Experimental design
The experimental fish originated from wild zebrafish (Uusi-Heikkilä et al., 2010), which had been reared in laboratory for 13 generations and from two different size-selected populations with two replicates both. Fish were selected for either large or small body size for five generations (Uusi-Heikkilä et al., 2015). The size-selective harvesting induced substantial phenotypic and genetic differences between the selection lines, which were consistent between the two selection-line replicates (Uusi-Heikkilä et al. 2015, 2017). Large-selected fish had lower juvenile growth rate than small-selected fish but reached higher maximum body size (L), had higher condition factor and reproductive success, and behaved differently (i.e., were more active, explorative, and bold; Uusi-Heikkilä et al., 2015; 2017). Fish used in this experiment had recovered (i.e., no harvesting) eight generations from size selection. Although some phenotypic differences might had eroded during the recovery period, there were still large-scale genetic differences between the selection lines (Uusi-Heikkilä et al., 2017) and certain behavioral and cognitional differences between the selection lines have been shown to remain after up to 10 generations of recovery (Roy et al., 2023; Sbragaglia et al., 2019). Selection line refers to the life-history type and we use these two terms interchangeably (small-selected fish=fast life-history type and large-selected fish=slow life-history type). Selection-line replicate refers to the two replicated populations within a selection line.
We used 10 females and 20 males from each selection-line replicate as parental fish. One female and two males were placed in a spawning tank and eggs were collected over five days. Embryos (and subsequently larvae and adult fish) were exposed to five different concentrations of MnSO4: 0.17, 0.5, 1.5, 3.5, and 7.5 mg L-1. Embryos of the control treatment were kept in tap water (approximately 0.02 Mn mg L-1). In preliminary experiments with concentrations of MnSO4 higher than 7.5 mg L-1, larval survival was extremely poor and therefore this was the highest concentration we utilized in this experiment. After hatching, larvae were moved to rearing tanks and fed daily with dry food (TetraMin) and Rotifers. Rearing temperature was kept in 26-27 °C and the water exchange took place once a week.
At age of 70 days post fertilization (dpf), fish were considered robust enough to survive from handling and measuring. For that, five individuals per selection-line replicate were moved to rearing containers, which were placed to 30 L aquaria with eight containers in each (Figure S1). Because of uneven hatching and larval survival rate, the number of containers slightly varied per selection line per concentration. From now on, fish in the rearing containers are referred to as “rearing group”:
Despite weekly water exchange, Mn concentrations measured from the rearing water were low compared to the nominal concentrations (Table 1) likely because Mn accumulated in fish and absorbed on the surfaces of the aquaria and filters. The observed Mn concentrations (0.19 and 0.41 mg L-1) in our highest exposure treatments were, however above the recommended chronic no-effect concentrations (Harford et al., 2015). Sulfate concentrations in our experiments remained well below the predicted no-effect concentration of sulfate (39 mg L-1) in soft freshwaters (Karjalainen et al., 2023).
More details about the experimental design can be found in the Supplementary Material.