Deficiency of vital organic nutrients in ecosystems limits brain development and fitness in wild fish

Deficiency of vital organic nutrients in ecosystems limits brain development and fitness in wild fish | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 9 April 2025 V1 Latest version Share on Deficiency of vital organic nutrients in ecosystems limits brain development and fitness in wild fish Authors : Libor Závorka 0000-0002-0489-3681 [email protected] , Johan Höjesjö , Stefan Auer , Benedikte Austad 0000-0002-1779-2396 , Francesco Dionigi , Pernilla Hansson , Shaun Killen , … Show All … , Stefano Mari 0009-0007-2964-7843 , Evelina Olsen , Matthias Pilecky 0000-0002-3404-5923 , Kurt Pinter , Alexandra Polonyiová , Tileuzhan Smagul , Patrik Stehlík , Simon Vitecek , Mourine Yegon , and Pavel Němec Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.174420773.34708385/v1 Published Journal of Experimental Biology Version of record Peer review timeline 391 views 253 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Animals in aquatic ecosystems impacted by global changes often face reduced availability of vital organic compounds, such as omega-3 polyunsaturated fatty acids (n-3 LC-PUFA), vital for brain development and cognition. Cognitive skills are crucial for buffering the impacts of environmental stress on fitness, yet the link between the quality of diet and fitness-enhancing behaviours of individuals in food webs altered by global change remains unclear. We examined how dietary n-3 LC-PUFA affect brain development, social dominance, and growth in territorial juvenile salmonids in a large-scale model of a natural pre-alpine stream. For this assessment we used wild fish whose diet quality was estimated using stable isotope analysis, and hatchery reared fish exposed to dietary treatments in a common-garden experiment. In both wild and common garden experiment fish, diets low in n-3 LC-PUFA led to a decreased content of n-3 LC-PUFA in brain tissue but did not affect brain size, morphology, or neuron count. Fish with lower brain n-3 LC-PUFA content exhibited reduced competitiveness in social interactions and suboptimal habitat use, resulting in slower somatic growth. Our findings indicate that the limited availability of key organic compounds may impair behavioural flexibility of top aquatic consumers, possibly with negative impacts on intra- and inter-specific diversity of the consumers. Deficiency of vital organic nutrients in ecosystems limits brain development and fitness in wild fish Libor Závorka 1 *, Johan Höjesjö 2 , Stefan Auer 3 , Benedikte Austad 2 , Francesco Dionigi 4 , Pernilla Hansson 2 , Shaun S. Killen 5 , Stefano Mari 1,6 , Evelina Olsen 2 , Matthias Pilecky 7 , Kurt Pinter 3 , Alexandra Polonyiová 4 , Tileuzhan Smagul 4 , Patrik Stehlík 4 , Simon Vitecek 1,8 , Mourine J. Yegon 1,3 , Pavel Němec 4 not-yet-known not-yet-known not-yet-known unknown 1WasserCluster – Biologische Station Lunz, Inter-University Center for Aquatic Ecosystem Research, Dr. Carl-Kupelwieser Promenade 5, 3293 Lunz/See, Austria 2Department of Biological and Environmental Sciences, University of Gothenburg, Medicinaregatan 7B, 413 90 Gothenburg, Sweden 3Institute of Hydrobiology and Aqautic Ecosystem Management, BOKU University, Gregor-Mendel-Straße 33, A-1180 Vienna, Austria 4Department of Zoology, Faculty of Science, Charles University, 128 43a Prague, Czech Republic 5School of One Health, Biodiversity and Veterinary Medicine, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow, United Kingdom 6Department of Functional and Evolutionary Ecology, University of Vienna Djerassiplatz 1 1030 Vienna, Austria 7University for Continuing Education Krems, Research Lab for Aquatic Ecosystem Research, Dr. Karl-Dorrek-Straße 30, A-3500 Krems, Austria 8University of Innsbruck, Department of Ecology, Technikerstrasse 25, 6020 Innsbruck, Austria Corresponding author: Libor Závorka, [email protected] Abstract Animals in aquatic ecosystems impacted by global changes often face reduced availability of vital organic compounds, such as omega-3 polyunsaturated fatty acids (n-3 LC-PUFA), vital for brain development and cognition. Cognitive skills are crucial for buffering the impacts of environmental stress on fitness, yet the link between the quality of diet and fitness-enhancing behaviours of individuals in food webs altered by global change remains unclear. We examined how dietary n-3 LC-PUFA affect brain development, social dominance, and growth in territorial juvenile salmonids in a large-scale model of a natural pre-alpine stream. For this assessment we used wild fish whose diet quality was estimated using stable isotope analysis, and hatchery reared fish exposed to dietary treatments in a common-garden experiment. In both wild and common garden experiment fish, diets low in n-3 LC-PUFA led to a decreased content of n-3 LC-PUFA in brain tissue but did not affect brain size, morphology, or neuron count. Fish with lower brain n-3 LC-PUFA content exhibited reduced competitiveness in social interactions and suboptimal habitat use, resulting in slower somatic growth. Our findings indicate that the limited availability of key organic compounds may impair behavioural flexibility of top aquatic consumers, possibly with negative impacts on intra- and inter-specific diversity of the consumers. Keywords: essential nutrients, global change, freshwater food webs, fishes, cognitive ecology Introduction The cognitive buffer hypothesis predicts that behavioural flexibility confers advantages in unpredictable and novel environments, improving animals’ capacity to cope with anthropogenic environmental changes (Sol et al. 2005). However, the benefits of high behavioral flexibility come with significant energetic and nutritional costs for the development and maintenance of neural system (Pilecky et al. 2021; Heldstab et al. 2022). The brains of juvenile animals and animals with indeterminate neuronal growth such as fishes exhibit a high plasticity in response to environmental pressures (Ebbesson & Braithwaite 2012; DePasquale et al. 2016). The plastic changes in brain development caused by environmental pressures such as elevated temperature (Závorka et al. 2020), high water turbidity (Pike et al. 2018), or reduced diet quality (Ishizaki et al. 2001; Mari et al. 2025) can influence cognitive performance and behaviour of fishes. However, it remains an open question of how such laboratory