FISHERY BULLETIN: VOL. 85, NO. 3 



relate its phase, if apparent, to otolith calcifica- 

 tion. Because diurnal variations in otolith calcifi- 

 cation show seasonality (Mugiya 1984), an ideal 

 way to examine such a phase relationship is to 

 determine the rates of calcium and matrix deposi- 

 tion on a single otolith simultaneously, using a 

 double-tracer method and in vitro, isolated sac- 

 culi from rainbow trout. Diurnal profiles of glu- 

 tamic acid and calcium uptake were examined in 

 the otoliths and the remaining saccular tissue. 

 Serum calcium and sodium concentrations were 

 also measured for diurnal variations. 



MATERIALS AND METHODS 



Rainbow trout, Sal mo gairdneri, 29-31 cm in 

 standard length, were obtained from a commer- 

 cial dealer and reared in a pair of outside ponds 

 supplied with 14'C running water. They were fed 

 trout food pellets once a day at around 0845 h. 

 Two females showed maturing ovaries, so their 

 data were omitted. No males were excluded be- 

 cause maturing testes have little, if any, effect on 

 the level of serum calcium. The experiment was 

 carried out in December 1984. Dusk occurred at 

 1600 h and dawn at 0700 h. 



At each sampling time, five or six fish were 

 gently netted one at a time. Blood was immedi- 

 ately collected from the caudal vessels by cutting 

 the tail of the fish and draining it into test tubes. 

 After centrifugation, the separated sera were 

 stored at -30°C for 6-24 hours and analyzed for 

 calcium and sodium concentrations by flame pho- 

 tometry using an atomic absorption spectrophoto- 

 meter (Hitachi- 518). 



After blood collection, the head was severed, 

 trimmed, and placed in an oxygenated Ringer so- 

 lution kept at 14'C. The sacculi were dissected 

 under a binocular microscope according to a pre- 

 viously described technique (Mugiya 1984). The 

 pair of sacculi were placed in the incubation 

 medium, and the next fish was netted. Time was 

 recorded to ensure that sacculi from each fish 

 were incubated for the same length of time. 



Isolated sacculi were placed in a glass vessel 

 and incubated in 50 mL of a Ringer solution 

 (Mugiya 1986 1 containing ^''Ca and '^H-glutamic 

 acid (New England Nuclear) at concentrations of 

 approximately 0.17 fxCi mL and 0.33 jxCi/mL re- 

 spectively. The incubation was carried out with 



oxygenation at 14°C for 2 hours. To determine 

 proper incubation times, the uptake of "^H- 

 glutamic acid by otoliths was plotted against 

 time; although in this preliminary experiment, 

 sacculi were incubated for periods of up to 

 3 hours, steady-state levels were obtained in less 

 than 2 hours. 



After incubation, sacculi were rinsed several 

 times in the radioisotope-free Ringer solution and 

 separated into otolith and saccular tissue frac- 

 tions under a binocular microscope. The sepa- 

 rated otoliths were lightly rinsed in water, placed 

 in individual counting vials, dried at 90°C 

 overnight and then weighed. The saccular tissue 

 was directly placed in the vial without a further 

 rinse and air-dried. These samples were solubi- 

 lized in a mixture of 0.2 mL perchloric acid and 

 0.2 mL hydrogen peroxide at about 80°C for 2 

 hours, and added to Scintisol EX-H (Wako) for 

 counting (liquid scintillation spectrometer, Aloka 

 LSC-673). 



Tritium and ^'^Ca activities were measured 

 simultaneously using two channels with nar- 

 rowed windows, 50-300 for '^H and 70-900 for 

 ^■"^Ca. Although the amount of 'H activity enter- 

 ing the Ca channel was found to be practically 

 negligible, '^'^Ca would certainly affect counts on 

 the H channel, despite the window conditions. 

 Therefore, counts on the H channel were cor- 

 rected by the equation: 



^H activity = H - 1/a Ca 



;i) 



where H and Ca represent counts on the H and Ca 

 channels respectively, and « (contamination 

 ratio) is experimentally defined as log 

 a = 0.1386R - 0.0488 where R is a ratio deter- 

 mined for the quenching level of each sample. The 

 validity of this correction was further checked by 

 another equation based on differences in the 

 physical half-life of the isotopes: 



^H activity 



CaoH2 - CaaHo 

 Cao - Ca2 



(2) 



-Reference to trade names doe.s not imply endorsement by the 

 National Marine Fisherie.s Service, NOAA, 



where Hq and Cao ai'e counts on the H and Ca 

 channels at time respectively, and H2 and Ca2 

 are those recounted a few months later. Because 

 these two methods of discrimination gave essen- 

 tially the same results, the data from Equation 

 (1) were presented in this study. 



Some rainbow trout have an aberrant otolith in 

 either or both of their sacculi (Mugiya 1972). If 



396 



