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.