measurements of brain and cognitive plasticity translate to behavioral flexibility in the natural environments and how brain plasticity might influence the capacity of fishes to maintain a high fitness in freshwater ecosystems altered by the global change. One of the key nutrients for vertebrate brain development are long-chain omega-3 fatty acids (n-3 LC-PUFA), which account for more than a third of the brain lipid mass and are crucial for signal transfer efficiency and neuronal plasticity (Pilecky et al. 2021). There are pronounced differences in the availability of n-3 LC-PUFA across food webs, with aquatic planktonic algae being the prime producer of n-3 LC-PUFA (Twining et al. 2016). Importantly, anthropogenic pressures such as climate change, habitat degradation, pollution, and eutrophication have been shown to disrupt the production of n-3 LC-PUFA in aquatic food webs and their transfer within and across ecosystems (Závorka et al. 2023, Shipley et al. 2024). There is a risk that the paucity of dietary n-3 LC-PUFA in aquatic ecosystems will lead to plastic changes in neural traits of fishes including reduction of brain size (Ishizaki et al. 2001), reduced neuronal numbers (Kawakita et al. 2006), and lower cerebral content of n-3 LC-PUFA (Závorka et al. 2021). All of these neural traits, including brain size (Triki et al., 2022), neuronal numbers (Marhounová et al., 2019), and n-3 LC-PUFA content (Lund et al., 2014), have been consistently shown to be positively associated with cognition in fishes. Therefore, we posit that reduced availability of n-3 LC-PUFA in aquatic food webs may impair fishes’ ability to cope with anthropogenic environmental changes due to direct effects dietary n-3 LC-PUFA deprivation on neural traits and corresponding effects on cognition and behaviour. Testing this prediction requires simultaneous assessment of ecological factors, individual’s fitness proxies ( e.g. , body growth rate), neural traits, and cognitive performance in a natural context, at a level of scrutiny rarely undertaken. The variation in the physical and social environment experienced by juvenile stream-dwelling salmonids, such as brown trout ( Salmo trutta ), provides an ideal system for investigating the impact of changing diet quality on neural traits, behavioral flexibility, and fitness in an ecologically relevant context. Juvenile stream salmonids exhibit intra-specific variation in their dietary reliance on n-3 LC-PUFA-rich aquatic invertebrates versus n-3 LC-PUFA-deprived terrestrial invertebrates (Závorka et al. 2022). These fish also face an ongoing decrease in n-3 LC-PUFA availability due to factors such as rising temperatures (Hixson & Arts, 2016) and biodiversity degradation (Shipley et al. 2024). Juvenile brown trout are territorial animals that establish a linear social hierarchy when competing for patches of suitable micro-habitats in their nursery streams (Kalleberg 1958, Bachman 1984). The probability of dominance in social interactions among size-matched individuals is directly influenced by cognitive performance, such as perception of contested habitat quality (Piccolo et al. 2014) or assessment of the opponent’s social position via social eavesdropping (Johnsson & Åkerman 1998, Johnsson et al. 2000). Dominant individuals in the social group occupy the most profitable habitats usually at the upstream end of a pool (Nakano 1995) and benefit from their social position in terms of increased fitness indicators, such as higher somatic growth (Höjesjö et al. 2002). Therefore, habitat use, social hierarchy position, and growth rate are suitable proxies to assess the ecologically relevant outcomes of the effect of dietary n-3 LC-PUFA on brain plasticity and cognition in wild trout. Here, we investigated the effect of dietary n-3 LC-PUFA on neural traits critical for the functioning of the brain ( i.e. , overall size and size of brain regions, neuron counts, and fatty acid content) in juvenile brown trout. We examined this by a combination of two complementary approaches: (1) inferring to the diet quality of wild fish using stable isotope analysis and by conditioning juvenile brown trout to diets containing low and high amounts of n-3 LC-PUFA in a common garden experiment; and (2) by testing at the individual level the link between neural traits of the brain and indicators of ecological performance ( i.e., social dominance, habitat use, somatic growth rate) in large seminatural mesocosms simulating a pre-alpine stream habitat. We predict that limited dietary intake of n-3 LC-PUFA would negatively affect the development of neural characteristics of the brain, leading to reduced ecological performance and fitness of juvenile trout. Material and methods The experimental work of this study consists of three major parts. First, the common garden experiment, where we exposed brown trout from different populations to diets with high and low n-3 LC-PUFA content ( Supplementary Material S1, Table S1 ). Second, a field survey of wild fish in streams, assessing the intake of aquatic prey naturally rich in n-3 LC-PUFA compared to terrestrial prey that is relatively poor in n-3 LC-PUFA (Závorka et al. 2022). This was achieved by analysing δ¹³C and δ¹⁵N in muscle tissue of the fish. These first two parts allowed us to test whether diet quality—either experimentally controlled or based on individual choice in the wild—correlates with neural traits of the brain. Finally, in the third part of the experiment, we used a large-scale model of a natural pre-alpine stream to test the effect of brain neural traits on the behaviour and ecological performance of fish from both the common garden experiment and the wild. Common garden experiment At the end of October 2022 juvenile brown trout (10 months post hatching) from 4 hatcheries maintaining genetically diverse brood stocks based on wild-caught fish from the Austrian part of the Danube catchment (Lercetau-Köhler et al. 2013; Schenekar & Weiss 2017), were transferred to the fish husbandry facility at WasserCluster Lunz, Austria (Supplementary Material S1, Table S1). In addition, we collected wild fish of the same age ( i.e. , 0+) from River Ybbs by electrofishing (EFKO 1500, Germany) and included them in the common garden experiment (Supplementary Material S1, Table S1). We choose the majority of fish of hatchery rather than wild origin for the common garden experiment, because the prolonged captivity required for the