L
Journal of Experimental Marine Biology and Ecology 245 (2000) 171–182
www.elsevier.nl / locate / jembe
Relationship between specific dynamic action and protein
deposition in calanoid copepods
* Peter Thor
Department of Life Sciences and Chemistry, Roskilde University, PO Box 260, DK-4000 Roskilde,
Denmark
Received 10 June 1999; received in revised form 20 September 1999; accepted 18 October 1999
Abstract
The link between specific dynamic action (SDA) and protein deposition was investigated in copepodites stage V of two calanoid copepod species, the neritic Acartia tonsa and the oceanic
Calanus finmarchicus. This was done by measuring respiration before, during, and after a specific
feeding period and measuring the incorporation of carbon into proteins. These were also measured on individuals incubated with cycloheximide, an antibiotic that inhibits protein synthesis. The cycloheximide treatment significantly diminished the magnitude of SDA in both A. tonsa and C.
finmarchicus, and inhibited carbon incorporation into protein in both species. This provides
evidence that the rate at which protein deposition takes place greatly affects the magnitude of SDA. The specific respiration rates of both starving and feeding copepods were generally higher in
A. tonsa than in C. finmarchicus. This influenced SDA, the magnitude of SDA normalised to an 8
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h feeding period being threefold higher in A. tonsa (78.7625.7 nlO2 mgC ) than in C.
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finmarchicus (27.5611.6 nlO2 mgC ). This difference may arise due to differences in energy allocation in the organisms of the copepodite V stage of the two species. In this stage C.
finmarchicus deposits large quantities of storage lipids, predominately wax esters, whereas A.
tonsa deposits proteins during somatic growth. 2000 Elsevier Science B.V. All rights reserved.
Keywords: Specific dynamic action; Protein deposition; Respiration; Acartia tonsa; Calanus finmarchicus; Protein synthesis inhibitor; Energetic costs
1. Introduction
In heterotrophic organisms feeding causes an increase in metabolic rate. This has been recorded for a wide range of aquatic animals such as brachiopods (Peck, 1996),
*Corresponding author.
E-mail address: [email protected] (P. Thor)
echinoderms (Vahl, 1984), ascidians (Petersen et al., 1995), fish and fish larvae (e.g. Jobling and Davies, 1980; Kiørboe et al., 1987), molluscs (Gaffney and Diehl, 1986; Carefoot, 1987), and crustaceans (Lampert, 1986; Carefoot, 1990a; Du-Preez et al., 1992) including copepods (Kiørboe et al., 1985). The phenomenon, termed ‘‘specific dynamic action’’ (SDA), is the result of an elevated energy demand for the integrated physical and physiological processes of feeding. The coincidence in time of SDA and increased filtration rate in the blue mussel Mytilus edulis led Bayne and Scullard (1977) to suggest that about 50% of the total SDA was caused by the energetic costs of filtration. However, comparison of metabolic rates of M. edulis feeding on inert particles and algal cells revealed that the energy required for filtering was low, constituting only a few percent of the total energy expenditure (Widdows and Hawkins, 1989). This is also thought to be true for filter-feeding crustaceans (Brendelberger et al., 1986; Strickler and Alcaraz, 1988). Indeed, despite a clear functional response of the clearance rate on the algal cell concentration there was no correlation between respiration rate and filtering rate in Acartia tonsa (Kiørboe et al., 1985). On the contrary, in feeding individuals metabolic rate varied with the rates of ingestion, assimilation and growth. Similar results were obtained with the daphnid Daphnia magna in which a close correlation between the metabolic rate and the assimilation rate was found (Lampert, 1986). Together these observations suggested that the increase in metabolic rate is linked to the processes of assimilation and growth.
Kiørboe et al. (1985) calculated the theoretical energetic costs of absorption, assimilation, and growth in A. tonsa using the macromolecular composition of cirriped eggs. They suggested that 50 to 74% of the measured SDA was caused by the costs of formation of biomass, and that the costs of absorption and assimilation was responsible for 18 to 28%. Thus, the formation of new biomass and the physiological processes leading to it, appear to be the most important factors governing energy expenditure of copepods during feeding.
Different macromolecules are formed during the formation of new biomass: amino acids may be incorporated into structural protein or enzymes, fatty acids may be incorporated into different kinds of lipids, and monosaccharides may be incorporated into polysaccharides such as chitin used in the formation of the exoskeleton (Yamaoka and Scheer, 1970). The energy demand for these processes will of course depend upon the biochemical pathways along which they occur, so formation of different macro-molecules demands different amounts of energy. Theoretical considerations indicate that protein synthesis has the highest energy demand of the processes involved in the formation of new biomass (Grisolia and Kennedy, 1966). Direct measurements of protein synthesis and metabolic rate in larval herring, Clupea harengus, indicate that the energetic demand for protein synthesis may account for almost 80% of the total energy consumption (Houlihan et al., 1995).
