DISCUSSION
Life-history type (i.e., selection line) affected fish performance under stressful environmental conditions. Large-selected fish, which had been selected for large body size, slow juvenile growth rate and old age at maturity (“slow life-history type”) seemed to tolerate better high MnSO4 concentrations than small-selected fish with small body size, fast juvenile growth rate and young age at maturity (“fast life-history type”; Uusi-Heikkilä et al., 2015). Large-selected fish had higher growth rate and condition factor in elevated MnSO4 concentrations than small-selected fish despite no significant differences in standard metabolic rate. They were also more active and had higher feeding probability. These results suggest, that potentially owing to different energy allocation strategies, personalities or stress coping styles, individuals with slow life histories may be better able to cope with chronic environmental stress. Therefore, we should react with caution to statements that a certain stress response is common across all individuals within a species (Balasch & Tort, 2019). Instead, it may depend on individual characteristics, such as life-history types or personalities (Caizergues et al., 2022; Prentice et al., 2022; but see Santicchia et al., 2020).
Although manganese accumulated in a similar manner to fish with both life-history types (Figure 3), the growth response differed between them. Fish with a selection history for slow life performed better than fish with a selection history for fast life in terms of growth (Figure 1). The difference was absent in the control treatment, which indicates recovery of this trait from the past selection but became visible when individuals experienced more stressful environments (i.e., elevated MnSO4 concentrations). Previous study has shown that large-selected fish exhibit more variation in growth and respond to starvation stress differently than small-selected fish (Uusi-Heikkilä et al., 2016). These observations imply that large-selected fish are more plastic and better able to cope with environmental stressors than small-selected fish. Indeed, it has been suggested by other studies that stress coping abilities are affected by the degree of individual phenotypic plasticity (Balasch & Tort, 2019).
The mechanisms behind the higher growth rate of large-selected fish may be related to differences in, for example, stress physiology and/or energy metabolism. We did not detect any significant trends in the standard metabolic rate, which neither differed between the life-history types nor was linearly affected by the MnSO4concentration. This finding could be related to the small sample size, high variation, as fish metabolic rates were measured in groups, and/or to the age of measured fish (i.e., they had already exceeded the period of fast growth). Furthermore, as the MnSO4concentrations could not be controlled in the respirometer chambers, the measurements were done in pure water. In addition to the metabolic rate, there might be other physiological traits (e.g., feed conversion ratio or metabolic efficiency) that could explain the higher growth rate of large-selected fish in high MnSO4 concentrations.
The effective Mn concentrations in our long-term experiment (measured mean concentrations in water 0.19 and 0.41 mg L-1, nominal concentrations 1.27 and 2.73 mg L-1) were higher than the predicted no-effect concentrations for aquatic organisms recommended (Peters et al., 2010; Harford et al., 2015). Acute effective or lethal concentrations of Mn are clearly higher, for example the effective concentration (EC10) of zebrafish embryos is 4.63 mg L-1 (Peters et al., 2010). Mn concentration of more than 4.5 mg L-1 (10 times higher than our highest measured concentration) has been shown to decrease growth in brown trout early stages (Salmo trutta ) and concentrations higher than 15.5 mg L-1 can be lethal (Stubblefield et al., 1997). Interestingly, even very high Mn concentration (100 mg L-1) did not affect brown trout hatching success (Stubblefield et al., 1997). Arola et al. (2017) demonstrated slightly lower concentrations (approx. 15-30 mg L-1) as LC50 value for whitefish (Coregonus lavaretus ) offspring, whereas Lewis (1976) noticed only 1 mg L-1MnSO4 to increase embryonic mortality in rainbow trout. Several studies have demonstrated different sublethal physiological effects of Mn in fish, for example altered hematological parameters (Aliko et al., 2018), such as decreased number of red blood cells and hemoglobin value (Sharma & Langer, 2014) even without mortality itself (Wepener et al., 1992). Furthermore, toxicity level of Mn is suggested to be associated with oxidative stress (Vieira et al., 2012; but see Baden et al., 1995). Increase in water temperature also increases the uptake of Mn in fish (Howe et al., 2004), also raising the potential effect of climate-induced changes in water temperature in this context.
