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Fishery Bulletin 89(2). 1991 



over a 24-hour period. Otolith and somatic growth were 

 defined by the rate of in vitro calcium deposition on 

 otoliths and RNA-DNA ratios in white trunk muscle, 

 respectively. Serum calcium concentrations were ex- 

 amined for diel variations based on previous work on 

 the relationship between serum calcium and otolith 

 growth (Mugiya 1984). 



Materials and methods 



We performed two experiments with food and diel 

 effects on somatic and otolith growth. Immature rain- 

 bow trout Oncorhynchus mykiss weighing 100-130g 

 were obtained from a trout farm and acclimated to ex- 

 perimental conditions for at least 4 weeks before use. 

 Throughout the acclimation and experimental periods, 

 they were maintained in a pair of outside concrete 

 ponds (3.8m 2 x 0.7m in depth) which were supplied 

 with running water at 14 ± 0.5°C, and fed commercial 

 trout pellets once a day at around 1500 hours, unless 

 otherwise stated. The ration fed was about 1.5% of 

 body weight. 



Starvation and refeeding 



This experiment was carried out in October 1988. Dusk 

 occurred at 1700 hours and dawn at 0545 hours. Sixty 

 fish were randomly divided into two groups (30 fish per 

 group), and separately assigned to one compartment 

 of the paired ponds. One group was starved for 5 days 

 and then fed. During these periods, they were sampled 

 on days 1 (43 hours from last feeding), 2, 3, and 5 after 

 starvation, and on days 1 (19 hours from the first 

 refeeding), 2, 3, and 4 after refeeding. The other group 

 was fed throughout the period and sampled as the con- 

 trol on the same time schedule, except on the third day 

 of starvation and on the second day of refeeding when 

 only the experimental group was sampled. Sampling 

 was conducted alternately between experimental and 

 control groups at 0945-1045 hours every day. At each 

 sampling, 4-6 fish were gently netted one at a time, 

 and immediately bled by cutting the tail of the fish. 

 After bleeding, a pair of sacculi containing otoliths 

 were isolated, placed in an incubation medium, and 

 then the next fish was netted. The remaining part of 

 the body was stored at -40°C and analyzed within 5 

 days for RNA-DNA ratios. Data from the control group 

 were pooled for statistical analyses, as little difference 

 was found among the sampling days. 



Diel variation 



This experiment was carried out in December 1988. 

 Dusk occurred in 1600 hours and dawn at 0700 hours. 



To examine the diel relationship between otolith and 

 somatic growth, 25 fish were stocked in each compart- 

 ment of the paired ponds, and 7 fish each were ran- 

 domly sampled at 6-hour intervals of 1000, 1600, 2200, 

 and 0400 hours over a 24-hour period. Sampling was 

 conducted alternately from one compartment at 1000 

 and 2200 hours, and from the other compartment at 

 1600 and 0400 hours. This sampling regime is recom- 

 mended for minimizing handling effects. After fish 

 were netted one at a time, blood was immediately col- 

 lected from the caudal vessels by cutting the tail of the 

 fish and draining it into test tubes. After centrifuga- 

 tion, the separated sera were stored at -40°C for 6-24 

 hours and analyzed for total calcium concentrations by 

 flame photometry using an atomic absorption spectro- 

 photometer (Hitachi #518). After the blood collection, 

 sacculi were isolated for incubation, and the remain- 

 ing part of the body was stored for RNA and DNA 

 analyses. In this experiment, the fish were starved 

 throughout the sampling day. 



Otolith incubation 



Otolith-containing sacculi were isolated according to a 

 previously described technique (Mugiya 1987). Isolated 

 sacculi were incubated in 50 mL of a Ringer solution 

 (Mugiya 1986) containing 45 CA (New England Nu- 

 clear) at a concentration of 1 x 10 4 Bq/mL. Incubation 

 was carried out with oxygenation at 14°C for 2 hours. 

 After incubation, sacculi were rinsed several times 

 in 45 CA-free Ringer solution and the otoliths were sep- 

 arated under a binocular microscope. The separated 

 otoliths were lightly rinsed in water, placed in each 

 counting vial, dried overnight at 80°C, and then 

 weighed. They were solubilized in a mixture of 0.2 mL 

 perchloric acid and 0.2 mL hydrogen peroxide (Mugiya 

 1987), and added to Scintisol EX-H (Wako Chem.) for 

 radioactive counting (liquid scintillation spectrometer, 

 Aloka LSC-673). The rate of otolith growth was evalu- 

 ated in terms of microgrammes of calcium deposited 

 per unit otolith weight. 



RNA and DIMA determinations 



RNA-DNA ratios were estimated in the white muscle 

 collected from an area of the dorsoanterior trunk, using 

 a modification of the Schmidt and Thannhauser method 

 (Buckley and Bulow 1987). Briefly, white muscle 

 (l.OOg) was homogenized with cold distilled water and 

 adjusted to 10.0 mL. A 1.4-mL aliquot of the homog- 

 enate was used for the estimation of nucleic acids. RNA 

 and DNA were purified with 0.6 N cold HC10 4 , and 

 then extracted with 0.3N KOH and 0.6N hot HC10 4 , 

 respectively. Both acids were quantified from the ab- 

 sorbance of the extracts at 260 nm. RNA and DNA 



