P . Thor J. Exp. Mar. Biol. Ecol. 245 2000 171 –182
173
2. Method
Copepodites stage V of A . tonsa were reared from eggs and copepodites stage V of C.
finmarchicus were caught by horizontal net tows in Raunefjorden, Norway 608 179 N, 58 109 E using a 200 mm mesh WP2 net equipped with a large nonfiltering cod end. The
copepods were held in 60 l tanks at 148C and 34‰ salinity and fed the algae
21
Rhodomonas baltica 6.91 mm ESD; 36.7 pgC cell ; Kiørboe et al., 1985; Støttrup et
al., 1986. The algae were cultivated in a continuous culture in 10 l flasks illuminated at
22 21
100 mE cm s
and diluted to approximately half density with B1 growth medium every other day to ensure exponential growth. After the experiments the prosome length
L, mm of all copepods was measured and the carbon content estimated from the
25 2.92
regression w ngC 5 1.11 ? 10 L
for A . tonsa Berggreen et al., 1988 and w
c c
mgC 5 2 363.6 1 0.217L for C . finmarchicus Thor, in press.
2.1. Respiration measurements Respiration was measured using a flow through technique Møhlenberg and Kiørboe,
1981 adapted to copepods. Copepodites were held in filtered seawater fsw for 24 h prior to the experiments. Fifteen to 25 individuals of A
. tonsa or one to two individuals of C
. finmarchicus were placed in small 400 ml glass chambers fitted with silicone stoppers. Flow through of seawater was maintained through stainless steel needles in the
stoppers, a 200 mm mesh preventing the copepods from entering the outflow. Polarographic oxygen electrodes were connected to the outflows with lengths of tubing
21
Tygon R-3603 never exceeding 5 mm. The water flow-rate was 12 to 18 ml min maintained with a peristaltic pump and the oxygen consumption of the copepods
lowered the oxygen content by 10 to 30. The oxygen electrodes were connected through an amplifier to a computer mounted with data acquisition hardware Computer
Boards CIO-DAS 802. The oxygen content of the outflowing water from six experimental chambers and one reference chamber without copepods was measured
simultaneously every 10 s, and every minute the averages of six consecutive measure- ments were stored.
Oxygen consumption was monitored for 15 to 30 h. After an initial phase of 3 h, in which an equilibrium between water flow and oxygen consumption was reached [the
21
95 flush-out time was 2 h Steffensen, 1989], 1000 mgC l of R
. baltica was added to the inflow. This resulted in an increase in oxygen consumption and when a new
equilibrium was reached the supply of algae was stopped and oxygen consumption was monitored until it reattained prefeeding levels.
Respiration r was calculated as: S v
]]
9
r 5 U
2 U
ref
U W
s c
21
where S is the solubility of oxygen in mlO l
assuming 100 saturation of the
2
inflowing water Green and Carrit, 1967, v is the flow-rate of water through the chambers, and W is the total carbon weight of the copepods. U is the output voltage of
c s
174 P
. Thor J. Exp. Mar. Biol. Ecol. 245 2000 171 –182
the amplified oxygen electrode signal at saturation, U is the electrode signal from the chamber containing the animals, and
U
s
]]
9
U 5 U
ref ref
U
s,ref
where, U is the output from the reference electrode during the measuring period and
ref
U is the output from the reference electrode at 100 oxygen saturation Fig. 1.
s,ref
The magnitude of SDA i.e. the total amount of oxygen respired during and after the
.21
feeding period, in nlO ind
was calculated by integrating the area between the
2
transposed U 9
line dotted line in Fig. 1 and the curve of respiration during and after
ref
feeding. Tests of performance and lag approx. 2 h of the system were carried out by modelling values of oxygen saturation in a one chamber flow through system
Steffensen, 1989. In total SDA was measured in 12 tests of A
. tonsa and eight tests of C. finmarchicus. 2.2. Carbon incorporation measurements
The copepodites were held in 0.2 mm filtered seawater fsw for 24 h prior to the experiment. Exponentially growing R
. baltica were diluted to one half with B1 growth
14 21
medium, inoculated with 330 mCi NaH CO l , and grown for 3 days. They were then
3
centrifuged 2000 rpm, 2 min and rinsed in fsw twice to remove extracellular isotopic activity, and the specific isotopic activity was determined by liquid scintillation
counting.
Fig. 1. Calculation of respiration rate and magnitude of SDA. U is the electrode signal from the chamber containing the animals, U is the electrode signal at 100 oxygen saturation before addition of copepods. U
s ref
is the output from the reference electrode during the measuring period, and U is the output from the
s, ref
reference electrode at saturation. Vertical line shows addition of copepods and horizontal bar shows feeding period. The magnitude of the SDA was calculated as the area between the transposed U
9 lower dotted line
ref
and U.
P . Thor J. Exp. Mar. Biol. Ecol. 245 2000 171 –182
175
During the experiment 432 replicates of 50 A . tonsa copepodite V or 433 replicates
21
of 15 C . finmarchicus copepodite V were fed labeled algae 1000 mgC l
for 2, 6, 12, and 24 h, respectively. Four32 A
. tonsa or 433 C. finmarchicus replicates, acting as
21
nonprotein synthesising controls, were incubated with cycloheximide 1 mg l and fed
labeled algae similarly. All samples of copepods were frozen in 120 ml fsw immediately after the experiments.
For analysis the samples were thawed and protein extracted chemically. The samples were homogenised and 300 ml methanol and 150 ml chloroform were added to extract
lipids Bligh and Dyer, 1959, the relative proportions of the extracting agents being 1:2:0.8 chloroform–methanol–water. After 10 min at 48C another 150 ml chloroform
and 150 ml distilled water was added and the samples were centrifuged at 1000 g for 5 min. The supernatant was then removed. To precipitate proteins the pellet was then
heated to 908C for 30 min in 500 ml 0.3 M trichloroacetic acid followed by ultracentrifugation 15 000 g, 15 min. The supernatant containing polysaccharides was
removed and the pellet containing the proteins was dissolved in 500 ml 1 M NaOH and removed for liquid scintillation counting.
Due to reported low extraction efficiencies of protein Roman, 1991 a test was conducted to compare the amount of protein in 12 extracted and 12 nonextracted
samples of homogenised shrimp Pandalus borealis. The protein in 120 ml extracted or nonextracted subsamples was solubilised with 1200 ml 0.5 N NaOH and the amount of
protein measured using the Bradford method for total protein with bovine serum albumin as standard Bradford, 1976. The protein extraction efficiency was 9064.7.
3. Results
