, can reduce the capacity of
consumers to synthetize and retain DHA for brain development.~

Dietary intake and retention of energy in form of lipids has a
substantial effect on the physiological development of consumers (Arts
et al. 2009). However, our results indicate, in agreement with previous
laboratory studies (Speake \& Wood 2005; Lund et al., 2012; Hall et al.
2014), that the availability of structural lipids, especially DHA, and
not the lipid energy reserve is the key for brain development in
vertebrate consumers. Our findings suggests that the specialization of
dietary niche in wild consumers on resources that have similar content
of energy, but differ in the content of n-3 LC-PUFA (Heissenberger et
al. 2010; Twining et al. 2019; Scharnweber et a. 2021), can lead to
intra-specific diversification of brain size. This is an important
finding because brain size is positively correlated to cognitive
capacity, especially among closely related species (Olkowicz~et al.
2016) and at the intra-specific level (Møller 2010;~Kotrschal et al.
2013). For example, guppies with larger brain size have shown higher
learning capacity (Kotrschal et al. 2013), and large brain have been
suggested to improve migration efficiency and offspring defence in barn
swallows~\emph{Hirundo rustica~}(Møller 2010). However, it should be
noted that while dietary supply of omega-3 LC-PUFA is determinant for
brain development of vertebrates (Pilecky et al. 2021), dietary
availability of these biomolecules can also influence other phenotypic
traits important for consumers fitness, such as mitochondrial efficiency
(Salin et al. 2021; Závorka et al. 2021);~metabolic rate (McKenzie
2001;~Twining et al. 2016), growth rate (Chaguaceda et al. 2020; Závorka
et al. 2021), and reproductive capacity (Brett et al. 2009; Scharnweber
\& Gårdmark 2020).~Therefore, further carefully designed experiments in
a realistic ecological context are needed to understand the trajectory
of how dietary omega-3 PUFA influences fitness of consumers in the wild.

Effects of diet quality on development of phenotypic traits necessarily
depends on temporal scales on which the consumption of low- and
high-quality diets occurs (Brett et al. 2009; Murray et al. 2014). For
example, a relatively short-term study (\emph{i.e.}, 8 weeks of dietary
treatment) by Závorka et al. (2021) on juvenile Atlantic
salmon~\emph{Salmo salar}~showed a strong shift of DHA content in the
brain, but no significant effect on brain size and performance in a
cognitive test. On the other hand, a longer study (\emph{i.e.,}~21 weeks
of dietary treatment) by Lund et al. (2012) on juvenile
pikeperch~\emph{Sander luciperca}~demonstrated that dietary n-3 LC-PUFA
deprivation induces leads to reductions of DHA content and size of the
brain resulting in lower cognitive capacity. The reliance on terrestrial
prey in our study was estimated based on bulk~d\textsuperscript{13}C
values of fin clips, which indicate dietary carbon on the temporal scale
of several weeks (Jardine et al. 2005; Layman et al. 2012). However, a
previous study has shown that the dietary shift of our focal species,
brown trout, in sympatry with invasive brook trout occurs early in
ontogeny and remains stable in later life-stages (Cucherousset~\emph{et
al.}, 2020). The increased reliance on low-quality terrestrial prey in
our study did not appear to be influenced by the availability of aquatic
prey, indicating that this dietary shift was likely caused by
behavioural changes of brown trout in sympatry with the invasive species
(Lovén Wallerius et al. 2017; Larranaga et al. 2018;
Cucherousset~\emph{et al.}, 2020). Thus, the intra-specific dietary
differences in our study likely represent a long-term specialization of
individuals that might have a permanent effect on supply of n-3 LC-PUFA
to their brain.

