Fish collection and rearing
We established lab populations of guppies from high- and low-predation populations pairs collected from the Aripo and Quare river drainages in 2012 and 2014, respectively. To maintain the genetic variation of the original wild-caught fish, we established 20 - 30 unique family lines for each population (i.e., for each generation a single female from each family was crossed to a single male from another family) (D. N. Reznick & Bryga, 1987). We used second-generation lab born fish from these unique family lines in this study to minimize environmental and maternal effects. At birth, we split second-generation siblings into rearing environments with (pred+) or without (pred-) predator chemical cues, and they remained in these environments until the completion of the experiment (as in Fischer et al., 2016). We used only mature males in this study. To maximize the range of genetic variation captured among focal fish, all males in a given experimental group (i.e. population and rearing environment) were from distinct families; however, our split-brood design allowed us to simultaneously leverage genetic similarity among siblings and control for the effects of genetic background on developmental responses. Figure 1 provides an overview of our experimental design and interpretation of comparisons.
All guppies were individually housed in 1.5 liter tanks on a 12:12 hour light cycle at Colorado State University. Fish were fed a measured food diet once daily, receiving Tetramin™ tropical fish flake paste and hatched Artemia cysts on an alternating basis. Prior to tissue collection for this study, behavioral and hormone data were collected in an identical manner for all fish and these results are reported elsewhere (Fischer et al., 2016b). All experimental methods were approved by the Colorado State University Animal Care and Use Committee (Approval #12-3818A).