Prior to the beginning of the study, turtles were all fed ad libitum to establish average daily

intake. During the study, turtles in the ad libitum group (AL) were offered an excess of food each

day for 12 weeks, turtles in the restricted group (R) were fed 50% of average initial AL intake

each day for 12 weeks, and turtles in the restricted-ad libitum group (R-AL) were fed the

restricted amount of food for five weeks and then were fed ad libitum for seven weeks. The

amount of food offered during food restriction was sufficient to maintain turtles on a positive

growth trajectory. Turtles were weighed and measured (straight carapace length) each week.

Tissue Collection

 At the conclusion of the twelve-week study, seven AL turtles, ten R turtles, and ten R-AL

turtles were weighed to the nearest 0.1 g and euthanized with an intramuscular overdose

injection of ketamine (Ketaset, 100 mg/kg body mass) in the right pectoral muscle. When each

turtle failed to respond to a pain stimulus, it was decapitated.

 A blood sample was collected from the decapitation site (as in Storey et al. 1993 and

Packard et al. 1997). The heart and a portion of the right lobe of the liver were excised, and

blood, heart, and liver samples were snap-frozen in liquid nitrogen no more than three minutes

after decapitation. Tissues were maintained at -80 C until they were homogenized as described

below.

Biochemical Assays

 Tissues from each individual turtle were analyzed for DNA and RNA concentrations.

Subsamples of frozen whole blood, heart, and liver were weighed, and DNA was isolated with

DNeasy kits (Qiagen Inc., Valencia, CA) using the manufacturer's protocol for animal tissues.

To isolate RNA, subsamples of frozen heart and liver tissue (different from those used for DNA

isolation) were weighed and then ground in liquid nitrogen using mortar and pestle. Subsamples

of blood (different from those used for DNA isolation) were weighed but not ground in liquid