**2.3 Iodine analyses**

36 Aquaculture

foodstuffs are commonly used to avoid iodine-deficiency pathologies such as hyperthyroidism and goiter. Nevertheless, environmental conditions such as poor water quality or malnutrition can lead to marked proliferation of thyroid tissue in some fishes (Hoover, 1984) indicating that care must be taken to maintain a minimum iodine level in artificial environments including zebrafish aquaculture facilities. Dietary supplements have been shown to prevent thyroid hyperplasia in the saltwater cyprinodont *Fundulus* (Pickford, 1954) and goiters in the Chinook salmon (Woodall & La Roche, 1964), but no study to date has effectively assessed the minimum iodine level for maintaining normal development and

While experimenting with varied diets in our facility, we observed that conditions of reduced iodine result in less robust somatic growth and an elevated incidence of jaw dysmorphogenesis. This prompted us to evaluate the developmental consequences of rearing zebrafish on a single live food or a mixed food diet with varying levels of iodine. Herein we demonstrate that a strict single-food *Artemia* diet, although high in the protein necessary for robust egg production, may also result in a morphological alteration of the zebrafish jaw architecture formed in response to swelling and hypervascularization of

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

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

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

thyroid health in zebrafish.

**2. Experimental methods** 

Ecosystems) in the afternoon.

**2.2 Dietary paradigm** 

*Artemia* feedings per day.

**2.1 Animal housing and maintenance** 

adjacent tissues.

Quantification of iodine in the foodstuffs was performed using ICP-MS as described previously for other heavy metals (Figueroa et al., 2008). Briefly, *Artemia* samples were cultured for 72 h, harvested and freeze-dried overnight in a lyophilizer (FreeZone 4.5 Liter Benchtop System, Labconco Corporation, Kansas City, MI). Three replicates of dried *Artemia*  and system water samples (~30 mg) were placed in septum sealed glass tubes, treated with HNO3 and microwave digested: power, 130 W; ramp, 1:00 min; hold, 1:00 min; temp, 75 °C. Subsequently, 0.1 mL of 30 % H2O2 was added and the samples were processed through the second digestion stage: power, 130 W; ramp, 1:00 min; hold, 1:00 min; temp, 110 °C. Samples were diluted with DDW to 10 mL and analyzed for total iodine content in continuous flow mode using an Agilent 7500ce ICP-MS (Agilent Technologies, Tokyo, Japan) equipped with a quadrupole mass analyzer.
