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Comparison of two methods for measuring the rates of oxygen consumption of small aquatic animals (Artemia)
$6.00+O.OO 0300-9629/93
1993 Vol.106A.No. 3.pp.551-555, Camp. Biochem.fhysiol.
0 1993Pergamon Press Ltd
Printedin Great Britain
COMPARISON OF TWO METHODS FOR MEASURING THE RATES OF OXYGEN CONSUMPTION OF SMALL AQUATIC ANIMALS (ARTEMIA) I. VA&I*,
A.
C.
TAYLOR~$
and F.
AMAT*
*Institute de Acuicultura de Terre de la Salle (C.S.I.C.), El2695 Ribera de Cabanes, Castel16n. Spain tDepartment of Zoology, University of Glasgow, Glasgow G12 8QQ. U.K. (Fax 041-330-5971) (Received 22 December 1992; accepted 29 January 1993) Abstract-l. Two methods for measuring rates of oxygen consumption of small aquatic animals namely, the Gilson differential respirometer and a micro-cathode oxygen electrode contained within a closed micro-respirometer have been compared. 2. The rates of oxygen consumption of Artemia nauplii, but not the adults, obtained using the Gilson respirometer were slightly higher than those obtained using the oxygen electrode technique. 3. The weight-specific rates of oxygen consumption of the nauplii were not affected by the density of animals within the oxygen electrode respirometer chamber. 4. The advantages and disadvantages of the two methods are discussed.
INTRODUflION
A wide variety of methods have been used to measure oxfgen consumption rates of small (S-10 mg dry wt) aq latic organisms. These include the classical manometric or volumetric systems, such as Warburg, Gilson and Cartesian Diver respirometers, as well as numerous types of respirometers which incorporate oxvgen electrodes to measure changes in oxygen ter sion within the respirometer chamber (see reviews of Gnaiger, 1983; Kaufmann et al., 1989). e3ne of the most sensitive closed respirometers is tht. Cartesian diver which enables oxygen consumption rates of animals weighing only a few pg to be measured (Linderstrom-Lang, 1943; Zeuthen, 1943, 19iO; Klekowski, 1971). Despite its sensitivity, the tee hnical difficulties associated with the use of this type of respirometer have resulted in it being a rather unpopular technique. The Warburg and the Gilson rerpirometers, although less sensitive, are often used in studies on small aquatic animals if the oxygen uptake rates are in the order of 1 pmol O,/hr (Gnaiger, 1983). The relative ease of use of the Gilson respirometer combined with the possibility of carryinl: out concurrent replicate determinations has contriruted to the popularity of this technique. For ex.tmple, Gilson respirometers have frequently been used to measure oxygen consumption rates of nauplii anl adult Artemia (Engel and Angelovic, 1968; Vos et A., 1979; Conte et al., 1980; Bernaerts et al., 1987; De Watchtter and Van Den Abbeele, 1991). Few studies have been carried out, however, to compare the values for rates of oxygen consumption obtained using different types of respirometric techniques. fTo whom all correspondence should be addressed.
The introduction of oxygen electrodes, especially micro-cathode electrodes having low rates of O2 consumption (Hale, 1983), has led to their use in both open and closed respirometers (Kaufmann et al., 1989). Although the sensitivity achieved using oxygen electrodes is considerably less than can be achieved with the Cartesian diver, their ease of use offers an attractive alternative for measuring oxygen consump tion rates of even very small aquatic animals. The present study was carried out to compare the respiration rates of both nauplii and adult Artemia obtained with the Gilson differential respirometer and with a micro-cathode oxygen electrode contained within a closed micro-respirometer. MATERIALS
Experimental
ANDMETHODS
animals
Artemia nauplii were obtained from dried cysts of
the parthenogenetic diploid strain collected from La Mata, Alicante, Spain. Cysts were hatched in 30% artificial sea-water (Tropic Marine) at 28°C under conditions of continuous illumination and aeration. After hatching, the nauplii were separated from their shells and any remaining unhatched cysts discarded (Amat, 1985). Culture conditions
Each batch of newly hatched nauplii was washed in thoroughly filtered (0.2 pm, Whatman WCN/filters) artifical sea water (salinity = 30%), transferred to clean filtered sea water and then acclimatized to 20 + 0.5”C for 24 hr in an incubator under constant aeration and illumination before oxygen consumption rates were determined. Additional batches of nauplii were transferred to 551
552
1. VARb
water having a salinity of 90% and reared in 2 1 glass flasks at 25 f 05°C under 12: 12 1ight:dark regime. Previous work has shown that the adult stage is reached quickly at this temperature with very low mortality rates (Browne et al., 1984). The animals were fed regularly on the alga, Dunaliella sp., and the medium changed every 2-3 days. Animals reached the adult stage after 8-10 days of culture when they had attained a length of 5.6-6 mm. The females became sexually mature between 1416 days of culture (oocytes were present in the ovarium), having reached a size of 8-9 mm. Animals above this size were not used because the determination of oxygen consumption rates would be complicated by the presence of cysts or embryos in the brood pouch. Prior to measuring the rates of oxygen consumption, the animals were