**2. Experimental methods**

#### **2.1 Animal housing and maintenance**

Adult WIK wild-type zebrafish were raised, maintained, and spawned in the Zebrafish Model Organism Laboratory, located at the University of Cincinnati (UC) Genome Research Institute according to previously published protocols (Craig et al., 2006; Kimmel et al., 1995; Westerfield, 2000). Zebrafish fry were reared in a multi-rack system from Aquatic Habitats (Apopka, FL) with automated control of water chemistry (YSI Model 5200 control system, Yellow Springs, OH) utilizing a 14-h light/10-h dark cycle. Water chemistry was maintained at the following conditions to ensure optimal fish health: temperature: 28.5 ± 1 oC; pH: 7.5 ± 0.2; ammonia: 0 ± 0.25 mg/l; nitrite: 0 ± 0.20 mg/l; nitrate: 5 ± 5 mg/l; total hardness: 102 ±17 mg/l; alkalinity 50 ± 17 mg/l. Conductivity was maintained at 750 ± 50 μS. Adult breeding stock fish were fed a standard diet of 3 day old HUFA-enriched (Super SELCO, Aquatic Ecosystems) *Artemia* in the morning plus a balanced flake food (Aquatox, Aquatic Ecosystems) in the afternoon.

#### **2.2 Dietary paradigm**

Fry were fed a mixed diet of *Paramecium*, rotifers, and Zeigler's larval diet (Zeigler Bros, Inc.) until they were capable of eating *Artemia* (approximately 1 month of age) and to ensure proper early development. Beginning at 1 month of age, fish were placed on either a live study diet of three *Artemia* feedings or a mixed control diet of two Aquatox flake and one *Artemia* feedings per day.

Dietary *Artemia* intake was measured by feeding *Artemia* to overnight fasted fish (17.2 mg per tank, 15 minutes feeding time to approach satiety). Fish were euthanized in concentrated 250 mg/l MS-222, eviscerated and the alimentary tract was removed. The gut was everted, and the total number of ingested shrimp counted under a stereomicroscope. Aquatox flake food uptake was similarly measured by feeding fish to satiety (4.7 mg per tank, given 15 minutes feeding time) and then vacuuming up the excess food with a siphon, drying and weighing the uneaten excess.