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 (608179N, 58109E) using a 200mm 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
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Rhodomonas baltica (6.91mm 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)c 51.11?10 L for A. tonsa (Berggreen et al., 1988) and wc (mgC)5 2363.610.217L 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
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95% flush-out time was 2 h (Steffensen, 1989)], 1000mgC 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:
where S is the solubility of oxygen in mlO2 l assuming 100% saturation of the inflowing water (Green and Carrit, 1967), v is the flow-rate of water through the
the amplified oxygen electrode signal at saturation, U is the electrode signal from the chamber containing the animals, and
Us
]]
9
Uref5Uref U s,ref
where, Uref is the output from the reference electrode during the measuring period and
Us,ref is the output from the reference electrode at 100% oxygen saturation (Fig. 1). The magnitude of SDA (i.e. the total amount of oxygen respired during and after the
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feeding period, in nlO2 ind ) was calculated by integrating the area between the transposed U9ref line (dotted line in Fig. 1) and the curve of respiration during and after 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
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medium, inoculated with 330mCi NaH CO l3 , and grown for 3 days. They were then 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. Us ref
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 (1000mgC l ) for 2, 6, 12, and 24 h, respectively. Four32 (A. tonsa) or 433 (C. finmarchicus) replicates, acting as
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nonprotein synthesising controls, were incubated with cycloheximide (1 mg l ) and fed labeled algae similarly. All samples of copepods were frozen in 120ml 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 150ml 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 500ml 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 120ml extracted or nonextracted subsamples was solubilised with 1200ml 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
After 24 h of food deprivation, the average respiration rate of A. tonsa was 9.4 nlO2 21 21
mgC h (Fig. 2), and during feeding it increased significantly to a maximum of 17.7 21 21
nlO2 mgC h (Student’s t-test: t853.76, P50.006). Thereafter respiration declined to prefeeding levels within 10 h. The respiration rate of food-deprived C. finmarchicus
21 21 21 21
was 3.5 nlO2mgC h , increasing significantly to a maximum of 9.8 nlO2mgC h during feeding (Student’s t-test: t757.15, P50.0002). The decline to the prefeeding level came within 7–9 h.
21 The magnitude of SDA following a feeding period of 8 h was 78.7625.7 nlO2mgC
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in A. tonsa and 27.5611.6 nlO2 mgC in C. finmarchicus (Fig. 3).
Incubation with cycloheximide had a significant influence on the magnitude of SDA (Fig. 3). The addition of algae did not result in a significant elevation of respiration rate above food-deprived levels (Student’s t-test, one-tailed t450.88, P50.43) in either A.
tonsa or C. finmarchicus (Student’s t-test, one-tailed t651.95, P50.09) and the magnitude of SDA of cycloheximide treated copepods was very small as compared to the nontreated copepods (Fig. 3). The magnitude of SDA of cycloheximide treated
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copepods was 6.3 nlO2 mgC in A. tonsa and 3.0 nlO2 mgC in C. finmarchicus. Thus, the magnitude of SDA was lowered by 93% in A. tonsa and 88% in C.
finmarchicus as compared to nontreated individuals.
Fig. 2. Acartia tonsa and Calanus finmarchicus. Respiration rate of copepods deprived of food for 24 h prior to the feeding period and maximal respiration rate of individuals during the feeding period. Vertical bars depict standard deviation. N56 for A. tonsa and N58 for C. finmarchicus.
and the magnitude of SDA (Fig. 5) in untreated A. tonsa. In the cycloheximide treated individuals there was only a small and insignificant increase with longer feeding time (linear regression: F654.44, P50.080).
The treatment with cycloheximide inhibited the incorporation of carbon into protein in both A. tonsa and C. finmarchicus (Fig. 6), with the amount of carbon incorporated after 24 h being reduced by 67% in A. tonsa and 69% in C. finmarchicus as compared to the nontreated individuals. The inhibition was significant in both species when comparing
Fig. 4. Acartia tonsa. SDA of copepods fed algae for different lengths of time starting at the point marked by the arrow. Each line represents a single trial.
the standard errors of the slopes of linear regressions (Zar, 1984) of the incorporation over the 24 h period (A. tonsa: Student’s t-test, t1555.75, P,0.01; C. finmarchicus: Student’s t-test, t2857.13, P,0.01).
Visual examination of the copepods during the experiments showed full digestive
Fig. 5. Acartia tonsa. Magnitude of SDA with respect to feeding time. Regressions are: SDA59.77t20.63,
2 2
Fig. 6. Acartia tonsa and Calanus finmarchicus. Incorporation of carbon into protein of individuals treated with the protein inhibitor cycloheximide compared to nontreated individuals. The regressions are: A. tonsa,
2 2 2
Inc50.75t20.74, r 50.89; Inccycl50.17t12.19, r 50.78; C. finmarchicus, Inc50.27t20.33, r 50.89;
2
Inccycl50.12t20.005, r 50.008. Vertical bars depict standard deviation; N52 for A. tonsa and N53 for C.
finmarchicus.
tracts of both treated and nontreated copepods indicating normal feeding activity. Thus, the inhibitory effect of the cycloheximide was not the result of inhibition of feeding (ingestion). Unfortunately, no data are available on a possible effect of cycloheximide on absorption and assimilation in crustaceans.