dietary treatment can have negative effects on the behaviour, physiology and survival of wild brown trout (Závorka et al. 2019, Johnsson & Näslund 2018). On the other hand, the use of a wild population allowed us to control for a potential difference in response of hatchery reared fishes to dietary intake of n-3 LC-PUFA ( e.g. , Betancor et al. 2016). The fish were kept in 500 L holding tanks with flow through water (2 L/minute – complete water change ~ 6 times a day) fed by a nearby spring. The temperature in the tanks corresponded to the ambient temperature of the source spring water, which had small daily fluctuations but varied across seasons from ~ 4°C in January/February to ~14°C in June/August. Upon arrival, fish were distributed according to their origin among 10 holding tanks with 32±2 individuals in each tank ( i.e., fish from different hatcheries were not mixed together and each population has occupied 2 holding tank). Fish were fed daily to apparent satiation via feeding belts that slowly delivered feed pellets between 9.00 and 16.00. During the first two weeks, the hatchery fish were fed the feeds from their hatcheries of origin, while the wild fish were fed a mixture of feeds from the experimental diets. From November 22 nd all individuals were fed exclusively on one of the two experimental diets (GARANT Aqua, Austria). We used two isocaloric diets, of near-identical nutritional value, but one diet was high in n-3 LC-PUFA (n3+ diet), and the other was limited in n-3 LC-PUFA (n3- diet). For details of the biochemical composition of diets and their resemblance to biochemical composition of natural prey of brown trout see Závorka et al. (2021) and (Supplementary Material S2). On February 15 th , 2023, all fish were tagged with 12 mm PIT-tags (Biomark, USA) and measured for their body mass and fork length to the nearest 0.1 g and 1 mm respectively. After tagging, individuals were re-distributed among the 10 holding tanks in groups of 32±2 size-matched individuals, forming now a mix of populations, but maintaining matching dietary treatments. A subset of 24 individuals from each population was used for observations in the stream mesocosms (Supplementary Material S1, Table S1). The rest of the individuals were used in another study (Mari et al. 2025), and then euthanised as a humane endpoint. Individuals used in the stream mesocosms were acclimatized to natural prey in their holding tanks for a period of ten days before the transfer, by adding live benthic macroinvertebrates collected by kick sampling in a nearby stream to the holding tanks (Seebach, see Supplementary Material S3, Table S3.1 for taxonomical composition of the prey). During the first five days, macroinvertebrates were provided together with the experimental feed, while during the remaining five days, the experimental feed was completely withheld, and the fish fed only on macroinvertebrates. Field survey Wild individuals of juvenile brown trout (age 0+ and 1+) from four different source populations (Supplementary Material S1, Table S1) were caught by electrofishing and transferred from their streams of origin to the husbandry facility at WasserCluster Lunz. Fish were tagged with 12 mm PIT-tags (Biomark, USA) upon arrival and kept in the same holding tanks used for the common garden experiment for ten days, and fed live benthic macroinvertebrates before being transferred to the stream mesocosm facility. Samples of potential prey of brown trout were collected at each sampling site as a baseline for stable isotope analysis (see below). Benthic macroinvertebrates were collected using kick sampling and terrestrial macroinvertebrates were collected by hand picking and dragging a net over the canopy surrounding the streams. Samples collected at each site were stored alive in an icebox and after determination in the laboratory kept frozen at − 70°C until further processing. Behavioural measurements in HyTEC The HyTEC flumes (Hydromorphological and Temperature Experimental Channels) (47.8562242N, 15.0366653E) are two 40 meters long gravel-bottom channels simulating natural habitat of a small pre-alpine stream (Auer et al. 2023). Each of the two channels was split transversally using a fence with a mesh size of ~0.5 cm² into four equivalent experimental enclosures (N = 8). Each enclosure was 7 m long and 1.5 m wide and separated from each other by 2 m long buffer zones (Supplementary material S3). The water discharge through each channel was constant at 25 L s⁻¹, and the average water temperature ranged from 8°C to 12°C throughout the study (Supplementary material S1, Table S1). The water level was adjusted by wooden panels placed at the downstream end of each enclosure. In early May 2023, before the start of the experiment, each enclosure of the mesocosms was inoculated with benthic macroinvertebrates collected from a nearby stream (see Supplementary Material S3, Table S3.1 for taxonomic composition). We stocked approximately 60,000 macroinvertebrates in each enclosure, resulting in a density of ~5,000–6,000 individuals per m², which corresponds to the lower end of natural macroinvertebrate densities in a pre-alpine stream (Leitner et al., 2015). This density was maintained throughout the duration of the study (Supplementary Material S3). Each enclosure hosted six size-matched juvenile brown trout from the same population of origin, which corresponded to the density of 0.6 fish per m 2 , which falls within the medium range of natural density of brown trout in nursery streams (Bohlin et al. 2002, Pinter et al. 2019). Wild individuals were distributed solely based on their body size (i.e., fish were size-matched within a given enclosure), whereas individuals from the common garden experiment were stocked within the given enclosure to be size-matched and according to their dietary treatment, with three individuals fed the n3+ diet and three individuals fed the n3- diet. The sex of individuals was determined after the experiment (see below) and thus could not be considered when distributing individuals among the experimental enclosures. Batches of experimental individuals were transported to the stream mesocosm facility 7 ± 2 days before the start of the experiment and released into separate acclimation pools to acclimatize to the water and habitat quality of the mesocosms (Supplementary material S3, Fig. S3.1). After the acclimation period, individuals were collected from the acclimation pool