2.2.2 Fish rearing
Marine medaka (O . melastigma ) were reared under the same conditions as the rotifer. Four-month-old fish that had been acclimated to feed on the rotifer (B .plicatilis ) were used for the experiments. None of the fish reached sexual maturity during the experiment. The average fish weight was 77.7 ± 11.2 mg in wet weight (WW) and 23.5 ± 3.7 mg in dry weight (DW). The ratio of DW to WW for the experimental fish was 30.3% ± 2.7% (n = 4). The carbon content of the fish was 46.7% ± 8.4% (n = 6) of the DW (see details in Text S2).
2.2.3 Short-term depuration experiments: examining the allocation of ingested food carbon by labeling fish food with 14C
Prior to the experiments, the experimental fish were placed in aerated FSW without the presence of food for 24 h for evacuation. Fish were then transferred individually into 50-mL feeding beakers with 25 mL aerated FSW. The three rations of14C-labeled rotifers were 1000, 1500 and 4000 ind./fish, corresponding to 2.2%, 3.2% and 8.6% of the fish DW, respectively (assuming that each rotifer contained 226 ng C, and its carbon content was a factor of 0.444 of its DW) (Hansen et al., 1997; Øie et al., 1997). The fish were fed different rations of rotifers for 20 min, a period shorter than the fish gut passage time (approximately 30–60 min) (see detailed methods in Text S3; data not shown). All rotifers were consumed during the feeding period, except in the ration of 4000 ind./fish, in which only 2650 rotifers (equal to 5.7% of the fish DW) on average were eaten by each fish.
After being fed the radioactive food, the fish were collected, rinsed with FSW, and immediately transferred to beakers with 25 mL of new FSW for depuration for 36 h. The seawater in the beaker was changed at 2, 4, 8, 16, 24, and 36 h. Three to five fish were collected at 0, 2, 4, 8, 16, 24, and 36 h for measuring14C in fish. Feces and water samples were collected from the depuration system for measuring 14C in the form of PC, CO2, DOC, and colloidal organic carbon (COC) at 2, 4, 8, 16, 24, and 36 h. Fish feces were collected by filtering all seawater in each beaker through a polycarbonate membrane (0.2 μm pore size, Millipore). 14CO2 in 15 mL of the filtrate was collected into 5 mL of 1 M NaOH according to a method described by Lee and Fisher (1992). Two portions of 3-mL aliquots of the filtrate were used for measuring DO14C and CO14C in the seawater according to the method described by Zhang and Wang (2004). The fish and feces were digested in 1 mL of 2 mol/L NaOH at 80°C overnight for 14C measurement according to the method described by He and Wang (2006). The detailed procedures for collecting and processing the samples are provided in Text S4.
To determine the radioactivity of 14C in all the above samples, a 4-mL liquid scintillation cocktail (OptiPhase Hisafe 3, Perkin-Elmer Life Science) was added to each sample. The sample was thoroughly mixed by vortexing and placed in the dark for more than 12 h.The sample was then was revortexed before being measured by a liquid scintillation counter (Beckman LS 1801). The 14C recovery rates in the fish (41% ± 5%, n = 3), feces (41% ± 5%, n = 3), DOC (75% ± 1%, n = 3) and CO2 (46% ± 0.0%, n = 3) were used to calculate the actual 14C in the samples.
The decreasing rate of 14C retained in fish (h-1) was calculated as the slope of the linear regression of the natural logarithms of 14C in fish with the time of depuration. The carbon AE was operationally calculated as the percentage of ingested 14C retained in the fish after depuration. The release rates of DO14C,14CO2 and P14C (µg C/mg DW/h) at each stage during the depuration were calculated by dividing the measured 14C in each form by the time interval of the stage and then normalizing this result to the DW of the fish. According to the mass balance, the apparent food carbon release (F 14C release) was calculated as the difference between the 14C ingested at the beginning and the 14C retained in fish at the end of the depuration. The sum of the actually collected14CO2, DO14C and P14C during depuration was taken as the measured food carbon release (F 14Crelease). The ratio ofF 14Crelease/F 14C releasewas used to indicate the 14C recovery of the total released 14C in seawater.