We caught wild juvenile sheepshead minnows (Cyprinodon variegatus) from South Carolina (SC), Maryland (MD) and Connecticut (CT) in mid-August in 2014. All fish were transferred to acclimation aquaria at 24°C at the NOAA Fisheries Science Center, Santa Cruz, California. These temperatures represent the range experienced by sheepshead minnows from SC, MD and CT during a normal non-breeding season. Daily care followed standard protocols (Cripe et al. 2009, Salinas and Munch 2012), including ad libitum feeding of TetraMin flakes (Tetra Holding, Blacksburg, VA, USA). Salinity was maintained at 20 ppt, but was reduced to 10 ppt for two days prior to egg collection. The photoperiod was 14L:10D. Each day we changed 10% of the total volume of water.

For the experiments of thermal transgenerational plasticity, all eggs were divided in half and transferred to either same temperature with parent or different temperature with parent: for example, if we collected eggs from 26°C parents, then a half of eggs were at 26°C and another half of eggs were at 32°C. Upon hatching we randomly selected up to four larvae from each treatment group. We measured standard body length from photographs of the fish obtained with a Canon 40D digital camera with Image J (Rasband 2016). At the end of experiment, we measured wet-mass, and them removed and weighted the testes and gonad.