Large-selected fish were not only able to grow faster but they were also able to potentially allocate more energy into fat production particularly in high concentrations indicated by higher condition factor (Figure 1B). This process was, however, severely disrupted in the highest concentration (7.5 mg L-1) where the condition factor of both large- and small-selected fish was five times lower than in other concentrations and could reflect altered metabolic homeostasis. In the highest concentration, there were more individuals with very low than high condition factor among both life-history types. One explanation underlying this bimodal distribution could be related to social structure in zebrafish shoals. In stressful conditions, the competition for food might become more intense as energy demand increases and create more pronounced social hierarchies where few individuals dominate the resources. Among large-selected fish, there seemed to be more of these potentially dominating individuals than among small-selected fish. Similar decreases in condition factor in fish exposed to high heavy metal concentrations have been reported earlier because of reduced feed intake or metabolic activity (Baudou et al., 2017; Eastwood & Couture, 2002; but see Dethloff et al., 2001; Farag et al. 1998;). While the effect of environmental toxins and heavy metals on fish condition has been well studied and demonstrated, less attention has been paid to the interaction between heavy metal concentrations and individual differences in life histories. Therefore, it is important to consider the heterogeneity of stress response among individuals in an environment where several human-induced selection pressures operate simultaneously and potentially antagonistically (e.g., size-selective harvesting and heavy metal exposure).
Energy demand typically increases under stress and growth may be compromised if the animal cannot balance their energetic requirements (Rueda-Jasso, 2004). Indeed, toxic agents may lead to an imbalance between energy supply and demand by negatively affecting feeding behavior either directly or indirectly (e.g., damaging sensory, and/or nervous system; Hoskins & Volkoff, 2012). Studies have reported reduced intake of food in cadmium-exposed fish together with altered swimming activity (Baudou et al., 2017; Ferrari et al., 2011; Sloman et al., 2003). In fish, appetite is commonly associated with increased swimming activity as they search for food. The lower feeding probability (Figure 2B) likely underlies, at least partly, the lower growth rate and condition factor of small-selected fish compared to large-selected fish in our study. Small-selected fish were also less active (Figure 2A), and this could indicate that they were not searching food as effectively as large-selected fish or alternatively they were saving energy. This, in turn, could indicate that they had lower appetite. However, differences in feeding behavior and activity between the two life-history types were already present in the control treatment. Large-selected fish have also been previously shown to be more active and explorative than small-selected fish (Uusi-Heikkilä et al., 2015), thus it seems that these behavioral differences are correlating with other morphological and life-history differences characterizing these different life-history types.
Behavioral responses to stress have been described as reactive (often characterized by freezing behavior) or proactive (e.g., highly active fight or flight behavior). In high MnSO4 concentrations, small-selected fish appeared to adopt a reactive behavior (less active, low feeding probability) whereas large-selected fish behaved evidently proactively (more active, high feeding probability). These behavioral types are considered as adaptations for life in unstable and stable environments, respectively; thus, reactive individuals are characterized by higher levels of physiological stress responses than proactive individuals (Cockrem, 2007). In vertebrates, the physiological stress response involves activation of the hypothalamic-pituitary-adrenal (HPA) axis, where exposure to stress stimulates secretion of glucocorticoids (e.g., Cockrem, 2007). In turn, glucocorticoid secretion elicits a cascade of physiological and behavioral processes that are essential to cope with stressful events (Landys et al., 2006; Wingfield & Ramenofsky, 1999). The stress-coping style hypothesis makes the specific predictions that proactive individuals should have lower baseline concentrations of glucocorticoids and a less reactive HPA axis. Although we did not measure cortisol concentrations of the fish in the present study, we have shown earlier that cortisol concentration in zebrafish correlates negatively with body size and feed intake (Merino et al., unpublished; Uusi-Heikkilä et al., 2018).
Finally, small-selected fish have been shown to differ genetically (both at sequence and gene expression level) from large-selected fish (Uusi-Heikkilä et al., 2015,2017). Although gene expression profiles of the experimental fish were not investigated in the present study, it is possible that there were differences in certain stress-related regulatory mechanisms between the life-history types leading to differences in the ability to compensate the effects of the stressor. This type of a response is often associated to chronic stress since heavy acute stressors may result in death, and mild ones in recovery.
In the present study, we demonstrated that life-history type may affect stress coping ability in fish. Considering the complex response to stress becomes important when human activities are imposing different selection pressures on wild animal populations. For example, fisheries is often size-selective and selects for small body size and fast life-history types (e.g., Jørgensen & Fiksen 2010; Olsen et al., 2004; Uusi-Heikkilä et al., 2015). Harvested populations may experience other human-induced selection pressures in their environment operating, for example, on stress coping abilities. Therefore, if a population mostly consists of individuals with fast life-histories and potentially low stress coping abilities, the two selection pressures operating antagonistically may exacerbate population decline. It is also good to keep in mind that human-induced selection pressures are often directional and may reduce plasticity in a population, which has been suggested to help coping with maladaptive stressors (Balasch & Tort, 2019). While complicating the predictions how organisms may respond to stress, these are important factors to consider when anticipating the effects of multiple, simultaneous human-induced stressors on heterogeneous populations.