using electrofishing, measured for their body mass (to nearest 0.1 g) and fork length (to nearest 1 mm), and distributed among one of eight experimental enclosures. The period of behavioral observation in each round of the stream mesocosm experiment lasted 10 days (Table S1). To simulate the fluctuation of water level typical for pre-alpine streams ( e.g. , Cane 1992), each round included an initial period with high water levels from day 1 to day 4, a low water level period from day 5 to day 6, followed by a high-water level period from day 7 to day 10. We considered behavioral data in the stream mesocosm enclosures only up to day 7, because on this day, one individual was removed from each enclosure by electrofishing to be used in another study (Austad et al., unpublished data ). The longitudinal position of individual trout in the stream was determined using active RFID telemetry with a portable antenna (HPR Plus reader with BP Plus Portable Antenna, Biomark, USA). The identity of the dominant individual in each enclosure was identified using a combination of stationary RFID telemetry with two stationary antennas (Multiple Antenna PIT tag reader, Oregon RFID, USA) and infra-red video cameras (RLC-810A, Reolink, China) equipped with additional infra-red spotlights (Supplementary Material S3). We were able to clearly determine the dominant individual in 35 out of 36 experimental enclosures. While we did not determine the dominant individual in one enclosure due to a missing record, social dominance appeared to be clearly established in all enclosures at day 7 (Supplementary Material S3, Fig. S3.3). Finally, at the end of each round on day 10, all individuals were collected by electrofishing, and their body mass and fork length were measured again. The individuals were then euthanized with an overdose of anaesthetics (2 mL.L -1 of 2-phenoxy ethanol), followed by spinal cord transection. Samples of dorsal muscle and brain tissue were collected for biochemical and histological analysis, and a fin clip was taken for genetic sex determination. The specific growth rate (SGR) of individuals was calculated using the equation from Brett & Groves (1979). The SGR during the mesocosm experiment was determined based on the initial and final body mass measured at the start and end of the stream mesocosm experiment. Similarly, the SGR of fish from the common garden experiment during the feeding treatment preceding the stream mesocosm experiment was calculated using the initial mass recorded on February 15, 2023, and the mass at the beginning of the stream mesocosm experiment. Brain analysis Immediately after the euthanasia, we opened the fish skull and collected midsagittal cut for fatty acid analysis. This fresh tissue was stored on dry ice and then at –70°C until further processing. The whole head with partially exposed brain was then fixed by immersion in 4% phosphate-buffered paraformaldehyde solution. After 24 hours, the brains were dissected, rinsed in phosphate buffer (pH = 7.4) to remove residual paraformaldehyde, and then stored in antifreeze solution (30% glycerol, 30% ethylene glycol, 40% phosphate buffer) at –20°C until further processing. Brains cellular composition was analysed at Charles University in Prague, Czech Republic. There, we divided the brains into four parts, namely the telencephalon, optic tectum, cerebellum and “rest of the brain” comprising the diencephalon, tegmentum and medulla oblongata. The remains of the left optic tectum were removed, and we further analysed only the right optic tectum and multiplied the results by two to get comparable data with the rest of the brain part. Brain parts were weighed to the nearest 0.001 g using a Mettler Toledo (Mettler Toledo, Columbus, Ohio) MX5 microbalance. Cell and neuron numbers in each of the above-mentioned brain parts were assessed using a modified isotropic fractionator technique (Marhounová et al. 2019) (see Supplementary Material S4 for further details). Number of cells and neurons was assessed in a subsample of 70 individuals from the common garden experiment and 39 wild individuals, evenly distributed across dietary treatments, sexes, and populations of origin. Fatty acids and stable isotope analysis We used bulk tissue analyses of δ 13 C and δ 15 N of trout muscle tissue and potential prey sources to estimate reliance of wild brown trout on terrestrial and aquatic prey in their stream of origin. Freeze-dried and homogenized samples of trout muscle tissue and prey sources were analysed at WasserCluster Lunz, Austria. Isotope ratios are reported relative to the international Vienna PeeDee Belemnite carbonate (δ 13 C) standard and air (δ 15 N). δ 13 C and δ 15 N values were mathematically corrected for the basal resource values at each sampling site (Fedorčák et al., 2025) to ensure comparability across streams of fish origin. Higher δ 13 C indicated a reliance on n-3 LC-PUFA-poor terrestrial prey (Závorka et al., 2022), correspondingly the local δ 13 C of terrestrial prey in the four sampled streams was, on average, 2.3‰ higher than that of aquatic prey. δ 15 N values served as an indicator of the trophic position of individuals, with higher δ 15 N indicating a higher trophic position (Bunn et al., 2013). Fatty acids were extracted and analysed from freeze-dried samples (3–10 mg dry mass) that were homogenized, sonicated, and vortexed (3 times) in a chloroform–methanol (2:1) mixture, following Pilecky et al. (2023). Total lipid mass ratios were determined by gravimetry. Fatty acids were derivatized to obtain fatty acid methyl esters (FAME) using toluene and 1% sulphuric acid in methanol (incubated for 2 h at 70 °C). FAME were separated on and quantified using a gas chromatograph (Thermo Scientific TRACE GC Ultra) equipped with a flame ionization detector (FID) and an Agilent HP-88 column (100 m, 25 mm i.d., 0.2 μm film thickness). Quantification of fatty acids were performed by comparison with a known concentration of the internal standard using Chromeleon 7. All the fatty acid values reported in this study are relative % of fatty acids with respect to total FAME. Sex determination Fin clips were used as a source of nuclear DNA for sex determination following a modified duplex PCR protocol. Following the manufacturer’s instructions, a NucleoSpin™ Tissue kit (Macherey-Nagl) was used to extract genomic DNA from adipose fin clips. Quality was controlled with spectrophotometry (NanoDrop, ThermoFisher