4. Discussion
Treatment with cycloheximide significantly diminished the magnitude of SDA in both
1991a,b). This was not seen in individuals treated with cycloheximide and they concluded that there was a cause-and-effect relationship between protein synthesis and SDA. Similar results have been obtained with herring larvae, Clupea harengus, where 79% of the total oxygen consumption seemed to be attributable to cycloheximide sensitive protein synthesis (Houlihan et al., 1995). This was also found in isolated hepatocytes from rainbow trout, Oncorhynchus mykiss (Pannevis and Houlihan, 1992). In the isopod Ligia pallasii, there was a significant relationship between the magnitude of SDA and amount of dietary amino acids (Carefoot, 1990a,b). However, Carefoot was able to connect SDA with protein synthesis only by assuming that the amino acids were primarily incorporated into protein. This was implicated by the correlation between the amount of dietary amino acids and growth rate. I have been able to focus on this connection directly by specifically manipulating the protein synthesis process with cycloheximide. The results clearly supported Carefoot’s findings. Also in calanoid copepods, protein synthesis comprises a very important energetic expense during feeding. Apparently this is true for both neritic and oceanic species.
A high magnitude of SDA has been linked to elevated energetic costs resulting from increased turnover of newly synthesised protein (Grisolia and Kennedy, 1966). Investigations of protein deposition indicate that protein synthesis may be much higher than the resulting protein growth because a considerable fraction of the protein synthesised is turned over quite rapidly (Waterlow, 1980; Reeds and Fuller, 1983). Taking the assumption of a relationship between protein turnover and SDA a step further, one could argue that SDA is caused mainly by protein turnover and that animals that do not experience this turnover have a smaller SDA. Judging from simultaneous measurements of growth and respiration at different feeding levels in A. tonsa, the overall physiological costs of growth are close to the calculated theoretical costs of biosynthesis (Kiørboe et al., 1985). The interpretation of this is that A. tonsa do not hold any energetic expenses for protein turnover. Nevertheless, a large part of SDA in A.
tonsa appears to be linked to protein metabolism which is further supported by studies of
carbon incorporation not only into protein but also into lipid and polysaccharide in both
A. tonsa and C. finmarchicus (Roman, 1991; Thor, in press).
The respiration rates of both food-deprived and feeding copepods were generally higher in A. tonsa than in C. finmarchicus. This significantly influenced SDA and the weight specific magnitude of SDA was three times higher in A. tonsa than in C.
finmarchicus. This may be related to differences in energy allocation between the
copepodite V stage of the two species. In this stage C. finmarchicus deposits large quantities of storage lipids predominately wax esters (Conover, 1988) whereas A. tonsa grows somatically primarily depositing proteins [approximately 56% of A. tonsa is protein, calculated with a C:N ratio of 4.1 (Berggreen et al., 1988) and a conversion factor of 5.8 from nitrogen to protein (Gnaiger and Bitterlich, 1984)]. This difference between the two species could be inferred from the three times higher incorporation of carbon into protein in A. tonsa than in C. finmarchicus in the present study (Fig. 6).
From Fig. 4 it seems that the maximum respiration rate in A. tonsa was first reached beyond a constant feeding period of 12 h. From this it might be speculated that A. tonsa reaches the maximum respiration rate after feeding continuously for 12 h on 1000mgC
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food abundance which affects their metabolic performance in general (Kremer and Kremer, 1988; Saiz et al., 1993; Tiselius, 1998). Therefore it is rather surprising that the metabolic rate adapts so slowly to changes in food availability. If this is indeed the case, then the patchiness of food in time and space must have a relatively high influence on the overall energetics in this species. The respiratory reaction to food was much faster in
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D. magna, only about 2 h at a food concentration of 2000 mgC l (Lampert, 1986). When the algal supply was stopped the respiration rate decreased to a low level within 4 h. In L. pallasii the reaction to food also came within 2 h (Carefoot, 1990a), but the elevated respiration rate continued for at least 12 h making the reaction to food in this species more alike that of A. tonsa and C. finmarchicus in my experiments.
Protein deposition was used as a measure of protein metabolism because of methodological considerations, and the measurement of incorporation of labeled carbon is by far the most easily applied method. This method does not give actual protein synthesis rates, but may still have application in the comparison of protein metabolism, assuming that the cycloheximide treatment only affects the synthesis of proteins and not the breakdown. Since cycloheximide specifically inhibits the large ribosomal unit during chain elongation (Vazquez, 1974; Pestka, 1977) this is probably the case.
Acknowledgements
I would like to thank Dr. Thomas Kiørboe, Dr. Benni W. Hansen, and an anonymous referee for revising the manuscript and Dr. Morten Foldager Pedersen for help with the statistical analysis. I would also like to thank Hans Wallin for manufacturing the respiration chambers and oxygen electrodes and for invaluable creativity during the design. [RW]
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