Despite the higher reliance on low-quality terrestrial prey and
corresponding decrease of EPA in muscle tissues, there was no
significant reduction of DHA in muscles of sympatric compared to
allopatric brown trout. A potential explanation is that sympatric brown
trout has adapted to the lower dietary intake of n-3 LC-PUFA by
increasing retention and/or internal synthesis of DHA (Murray et al.
2014). Such adaptation has been observed in populations of three-spined
stickleback (Ishikawa~\emph{et al.}, 2021) and European perch
(Scharnweber~\emph{et al.}, 2021) foraging on low-quality prey from
littoral lake habitat that is deprived of n-3 LC-PUFA. Similarly, tree
swallows have been shown to increase synthesis of DHA when consuming n-3
LC-PUFA deprived terrestrial macroinvertebrates (Twining~\emph{et al.},
2018). However, our results indicated limits of this adaptation as the
relative content of DHA in sympatric trout decreased with increasing
reliance on terrestrial prey. This suggests that the combination of
external stressor (\emph{i.e.}, interaction with an invasive species)
and high consumption of n-3 LC-PUFA deprived prey can affect the
capacity of wild consumers to maintain an optimal biochemical
composition of their tissues. Synthesis of DHA from short-chain
precursors, such as ALA, contained in low-quality terrestrial diet comes
at substantial energetic costs that can result in reduced somatic growth
rate (Murray et al. 2014; Twining et al. 2016; 2018; Závorka et al.
2021).~Our findings thus~accentuate the suggestion that the trade-off
between internal synthesis and dietary acquisition of n-3 LC-PUFA might
be an important driver of phenotypic adaptations across~vertebrate
consumers (Twining et al. 2021).~We also found that larger (and
presumably older,~\emph{i.e.,}Bowker 1995; Öhlund et al. 2008)
individuals across populations relied more on terrestrial prey and had
lower EPA and DHA contents in muscles. This age-dependent effect can be
explained by the fact that older consumers require less n-3 LC-PUFA than
younger consumers (Brenna 2011; Chaguaceda et al. 2020), because the key
period of vertebrate brain development occurs during early ontogenetic
stages (Innis 2007; Brenna 2011; Lund et al. 2012).~{}

In conclusion, this field study demonstrates the largely overlooked
importance of dietary intake of n-3 LC-PUFA for brain development of
wild vertebrate consumers, which have so far been recognized just under
laboratory conditions (Pilecky et al. 2021). We have shown the link
between n-3 LC-PUFA deprived diets and consumer's brain size in the
context of competition with an invasive species. However, the ongoing
global change is predicted to limit availability of n-3 LC-PUFA for
vertebrate consumers on a large spatial scale (Hixson \& Arts, 2016;
Heilpern et al. 2021) and it is still poorly understood how wild
vertebrate consumers respond to this dietary deprivation of n-3 LC-PUFA.
Our results indicate that it can have negative impacts even on species
which are pre-adapted to dietary scarcity of these important
biomolecules (\emph{i.e.}, salmonids often consume high proportion of
low-quality terrestrial prey, Syrjänen et al. 2011; Evangelista et al.
2014). Dietary intake of n-3 LC-PUFA have a high potential to affect
fitness of vertebrate consumers (Twining et al. 2021; Pilecky et al.
2021), and therefore further studies are needed to understand how the
availability n-3 LC-PUFA in natural diets affects development of brain,
behaviour, physiology and life-history of vertebrates.~

\emph{}

Acknowledgements

Authors are grateful to Samuel-Karl Kämmer, Katharina Winter, and Niklas
Wengström for their assistance with collection and processing of
samples. Authors have no conflict of interests to declare.

Data archiving

Should the manuscript be accepted data will be archived at figshare.com
(doi:) and in Supplementary material.

Ethics statement

These experiments were approved by the Ethical Committee for Animal
Research in Göteborg (license dnr 5.8.18-04106/2018) and comply with
current laws in Sweden.

Funding statement

Study was funded by the Austrian Science Fund, FWF, Lise Meitner Program
(Project M 2742-BBL), Carl Tryggers foundation (CT 16:221).

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