Scientific), and quantification was conducted fluorometrically (Qubit 2.0, ThermoFisher Scientific), before dilution to 20 ng μL -1 . Duplex PCR amplified the male-chromosome gene, sdY , with 18S as a positive amplification control, and agarose gel (2%) electrophoresis was used to visualise resulting products (Koene et al. 2025 ). not-yet-known not-yet-known not-yet-known unknown Data processing and analysis All analyses were conducted in R v.4.4.2 “Pile of Leaves” (http://www.R-project.org/). See (Table 1) for the structure of models testing the effect of diet quality on brain neural traits and the effect of the neural traits on the ecological performance of fish in the stream mesocosms. The effect of diet quality on neural traits was tested by linear models (LM) and controlled for the fish population as co-variable. Effect of neural traits on the probability of becoming dominant among the group of conspecifics in the enclosure of stream mesocosms was evaluated by generalized linear mixed models (GLMM) and generalized linear models (GLM) for data with a binomial distribution (1 = dominant, 0 = subdominant). Effect of brain traits and social dominance on specific growth rate of body mass (SGR mass) was tested by linear mixed models (LMM). Initial models for dominance and SGR mass included the fish origin (i.e., hatchery or wild) and interaction between the neural trait and the fish origin, and population as a random factor. Non-significant interaction terms and random factors with predicted variance near zero were removed from the final models. To assess traits of an individual relative to conspecifics from the same experimental enclosure in the stream mesocosm, we used ranking instead of the absolute value of the trait. This approach has yielded best model fit, and distribution of residuals for our data compared to other normalization methods. It allowed us to assess performance of individuals relative to conspecifics facing the same conditions such as water temperature, prey availability, weather, position of the enclosure within the HyTEC flumes, and distribution of phenotypic traits within the social group. For example, Body size rank was based on the fork length of an individual at the beginning of stream mesocosm experiment relative to the other fish within the same enclosure of the stream mesocosm ranging from 1 (for the largest) to 6 (for the smallest) individual in the enclosure. Only enclosures with data for the given trait available for at least 4 individuals have been included. The same ranking logic has been used to establish the n-3 LC-PUFA rank, n-6 PUFA rank, TelW rank (i.e., the telencephalon weight rank), BRW rank (i.e., the whole brain weight rank). The weight of telencephalon was closely related to the total weight of the brain (see Supplementary Material Tables S5.4 and S5.5). Therefore, TelW rank was based on residuals from log-log linear regression between the weight of telencephalon and the weight of the whole brain minus the weight of telencephalon. The significance of the final models was evaluated using ANOVA tables using Type II and III sums of squares for models without and with significant interactions, respectively. Deviations from the assumptions of the models were diagnosed by visual inspection of the distribution of model residuals. The assumptions of the models were satisfactorily met in all cases. Differences among groups were analysed using Tukey’s HSD post-hoc test. P-values of the models were corrected for multiple comparison using the Bonferroni correction, treating all models based on the same subset, structure of explanatory variables, and type of response variables as multiple comparison (Table 1). Table 1 Summary of final models reported in Results. Diet: common garden dietary treatment (category with two levels: n3+ or n3- diet); FL – fork length at the end of experiment; BRW, TelW, CbW, OTW, RoBW: weight of the whole brain, telencephalon, cerebellum optic tectum, and the rest of the brain respectively; BRN, TelN, CbN, OTN, RoBN: number of neurons in the whole brain, telencephalon, cerebellum optic tectum, and the rest of the brain respectively; SFA, MUFA, n-6 PUFA, n-3 LC-PUFA relative content (%) of saturated fatty acids, mono unsaturated fatty acids, omega-6 polyunsaturated fatty acids, and omega-3 long chain polyunsaturated fatty acids respectively; δ13C and δ15N muscle isotopic values adjusted for the stream baseline; sex (category with two levels: male or female); origin (category with two levels: common garden or wild); population (category with: nine levels, four in wild fish and five in common garden fish). Brain quality LM wild SFA, MUFA, n-3 LC-PUFA, n-6 PUFA, BRW δ13C + δ15N + sex + FL + population common garden diet + sex + FL + population wild TelW, CbW, OTW, RoBW, BRN δ13C + δ15N + sex + FL + BRW + population common garden diet + sex + FL + BRW+ population wild TelN, CbN, OTN, RoBN δ13C + δ15N + sex + FL + TelW or CbW or OTW or RoBW+ population common garden Diet + sex + FL + TelW or CbW or OTW or RoBW+ population Social dominance GLM (binomial) all individuals Dominant in the group (1 or 0) n-3 LC-PUFA rank, or n-6 PUFA rank, or rTelW rank, or rBRN rank * origin + sex + Body size rank + (1| population) Growth LMM all individuals SGR mass Dominant * origin + sex + Body size rank+ (1| population) subdominant n-3 LC-PUFA rank, or n-6 PUFA rank, or rTelW rank, or rBRN rank * origin + sex + Body size rank+ (1| population) Habitat use all individuals Longitudinal position in the mesocosm Dominant * Day + sex+ Body size rank + origin + (1|Fish ID) + (1| population) subdominant n-3 LC-PUFA rank, or n-6 PUFA rank, or rTelW rank, or rBRN rank * Day + origin + sex+ Body size rank + (1|Fish ID) + (1| population) Effect of dietary intake of n-3 LC-PUFA and neural traits In the common garden experiment, the diet rich in n-3 LC-PUFA led to an increase of n-3 LC-PUFA (Table 2, Fig. 1a), a decrease of n-6 PUFA (Table 2, Fig. 1c), but had no effect on MUFA and SFA content of trout brain (Table 2, Fig. 1e, g). Similar to the fish from the common garden experiment, we found that wild fish consuming a high proportion of aquatic prey (rich in n-3 LC-PUFA) had higher n-3 LC-PUFA (Table 2, Fig. 1b) and lower n-6 PUFA (Table 2, Fig. 1d) content in their brains compared to conspecifics consuming more terrestrial prey. Higher intake of aquatic prey also led to a decrease of MUFA in the brain of wild brown trout (Table 2, Fig. 1f), but no effect of diet quality on SFA concentration in brain of wild fish were observed (Table 2, Fig. 1h). There was no significant effect of diet quality on the brain size, morphology and neuron numbers of fish from common garden experiment nor from the wild (Table 2, Supplementary Material S5, Table S5.1). We found no effects of trophic position ( i.e , δ 15 N) on any measured neural trait of wild fish (Supplementary Material S5). Table 2 – Effect of dietary intake of n-3 LC-PUFA (explanatory variable for common garden fish = diet, and for wild fish = δ 13 C) on the fatty acid composition of the fish brain. Bold p-values are significant after Bonferroni correction for multiple hypothesis testing. common garden n-3 LC-PUFA 126 16.88 < 0.0001 *** n-6 PUFA 126 94.51 < 0.0001 *** MUFA 126 4.08 0.0456 SFA 126 0.69 0.4073 wild n-3 LC-PUFA 110 19.07 < 0.0001 *** n-6 PUFA 111 9.18 0.0031 * MUFA 111 11.53 0.001 ** SFA 111 5.16 0.0252 common garden Brain mass 133 1.21 0.2731 Brain neurons 68 1.11 0.2954 Tel mass 133 0.70 0.4052 Tel neurons 70 0.09 0.7635 wild Brain mass 119 2.48 0.1185 Brain neurons 39 0.27 0.6050 Tel mass 119 0.54 0.4639 Tel neurons 39 1.46 0.2364 Figure 1 – Effect of diet quality on the fatty acid content in the trout brain. Boxplots illustrating differences in fatty acid content among treatment groups in the common garden experiment (n3+ in red, n3- in green). The central lines represent the median, the box limits correspond to the 25th and 75th percentiles, and the whiskers extend to the 95th percentiles. Filled circles represent individual data points. Scatter plots with regression lines show the correlation between δ13C of muscle tissue and fatty acid content in the fish brain. Shaded areas indicate standard errors for statistically significant relationships. Effects of neural traits on social dominance, somatic growth, and habitat use Trout with higher n-3 LC-PUFA and lower n-6 PUFA content in their brains were more likely to socially dominate the group in the experimental enclosure of the stream mesocosm. This effect remained significant even after controlling for body size of individuals (Table 3, Fig. 2ab, Supplementary Material S6, Table S6.1). In contrast, brain mass and telencephalon mass, after controlling for individual body size, showed no effect on social dominance (Table 3, Fig. 2cd, Supplementary Material S6, Table S6.1). The dominant individuals clearly benefited from their position within the social group by having faster somatic growth rate (Table 3, Fig. 3a). In addition, subdominant trout with larger brain sizes exhibited faster growth compared to other subdominant individuals in the group (Table 3, Fig. 3d). The higher content of n-6 PUFA in the brain was associated with faster somatic growth among subdominant individuals, but only in the fish from the common garden experiment and not in those brought directly from the wild (Table 3, Fig. 3c). The growth rate of subdominant individuals was not related to the n-3 LC-PUFA content of their brain (Table 3, Fig. 3b) or to the mass of the telencephalon (Table 3, Fig. 3e). We found no association between neural traits and somatic growth rate during the common garden experiment preceding the stream mesocosm test (Supplementary Material S6, Table S6.3). Active telemetry data showed that, dominant individuals were detected farther downstream compared to the group average, particularly on day 7 (Table 4, Supplementary Material S7, Fig. S7.1). Similarly, subdominant individuals with higher n-3 LC-PUFA content in the brain were detected farther downstream, particularly on day 7 (Table 4, Supplementary Material S7, Fig. S7.2). Subdominant individuals with higher n-6 PUFA content in the brain were also detected farther downstream, particularly on day 4 (Table 4, Supplementary Material S7, Fig. S7.3). Brain mass and telencephalon mass had no effect on the position of subdominant individuals in the stream mesocosm (Table 4). Table 3 – The effect of brain traits on social dominance and somatic growth of individuals in stream mesocosms. Asterisks and bold type indicate the significance of p-values after Bonferroni adjustment for multiple comparisons. Dominance n-3 LC-PUFA Rank 148 7.10 0.0077 * n-6 PUFA Rank 148 5.62 0.0177 Brain mass Rank 158 0.56 0.4531 Tel mass Rank 158 0.23 0.6311 Body size Rank 158 15.40 < 0.0001 *** Mass SGR Dominance 174 6.53 0.0106 * Mass SGR of subdominant n-3 LC-PUFA Rank 119 0.73 0.3934 n-6 PUFA Rank : Origin 119 7.61 0.0058 * n-6 PUFA Rank (experimental) 57 10.61 0.0011 * n-6 PUFA Rank (wild) 62 0.36 0.5484 Brain mass Rank 122 8.78 0.0030 * Tel mass Rank 122 2.06 0.1508 Table 4 – The effect of brain traits on position of individuals in the stream mesocosms. Asterisk and bold type indicate significance of p-value after Bonferroni adjustment for multiple comparisons. Stream position Dominance 2054 0.54 0.4616 Dominance : Day 11.83 0.0371 * Stream position of subdominant n-3 LC-PUFA Rank 1527 0.09 0.7596 n-3 LC-PUFA Rank : Day 33.84 < 0.0001 *** n-6 PUFA Rank 1527 1.46 0.2268 n-6 PUFA Rank : Day 14.78 0.0113 * Brain mass Rank 1564 0.55 0.4548 Tel mass Rank 1525 0.40 0.5259 Figure 2 — Logistic regression lines representing the relationship between neural trait values relative to other individuals in the enclosure and the probability of becoming dominant within a group of conspecifics. Neural traits include (a) brain content of n-3 LC-PUFA, (b) brain content of n-6 PUFA, (c) whole brain mass, (d) telencephalon mass, and (e) body size. Solid lines represent relationships for experimental fish from the common garden experiment, while dashed lines represent relationships for wild fish. Data points for individuals from the common garden experiment and the wild are depicted as circles and triangles, respectively. Shaded areas indicate standard errors for statistically significant relationships. Figure 3 – Effect of neural traits on the specific growth rate (SGR) of body mass in dominant individuals (green) and the rest of the group (orange) in the stream flume mesocosms. (a) Boxplots illustrating differences in SGR of body mass between dominant and subdominant individuals originating from the common garden experiment and the wild. (b–e) Scatter plots with regression lines showing the correlation between SGR of body mass and neural trait values relative to other individuals in the enclosure. Solid lines represent relationships for experimental fish from