A .C. Brown, N.B. Terwilliger J. Exp. Mar. Biol. Ecol. 241 1999 179 –192
181
lacks subunit six, which is present in the adult Terwilliger and Terwilliger, 1982. Functionally, the intrinsic oxygen affinity of the larval juvenile 25S hemocyanin is half
the oxygen affinity of the adult 25S hemocyanin under identical experimental conditions Terwilliger et al. 1986. The oxygen affinities of the stage specific 25S hemocyanins
show differential sensitivity to the effects of calcium and magnesium ion concentrations Terwilliger and Brown, 1993. In the whole hemolymph of animals from 100 SW and
normal temperatures ¯ 108C, however, the apparent oxygen affinity is indistinguish- able between the stages Brown and Terwilliger, 1998, due at least in part to differences
in hemolymph ion concentrations.
All of these factors, developmental stage, habitat, ionic and osmotic regulatory patterns, and hemocyanin oxygen transport function, will affect the overall metabolism
of an animal exposed to changes in salinity and temperature. Measurement of oxygen uptake, as an estimate of metabolism of an organism, assuming aerobic metabolism, is a
valid method of assessing the organismal response to changes in the environment Cameron, 1989. In order to determine the extent of physiological stress that a certain
combination of external parameters imposes on an organism it is useful to look at oxygen uptake and some of the functional features of the respiratory protein in oxygen
transport. This study examines oxygen uptake of four life stages of C
. magister exposed to various salinities and temperatures that were determined to mimic the habitat changes
experienced by the life stages due to semidiurnal tides. The results are discussed in conjunction with C
. magister hemocyanin oxygen dissociation properties Terwilliger and Brown, 1993; Brown and Terwilliger, 1998 and blood gas parameters Johansen et
al., 1970. Estimated cardiac output necessary to fuel the apparent oxygen uptake is also discussed as a measure of performance of the circulatory system of the different
developmental stages.
2. Materials and methods
2.1. Animals and habitat conditions Cancer magister megalopas, juveniles and adults were caught and maintained as
previously described Terwilliger and Brown, 1993. Mudflat and SW salinity and temperature were measured using a refractometer American Optical and a thermometer
at various tide stages in the Coos River estuary.
2.2. Oxygen uptake Oxygen uptake was measured in closed respirometers: 80 and 100 ml were used for
the megalopas and first instar juveniles, 400 ml for the fifth instar juveniles and 5.5 l for the adult crabs. Oxygen uptake measurements were made during acute exposure 8 h of
megalopas and crabs to filtered SW of 32, 24 and 16 ppt hereafter referred to as 100, 75 and 50 SW, respectively at either 10 or 20
8C. Seawater dilutions were made by diluting filtered 100 SW with distilled water and checking the final salinity with a
refractometer. Temperature was maintained throughout the measurement of oxygen
182 A
.C. Brown, N.B. Terwilliger J. Exp. Mar. Biol. Ecol. 241 1999 179 –192
uptake by immersing the respirometers in a thermostated recirculating water bath. All oxygen uptake measurements were made during natural daylight hours. Respirometers
were shielded in styrofoam coolers so crabs were not exposed to visual stimuli during the measurement period and the illumination was dim natural daylight.
The rate of change of oxygen concentration in the sealed respirometer was measured with either a YSI Model 5739 oxygen probe and a YSI Model 57 dissolved oxygen
meter or a YSI Model 5420A oxygen probe stirring boot removed and a YSI Model 54 dissolved oxygen meter. Each chamber was stirred to mix the water and avoid depletion
of oxygen adjacent to the electrode. The crabs were shielded from the magnetic stirbars by a mesh frame over the stirbars. Control respirometers, without crabs, were also
measured to account for any change in oxygen concentration due to microorganisms or other potential chamber effects. There were no significant changes in oxygen con-
centration in the controls.
Food was withheld from the animals for 24 h prior to measurement of oxygen uptake rates to ensure that the animals were in a post-absorptive state and to reduce fouling of
the respirometers. Animals were transferred from the holding aquaria 30–33 ppt at 9–15
8C, with a natural light:dark cycle into the respirometers immediately prior to the experiments. Animals remained in the respirometers for the entire 8-h measurement
period. The crabs were able to move freely around the respirometer chambers. The megalopas swam nearly continuously, while there were brief periods of locomotor
activity in the other stages. During this time the respirometers were reoxygenated at regular intervals by bubbling air through the chambers. This was done to ensure that the
oxygen levels in respirometers would not become limiting. The oxygen uptake was measured during the first, third, fifth and eighth hour Fig. 1a. For the fifth instar
juvenile and adult crabs, the 1-h measurement periods were interspersed with aeration times Fig. 1b and c. Within these shorter periods the rates measured for each interval
were pooled to give an average rate of oxygen uptake for the hour. For each life stage examined there was a small, but statistically insignificant decline in oxygen uptake over
the 8-h acute exposure to the various salinity and temperature combinations. Therefore, the rate of oxygen uptake during the final hour of exposure was used for further
consideration.
A wet weight for each individual was determined at the end of the 8-h experiment, after the animal was blotted on paper towels to drain the branchial chambers. Megalopas
ranged from 0.033 to 0.055 g 3–4 mm carapace width, first instar juveniles were 0.065 to 0.125 g 6–8 mm carapace width, fifth instar juveniles were 2.53 to 5.43 g 25–32
mm carapace width and adults were 278.7 to 495.4 g 120–144 mm carapace width.
2.3. Calculation of cardiac output Cardiac output for each developmental stage in the different salinity and temperature
treatments was calculated using the Fick principle: V
O
2
]]]] V
5 1
b
C 2 C
aO vO
2 2
21 21
where V is the cardiac output in ml hemolymph g h
; V is weight specific rate of
b O
2
A .C. Brown, N.B. Terwilliger J. Exp. Mar. Biol. Ecol. 241 1999 179 –192
183
Fig. 1. Schematic diagram of the timing of measurement of oxygen uptake and aeration intervals. a Measurement and aeration periods from 0 to 8 h during acute exposure to salinity and temperature treatments.
b Measurement periods within each hour measurement interval for 5th juveniles. c Measurement periods within each hour for adults.
21 21
oxygen uptake in ml O g h
; C and C
are the oxygen content of the post and
2 aO
vO
2 2
21
prebranchial hemolymph in ml O dl
hemolymph. Values for C and C
were
2 aO
vO
2 2
derived from the following: 1 oxygen carrying capacity of the hemolymph of the different life stages of C
. magister Brown and Terwilliger, 1998, 2 averaged in vitro hemocyanin oxygen dissociation curves determined at ion concentrations equivalent to
those in megalopas and crabs in 50 and 100 SW at 10 and 20 8C Terwilliger and
Brown, 1993 and 3 values for P 91 mmHg and P
21 mmHg from Johansen
aO vO
2 2
et al. 1970. It should be noted that these P values are an extrapolation to the internal
O
2
conditions of the crabs and megalopas in this study. These values could change with developmental stage, and in response to changes in environmental salinity and
temperature. The results of the calculations are nonetheless informative in terms of identifying potential limitations or capacities of the respiratory and circulatory systems.
2.4. Data analysis The effects of salinity and temperature on the different stages were tested by analysis
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.C. Brown, N.B. Terwilliger J. Exp. Mar. Biol. Ecol. 241 1999 179 –192
of covariance ANCOVA. Multiple comparison of means were made using the Tukey– Kramer method to determine the minimum significant difference MSD. Statistical
significance was accepted at P ,0.05. Statistical analyses were done using
SYSTAT
version 4.1 Systat.
3. Results
