Discussion
Dietary reliance of brown trout on terrestrial prey increased in sympatry with invasive brook trout. The increased reliance on terrestrial prey did not appear to be influenced by the low availability of aquatic prey, indicating that it was likely caused by the behavioural changes of sympatric brown trout exposed to the invasive species (Lovén Wallerius et al. 2017; Larranaga et al. 2018; Cucherousset et al. , 2020). Higher reliance on terrestrial prey of sympatric brown trout resulted in lower relative content of EPA in their muscle tissues. In the sympatric populations, the relative content of ALA increased and the relative content of DHA decreased in tissues with increasing reliance on 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, i.e., Bowker 1995; Öhlund et al. 2008) individuals relied more on terrestrial prey and had lower relative content of EPA and DHA in muscles. We found that the relative content of DHA in muscle tissues was positively related to the total brain volume. However, brain volume did not differ between sexes and allopatric and sympatric populations. The brain morphology was not explained by any variable considered. These findings demonstrate that the variation in diet quality can cause divergence of n-3 LC-PUFA content in tissues of wild fishes and that DHA content in muscle tissues is a predictor of the brain size.
Lipids are the densest form of energy and key to dietary energy transfer from producers to consumers (Parrish 2009). Therefore, the dietary intake and subsequent 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 (Ishizaki et al., 2001; Lund et al., 2012), that the availability of structural lipids, especially of DHA, and not the energy reserve is key for brain development in wild fishes. These findings suggests that the specialization of dietary niche on resources that have similar content of energy, but differ in the content of n-3 LC-PUFA (Heissenberger et al. 2010; Scharnweber et a. 2021), can lead to diversification of brain development. This important finding indicates that diet quality induced differences in brain development can eventually also influence the cognitive capacity of individuals as small brain volume has negative effects on learning (Marhounová et al. 2019; Kotrschal et al. 2013) and on foraging behaviour (Wilson & McLaughlin 2010). Omega-3 LC-PUFA-deprived diets have also been shown to reduce mitochondrial efficiency of muscles (Salin et al. 2021; Závorka et al. 2021) and reduce n-3 LC-PUFA in fish eggs (Hou & Fuiman 2020). Therefore, carefully designed future experiments in a realistic ecological context are needed to understand the trajectory of how dietary omega-3 PUFA impacts consumer fitness.
Effects of diet quality on the physiology and brain development of fishes necessarily depends on temporal scales on which the consumption of different dietary resources occurs (Murray et al. 2015; Hou & Fuiman 2020). The relatively short study (i.e. , 8 weeks of dietary treatment) by Závorka et al. (2021) on juvenile Atlantic salmonSalmo 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 (i.e., 21 weeks of dietary treatment) by Lund et al. (2012) on juvenile pikeperchSander 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 δ13C 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 brown trout sympatric with invasive brook trout occurs early in ontogeny and remains stable across all life stages (Cucherousset et al. , 2020). Thus, it is possible that the observed diet differences in our study represent a long-term dietary specialization of individuals. Brown trout could also acquire and retain DHA from seasonal resources not included in our prey analysis, for example via egg predation in autumn (Aymes et al. 2010; Näslund et al. 2015), but internal synthesis from precursor molecules (i.e., ALA, but mainly EPA) contained in prey macroinvertebrates was likely the key source of DHA (Heissenberger et al. 2010; Twining et al. 2019; Guo et al. 2021). Interestingly, despite higher reliance on 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 via elevated retention and/or synthesis of DHA (Ishikawa et al., 2019). However, our results also indicated limits of this adaptation as the relative content of DHA in sympatric individuals decreased with increasing reliance on terrestrial prey. Therefore, individuals that relied mostly on terrestrial prey were clearly unable to compensate the impact of poor diet quality on the biochemical composition of their tissues. This is not surprising, because the synthesis of DHA from dietary precursors comes at a substantial energetic cost that can lead to reduced growth rate (Murray et al. 2014; Závorka et al. 2021). The increased metabolic cost due to the elevated DHA synthesis could possibly explain the lower growth rate and higher mortality of brown trout sympatric with brook trout observed in previous studies (Öhlund et al. 2008; Závorka et al. 2017).
Sexual differences did not influence the reliance on terrestrial prey, the relative content of n-3 LC-PUFA in muscles, and total brain volume and morphology. The lack of sexual differences in brain size morphology contrasts with findings of Kolm et al. (2009) who showed that females of stream resident brown trout have overall larger brain than males. In contrast to the landlocked populations of our study, Kolm et al. (2009) sampled individuals in a costal stream, which mostly contained anadromous individuals. Therefore, the gene flow between anadromous and residential part of the population (Nevoux et al. 2019) and the higher availability of marine derived n-3 LC-PUFA delivered to the stream by anadromous spawners (Näslund et al. 2015) could possibly lead to the sexual divergence in brain morphology that does not occur in landlocked populations. The lack of sexual difference in n-3 LC-PUFA muscle content could be explained by the fact that our study was conducted in early summer when investment to n-3 LC-PUFA rich gonads is low compared to late autumn when the spawning season occurs (Jonsson & Jonsson 1997). The size range of individuals in this study corresponds to subadult and adult life-stage (Öhlund et al. 2008) when the capacity to retain and/or synthetize n-3 LC-PUFA internally decreases compared to the early ontogenetic stages (Chaguaceda et al. 2020). However, we still found that larger individuals relied more on terrestrial prey and had lower EPA and DHA contents in muscles. This confirms that larger and older individuals may require less EPA and DHA than juvenile freshwater fishes (Tocher 2010; Chaguaceda et al. 2020).
In conclusion, this field study demonstrates the largely overlooked importance of dietary intake of n-3 LC-PUFA for brain development of wild fishes, which have so far been recognized mainly under laboratory and aquaculture conditions (Tocher 2010; Pilecky et al. 2021). We demonstrated effects of reduced dietary intake of n-3 LC-PUFA induced by co-existence with an invasive species, however, other anthropogenic factors can alter the availability of n-3 LC-PUFA for stream dwelling fishes and other consumers even more profoundly. For example, climate change is predicted to decrease the availability of n-3 LC-PUFA in aquatic food-webs (Hixson & Arts, 2016). It still remains to be determined how wild animals respond to diet quality shifts, but our results indicate that the reduction of dietary n-3 LC-PUFA can have negative impacts even on species which are adapted to n-3 LC-PUFA deprived prey (Syrjänen et al. 2011; Závorka et al. 2021). Dietary intake of omega-3 LC-PUFA have a high potential to affect fitness of consumers (Twining et al. 2021; Pilecky et al. 2021), and therefore further studies are needed to understand how the availability n-3 LC-PUFA affects brain development, behaviour and physiology of wild fishes and other animals.