the common garden experiment, while dashed lines represent relationships for wild fish. Data points for individuals from the common garden experiment and the wild are depicted as circles and triangles, respectively. Shaded areas indicate s.e. for statistically significant relationships. Discussion Our study demonstrates a clear positive association between dietary intake of n-3 LC-PUFA and the probability of attaining social dominance within a group of conspecifics, as well as the associated benefit of faster somatic growth in territorial juvenile fish. The positive effect of diet quality on the behavioral performance of individuals appeared to be directly linked with higher n-3 LC-PUFA and lower n-6 PUFA contents in the fish brain, but not with brain size, the size of its regions, or neuronal numbers. We showed that the observed patterns linking diet, neural traits, behaviour, and the fitness proxy ( i.e. , somatic growth) in near-natural habitat are consistent in both wild fish and fish exposed to high and low n-3 LC-PUFA diets in a common garden experiment. These results strongly suggest that the dietary intake of n-3 LC-PUFA-rich prey is essential for optimal brain function and fitness-enhancing behaviors, which confer competitive advantages in social interactions to top aquatic consumers. This means that the ongoing decrease n-3 LC-PUFA-rich prey is likely to have a negative impact on the capacity of fishes to maintain a high fitness in freshwater ecosystems altered by the global change. Importantly, our data shows that dietary fatty acids do not appear to significantly influence of neurogenesis, neuron survival, or the average sizes of neuronal and glial cells. Thus, the positive effects of high dietary n-3 LC-PUFA intake on cognitive and social skills were not linked to neural correlates traditionally associated with differences in information processing capacity, such as increased brain size (Sol et al., 2022), greater neuron counts (Herculano-Houzel, 2017; Němec and Osten 2020; Kverková et al., 2022), or high neuron packing densities (Dicke & Roth, 2016). Instead, these effects appear to arise from biochemical changes in the brain. Social interaction is controlled by a complex network of interacting neural systems, such as mirror neuron(-like) systems (Jeon & Lee 2018), which heavily depend on dopaminergic, serotoninergic and oxytocinergic signalling (Burbano Lombana et al. 2021). Indeed, n-3 LC-PUFA have been associated with increased sensitivity of neuronal receptors (G-coupled protein receptors) to neurotransmitters, including dopamine and serotonin (Pilecky et al., 2021), while n-3 LC-PUFA deficiency leads to degradation of dopaminergic and serotoninergic neurons (Cardoso et al. 2014). Therefore, it is conceivable that the faster processing of information and increased social competence by individuals with higher n-3 LC-PUFA content in the brain was the likely cause of their competitive advantage in the interactions with conspecifics (Desjardins & Fernald 2008; Watanabe & Yamamoto 2015; Mari et al. 2025). In contrast to n-3 LC-PUFA, high brain levels of n-6 PUFA can have pro-inflammatory effects (Calder et al., 2005), impair spatial orientation and memory, and promote anxious, less active behaviours (Nguyen et al., 2014; Delpech et al., 2015). This is in line with our finding that n-6 PUFA in the fish brains was negatively correlated to the probability of socially dominate the group of conspecifics. We found that dominant individuals exhibited a faster somatic growth rate compared to the rest of the group, suggesting that their social rank provided a fitness advantage. The positive effect of n-3 LC-PUFA on individual performance was thus closely linked to their ability to establish a dominant position within the group, likely driven by enhanced behavioural flexibility and problem-solving capacity in individuals consuming an n-3 LC-PUFA-rich diet (Mari et al., 2025). The design of our experiment and statistical model have effectively controlled for the initial body size of individuals, which in juvenile salmonids positively correlates with muscular endurance (Ojanguren & Brana 2003) and social dominance (Johnsson & Åkerman 1998). However, prior research indicates that behavioural flexibility such as the knowledge of the contested habitat (Johnsson & Forser 2002, Kvingedal & Einum 2011) of the social statues of the opponent i.e. , acquired through the social eavesdropping (Johnsson & Åkerman 1998) can be more crucial than physical performance per se in social interactions among size matched individuals. We found no association between neuronal traits and the somatic growth trajectories of fish prior to the behavioural test in the stream mesocosm experiment during the common garden study (see Supplementary Material S6, Table S6.3). Thus, we can confidently exclude the possibility that the growth rate of individuals during the mesocosm experiment was primarily driven by accumulated energetic reserves from the period before the mesocosm experiment. Instead, the faster growth of dominant individuals appeared to be facilitated by their access to microhabitats in the mesocosms that support energy-saving behaviours (Henke-von der Malsburg et al. 2020), such as proximity to shelter and a vantage point overlooking prey-rich sections of the flume. Indeed, dominant individuals with high n-3 LC-PUFA content in their brain clearly utilized the habitat in the central part of the stream mesocosm with two shelters available within the distance of 0.5 m and with the most prey rich habitat upstream of them allowing them to adopt energy saving sit-and-wait foraging strategy (Nakano 1995; Tunney & Steingrímsson 2012). Subdominant individuals with high content of n-3 LC-PUFA in brain were able to utilize similar microhabitat as the dominant fish, but did not benefit from this position in terms of higher somatic growth. Possibly because, their capacity to forage was suppressed by the dominant individual (Stradmeyer et al. 2008). This finding suggests that the link between neural traits, habitat use, and fitness may vary depending on the social rank of an individual (Milewski et al. 2022). Subdominant individuals originating from the common garden experiment with higher n-6 PUFA content in the brain tended to occupy the downstream part of the enclosure and exhibited greater somatic growth compared to conspecifics with low n-6 PUFA content in the brain. A high n-6 