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\acceptedDD MMMM YYYYINTRODUCTION
Collectively, the processes of perception, learning and memory that
permit decision making constitute cognition (Shettleworth, 2010a).
Cognitive performance impacts fitness and welfare of animals by
influencing their capacities to forage, find potential mates and avoid
predators (Boogert et al., 2018; Fuss, 2021). To collect and process the
multitude of sensory inputs from their surroundings, fish have evolved
sophisticated brains that allow them to distinguish between predator and
prey, assess competitors, discern their own movements through their
environments, evaluate risk, and decide whether and where to hide (Fernö
et al. 2020). Although not yet well studied, brain development has been
found in some fish species to be reliant upon the quality of their diet
during larval stages (Hou and Fuiman, 2020). In particular, omega-3
long-chain (≥ 20 C) polyunsaturated fatty acids (n‑3 LC‑PUFA),
especially docosahexaenoic acid (DHA 22:6n‑3), are important for neural
development, structure and function in vertebrates (Pilecky et al.,
2021; Twining et al., 2021; Závorka et al., 2023). Because vertebrates
have limited capacity to biosynthesise n‑3 LC‑PUFA, they must be
acquired, at least in part, via the diet (Twining et al., 2016).
Laboratory studies have shown a positive association between n‑3 LC-PUFA
enrichment and brain growth in freshwater and marine fishes (Lund et
al., 2016; Ishizaki et al., 2001). In many animals, including fishes,
cognition has been linked to relative brain size (Kotrschal et al.,
2013; Triki et al., 2021). Brain size is associated with
fitness-influencing behaviours such as prey capture (Edmunds et al.,
2016) and predator avoidance (Kondoh, 2010). However, fish brain
morphology varies widely across species (Triki et al., 2021), and so
relative brain size alone may not always be the best proxy for fish
cognition (Marhounová et al., 2019; Závorka et al., 2022b). In addition,
different cognitive skills are associated with the relative sizes of
specific brain regions: for example, learning and cognitive flexibility
in response to visual stimuli, which are often investigated in animal
cognition studies, are regulated by the telencephalon and optic tectum
in guppies (Poecilia reticulata ) (Triki et al., 2022). Therefore,
there is a need for experimental work integrating the impacts of diet
quality on brain size, morphology and biochemical composition, and the
cognitive and behavioural abilities they promote.
For stream-dwelling fishes, the combination of prey from freshwater
aquatic and terrestrial origins provides for a varied dietary intake of
essential nutrients (Sánchez-Hernández et al., 2018; Syrjänen et al.,
2011). Of the invertebrates typically consumed by juvenile salmonids,
freshwater prey tends to be relatively rich in a n‑3 LC-PUFA,
eicosapentaenoic acid (EPA 20:5n-3), which can be converted to DHA at
relatively low metabolic cost, while prey of terrestrial origin tends to
be richer in the short-chain precursor, α-linolenic acid (ALA 18:3n‑3),
which can only be converted to DHA at high metabolic cost (Pilecky et
al., 2021; Twining et al., 2021). The energetic costs of short- to
long-chain conversion may constrain the rate of DHA biosynthesis and
thus the ability to allocate DHA to the brain in consumers of diets low
in LC-PUFA (Pilecky et al., 2021; Závorka et al., 2021). Furthermore,
because n‑3 and n‑6 precursor fatty acids compete for the same enzymes,
both synthesis pathways from α-linolenic acid (ALA 18:3n‑3) to
docosahexaenoic acid (DHA 22:6n‑3) and linoleic acid (LIN 18:2n‑6) to
arachidonic acid (ARA 20:4n-6) should be considered when investigating
bioconversion of DHA precursors (Geiger et al., 1993): biosynthesis of
ARA is, ipso facto , powerful evidence of even greater n‑3 LC-PUFA
conversion than what may be deduced from DHA alone (Sprecher, 2000;
Hastings et al., 2001).
