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106 BESTGEN AND BUNDY TABLE i.-Summary of objectives and protocols for experiments with young Colorado squawfish. Photoperiod was 14 h light; 10 h dark for all experiments, dissolved oxygen was 5-6 mglL, and fish in all experiments were fed at the prescribed rate twice per day, Fish age (d) Artemia nauplii Experiment Objective Treatment at initiation per feeding Age validation; Detennine otolith microstructure Constant 22DC or :t2.5'C Ad libitum , otolith growth and growth in specimens up to fluctuations around 18, 22, or 165 d old 26DC Growth effects Experiment 1 Determine otolith growth rates Slow growth: 200 fishl2 L; Slow, 3D/fish; in fast- and slow-growing fish fast growth: 100 fishl2L; fast, 60/fish up to 122 d old constant 26DC Experiment 2 Detennine effects of starvation on Starve for 6 d between two 6-d 11 Ad libitum otolith and somatic growth of feeding periods; temperature individual larvae constant at or fluctuating around 22DC Experiment 3 Determine effects of starvation Larvae fed after 0 (control), 6 Ad libitum period on otolith and somatic 5, 10, 15, or 17,5-d starvation growth periods; constant 21 DC Experiment 4 Detennine effects of temperature Larvae reared at 18DC or 25DC 14 Ad libitum increase and tne time of first with and without food feeding on otolith growth over 7 d gins at hatch; (2) increment deposition is daily in otoliths of fish reared in constant and fluctuating temperatures; (3) starvation reduces increment contrast and otolith growth; (4) otolith growth (in- crement width) increases when fish begin feeding and when they are subjected to higher temperature; and (5) otolith growth rates are directly propor- tional to somatic growth rates. Methods Age Validation Colorado squawfish embryos were obtained from Dexter National Fish Hatchery and Tech- nology Center, Dexter, New Mexico, I d after eggs were fertilized. Groups of approximately 200 I-d- old Colorado squawfish embryos were randomly assigned to treatments (Table I) to assess timing of otolith formation and effects of constant tem- perature (220C) and daily temperature fluctuation (::'::2SC from 18, 22, or 260C) on otolith micro- structure and otolith growth. Temperature fluctu- ations mimicked the natural cycle: lowest from 0100 to 0700 hours; warming at a constant rate from 0700 to 1300 hours; highest from 1300 to 1900 hours; and cooling at a constant rate from 1900 to 0100 hours. Embryos were acclimated to test temperatures at rates of about 20C/h and were incubated and reared in 2.0-L aquaria (20 em X 10 em X 12.5 em deep). Water flowing through each aquarium prevented accumulation of wastes. Fish were always sampled between 1200 and 1400 hours and samples were preserved in 100% ethanol prior to and just after hatching (5 d postfertilization at 22 and 260C, 6 d at 180C) and at weekly to monthly intervals thereafter. Timing of initial oto- lith microstructure formation and otolith size at time of first increment deposition were determined from samples collected before and immediately af- ter hatching. Artemia nauplii were the only food provided during this and other experiments re- ported here (Table 1). For age validation, 3-5 individuals were ran- domly selected from a larger lot of specimens pre- served at intervals and were measured for standard and total lengths to the nearest 0.1 mm. Preserved specimens shrank less than 3% (Bestgen 1996) and no correction for shrinkage was made because wild fish would be preserved similarly. Otoliths were removed from specimens and mounted on num- bered microscope slides in either immersion oil or thermoplastic cement. Maximum otolith diameter and radius were measured with a compound mi- croscope and a calibrated ocular micrometer at 100X or 400X magnification. Otoliths mounted in immersion oil were examined without further preparation; those in thermoplastic cement were ground on one side with wet-dry sandpaper and lapping film and covered with immersion oil; all otoliths were examined at 400x or I,OOOX mag- nification. Sagittae became irregularly shaped and their in- crements were difficult to distinguish after fish were 30 d old, so only Iapilli, which were more consistently oval, were analyzed for older fish. In- ,