PUFA content in the brain should not benefit cognitive skills of an individual, but it is often associated with an increased capacity for fatty acid conversion (Pilecky et al., 2021). Therefore, this increased metabolic activity caused by fatty acid conversion in fish originating from the common garden experiment may reflect a greater investment in somatic growth alone rather than in cognitive functions themselves (Colombo et al., 2023). Furthermore, overall brain size in subdominant individuals was positively correlated with somatic growth, but there was no clear effect on habitat use. These findings suggest that anatomical and biochemical neural traits may influence the habitat use and fitness of individuals through various cryptic behavioural and physiological effects. Due to the limited number of studies investigating the fitness value of cognition in natural habitats at intra-specific level, some of these behavioural and physiological effects still remain poorly understood (Morand-Ferron 2016; Logan et al. 2018). Our study revealed that, in wild fish, the high intake of n-3 LC-PUFA—crucial for cognitive development in juvenile trout—primarily stems from their reliance on aquatic prey, not from the trophic position of the fish. This is supported by the positive correlation between n-3 LC-PUFA content in the brain and δ 13 C values in fish muscle tissue, alongside the absence of an association between n-3 LC-PUFA and δ 15 N (Bunn et al., 2013). This suggests that, at least in riverine ecosystems, shifts in the flow of subsidies between stream and riparian systems (Larsen et al., 2016) and the reliance of individuals on terrestrial versus aquatic prey sources influence the cognition and social structure of top consumers. However, changes in food chain length (Ward & McCann, 2017), are less likely to influence the cognition of top consumers and their ability to maintain high fitness in the ecosystems impacted by anthropogenic pressures. In conclusion, our findings indicate that insufficient dietary intake of n-3 LC-PUFA impairs the behavioural flexibility of top aquatic consumers, specifically limiting their ability to establish and maintain social dominance and optimal habitat use in natural environments. The direct link we found among n-3 LC-PUFA levels, behavioural performance, and growth suggests that nutritional stress could act as a previously underappreciated mechanism affecting population dynamics in changing environments. This is particularly relevant as human impacts on freshwater ecosystems frequently lead to diminished availability of dietary n-3 LC-PUFA and other vital organic compounds through multiple pathways, including altered algal communities, reduced aquatic-terrestrial connectivity, and warming temperatures (Závorka et al., 2023; Shipley et al., 2024). Our results indicate that such changes could create a negative feedback loop where reduced n-3 LC-PUFA availability impairs the very cognitive abilities that animals need to cope with environmental change. This mechanism may help explain why some populations show limited resilience to anthropogenic pressures, even when traditional metrics like prey abundance appear sufficient. Understanding these nutrition-mediated constraints on behavioural flexibility will be crucial for predicting how aquatic consumers will respond to ongoing environmental change and exposure to several simultaneous anthropogenic stressors (Jackson et al., 2021). Acknowledgements We thank following colleagues and students for their assistance with experimental work, and sample analysis: Peter J. Koene, Šimon Houška, Simon Lafont, Marco Reiterlehner, Lukas Wimmer, Johanna Harm, Theresa Reichenpfader, Martin J. Kainz, Lukas Hochbauer, Michi Grohmann, Hannes Hagger, Steven Weiss, and Günter Unfer. This research was funded in whole or in part by the Austrian Science Fund (FWF) [10.55776/P35515]. Authors contribution Conceptualization and Methodology - L.Z., S.S.K., S.V., J.H., P.N.; Data Validation - L.Z., S.A, P.H., S.M., E.O., M.P., P.S., S.V., M.J.Y.; Formal analysis and Visualization - L.Z.; Investigation - L.Z., S.A, B.A., F.D., P.H., S.M., E.O., M.P., A.P., T.S., P.S., S.V., M.J.Y . , P.N.; Resources - L.Z., J.H., K.P., S.V., P.N.; Writing – original draft preparation - L.Z.; Writing – review and editing – all co-authors; Supervision - L.Z., J.H., P.N.; Funding acquisition - L.Z., J.H. not-yet-known not-yet-known not-yet-known unknown Competing interests Authors have no competing interests to declare. Ethics approval These experiments were approved by the Austrian Federal Ministry of Education, Science and Research (license GZ: 2023-0.053.856) and comply with current laws in Austria and EU. References Auer, S., Hayes, D. S., Führer, S., Zeiringer, B., & Schmutz, S. (2023). 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Keywords cognitive ecology essential nutrients freshwater food webs global change salmonid fishes Authors Affiliations Libor Závorka 0000-0002-0489-3681 [email protected] WasserCluster Lunz - Biological Station View all articles by this author Johan Höjesjö University of Gothenburg View all articles by this author Stefan Auer BOKU View all articles by this author Benedikte Austad 0000-0002-1779-2396 University of Gothenburg View all articles by this author Francesco Dionigi Charles University View all articles by this author Pernilla Hansson University of Gothenburg View all articles by this author Shaun Killen University of Glasgow View all articles by this author Stefano Mari 0009-0007-2964-7843 WasserCluster Lunz View all articles by this author Evelina Olsen University of Gothenburg View all articles by this author Matthias Pilecky 0000-0002-3404-5923 Donau-Universitat Krems View all articles by this author Kurt Pinter BOKU View all articles by this author Alexandra Polonyiová Charles University View all articles by this author Tileuzhan Smagul Charles University View all articles by this author Patrik Stehlík Charles University View all articles by this author Simon Vitecek WasserCluster Lunz - Biologische Station GmbH View all articles by this author Mourine Yegon WasserCluster Lunz View all articles by this author Pavel Němec Charles University View all articles by this author Metrics & Citations Metrics Article Usage 391 views 253 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Libor Závorka, Johan Höjesjö, Stefan Auer, et al. 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