Apart from diet, greater environmental complexity or enrichment can
increase brain size and cognitive ability in fish (Ebbesson and
Braithwaite, 2012; Salena et al., 2021): evidence of plasticity-inducing
effects of habitat complexity on relative brain size exists for related
salmonids, chinook salmon Oncorhynchus tschawytscha (Kihslinger
et al., 2006) and Atlantic salmon Salmo salar (Näslund et al.,
2012), as well as brown trout themselves (Závorka et al. 2022b). Most of
the conversion of short-chain to long-chain fatty acids occurs in the
liver, from which DHA is distributed to the brain (Rapoport et al.,
2007; Wang et al., 2017), where it may be incorporated into polar lipids
(PL – mainly phospholipids) that increase neuronal membrane fluidity
and accelerate the formation of synapses and synaptic vesicles as
vehicles for neurotransmitters (Pilecky et al. 2021). DHA and its
precursors that are not currently needed for a specific physiological
function are stored in form of neutral lipids (NL, mainly
triacylglycerols) or allocated to other tissues, such as gonads or
gametes for the next generation (Rigaud et al., 2023; Závorka et al.,
2023; Zhu et al., 2019). Uneven distribution of DHA, and its precursors,
whether biosynthesised or dietary, may be caused by activation of
storage lipids and rerouting of the required fatty acids to the brain
(Lacombe et al., 2018; Pifferi et al., 2021). The combination of
biosynthesis and priority routing of DHA and its precursors may
represent a compensatory mechanism for those animals with diets depleted
in such nutrients.
Brown trout Salmo trutta are a widespread species throughout
Europe, and introduced worldwide, that exhibit enormous genetic,
morphological and life-history variation and sexual dimorphism
(Klemetsen, 2013; Koene et al., 2020; Reyes-Gavilán et al., 1997). Most
variants typically are born and spend their early years as juveniles in
low-productivity streams (Ferguson et al., 2019), which offer both
physical habitat complexity and instability (Guo et al. 2018) and the
opportunity for social complexity in the form of dominance hierarchies
in response to competition for preferred microhabitats (Sloman et al.,
2000). Juvenile brown trout often lack morphological features that
correspond to competitive ability that allows them to assess one another
at the onset of each potential dyadic conflict (sensu Kodric-Brown and Brown, 1984), beyond mere differences in size (Jacob et
al., 2007). So, recognition of individual rivals, memory of previous
confrontations, and inference of competitive ability become important
cognitive competencies when a potential opponent’s fighting capabilities
can otherwise be assessed only through escalated contests (Drew, 1993).
To test the effect of dietary quality on memory and inference abilities
that are crucial for juvenile brown trout during conflicts (Johnsson andÅkerman, 1998; Grosenick et al., 2007; Shettleworth, 2010b), we
conducted an experiment in which juvenile brown trout raised in either
complex or simple habitats and on either high or low n‑3 LC-PUFA diets
were subjected to a series of agonistic dyadic trials. We hypothesised
that larger brains, generally, larger optic tecta and telencephala,
specifically, and greater DHA content of brain polar lipids are
associated with greater cognitive performance; and that habitat
complexity and dietary DHA deficiency stimulates increased compensatory
DHA biosynthesis and routing to brain polar lipids, the energetic cost
of which is reflected in lower somatic growth. We tested the predictions
that: 1) trout deprived of n‑3 LC-PUFA and in a complex habitat would
show lower somatic growth than those raised on a high n‑3 LC-PUFA diet
and in a simple habitat; 2) trout raised on a high n‑3 LC-PUFA diet and
in a complex habitat would show greater cognitive performance than those
raised on a low n‑3 LC-PUFA diet and in a simple habitat; 3) trout
raised on a high n‑3 LC-PUFA diet and in a complex habitat would have
larger relative brain size, and larger optic tecta and telencephala,
than those raised on a low n‑3 LC-PUFA diet and in a simple habitat; 4)
that larger brains, and larger optic tecta and telencephala, would be
associated with greater cognitive performance; and 5) trout deprived of
n‑3 LC-PUFA and in a complex habitat would biosynthesise DHA, and route
DHA to brain polar lipids, to a greater extent than those raised on a
high n‑3 LC-PUFA diet and in a simple habitat.
There has been a suggestion that sex can play a role in routing and
synthesis of n‑3 LC-PUFA in fish, with female Eurasian perch Perca
fluviatilis showing significantly higher proportions of n‑3 LC‑PUFA in
muscle tissue (Scharnweber and Gårdmark, 2020), and rates of n‑3 LC-PUFA
biosynthesis before spawning (Rigaud et al., 2023), than males. Although
sex differences are normally unexpected in brown trout until they reach
maturity (Reyes-Gavilán et al., 1997), we considered the potential
impact of sex in all of our analyses.