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 14C ’release). The
ratio ofF 14C ’release/F 14C releasewas used to indicate the 14C recovery of the total
released 14C in seawater.