washed thoroughly in water (salinity = 90%) and transferred to clean filtered water and acclimated to the same conditions for 24 hr without being fed. Measurements of oxygen consumption rates Two methods for measuring rates of oxygen consumption rates were used, namely the Gilson differential respirometer and an oxygen electrode contained within a micro-respirometer. Gilson (Gilson Medical Electronics, Wisconsin, U.S.A.). Before they were transferred to the flasks the animals were again washed with filtered sea-water at the appropriate salinity to ensure that bacterial contamination was kept to a minimum. A 3 ml suspension of nauplii or adults was placed into a series of 15 ml flasks and left ,at the experimental temperature for 30 min during which time the flasks were continuously shaken at the minimum speed available (45 oscillations per min). The number of nauplii added varied between a minimum of 520 and a maximum of 1450 depending on the experimental temperature. It was found to be essential to use higher numbers of nauplii at low temperatures to obtain a clear change in the level of the manometric fluid during the period of observation. Similarly, between two and six adults were used depending on the experimental temperature, although in most experiments only three individuals were used. Two control flasks containing no animals were used for each experimental run. After equilibration, oxygen consumption was measured every 30-45 min over a period of 24 hr. At the end of each experiment individuals were removed from the flasks, the nauplii counted and the total body lengths of the adults measured. Flasks were washed with concentrated detergent (PCC-54, Pierce Chemical Company, U.S.A.) to minimize microbial contamination. Rates of oxygen consumption of the nauplii and adult Artemia were determined at temperatures of 15, 20, 25, 30 and 35°C. Weight specific rates of oxygen consumption (~1 Or mg-’ h-i) were calculated by dividing the total oxygen consumption of each batch of animals by the number of individuals in the flask
et a/.
and by their mean dry weight. The total dry weight of the adults in each flask was obtained after ovendrying (24 hr at 60°C). For the nauplii, the mean dry weight of a 24 hr nauplius (2.18 f 0.07 p(p) obtained by Varo et al. (1991) was used in these calculations. Finally, the weight specific rates of oxygen consumption were expressed as pmol Or mg-’ h-’ (ko,). Oxygen electrode. A micro-cathode oxygen electrode contained within a closed glass micro-respirometer (RC-300 respiration cell and 1302 electrode, Strathkelvin Instruments, Glasgow, U.K.) was used to determine the rates of oxygen consumption of both nauplii and adult Artemia (Fig. 1). The oxygen electrode was connected to an oxygen meter (Model 781, Strathkelvin Instruments) whose output was displayed on a pen recorder. The advantage of using a micro-cathode oxygen electrode is that it has an extremely low. rate of oxygen consumption (approximately 3 x 10-6~mol O,/hr, at 20°C with 20pm polypropylene membranes) due to the small surface area of the cathode. Since, with these membranes, the micro-electrode does not cause significant local depletion of oxygen, it was not necessary to stir the water in the respirometer chamber. This was very important because preliminary experiments had shown that even gentle stirring had an adverse effect on the behaviour of the animals in the respirometer and, in the case of the nauplii, may cause very high mortalities. During these experiments we also observed that the swimming movements of the animals kept the water in the chamber well mixed making any additional stirring unnecessary. The oxygen electrode was calibrated before each experiment using a solution of sodium sulphite and 0.01 M sodium tetraborate (Po, = 0 Torr), and aer-
Oxygen electrode
1
1cm
4
Fig. 1. Diagram of the RC-300 respirometer cell. The oxygen electrode is contained within the central holder.
Respiration of Arkmiu 1.0
1.2-
0.8
1.0-
0.8
L
553
0.8-
';a E 0"
0.6.
I. $
0.0
4 0
100
200
300
Number
400
500
600
of ncluplll
Fig. 2. Relationship between number of nauplii in the resp rometer chamber and the rate of weight-specific oxygen consumption (lir,,) of an individual nauplius. Recordings wen carried out at a salinity of 30% and a temperature of 20°C.
atec , filtered water at the salinity of the culture and at tie experimental temperature used. Although the volume of the respirometer chamber can he varied between 0.3 and 1 ml, in all experiments a chamber volume of 1 ml was used.
The nauplii were maintained for 30 min in clean filtered sea-water at the experimental temperature befclre a 1 ml suspension was transferred to the respirometer chamber. The number of nauplii added varied between 100 and 500 although, in most of the experiments, between 150 and 300 nauplii were used. Preliminary experiments were carried out to study the effec:t of the density of the nauplii within the chamber on oxygen consumption rate. The number of nauplii usetl varied from 40 to approximately 600 in 1 ml of watl:r. A similar protocol was used for the adults, but onl) one or two animals were placed in the chamber. At the end of each experimental run, the nauplii were: counted and the dry weights determined as abo r’e.Control experiments in which no animals were present in the chamber were run at the end of each expcsrimental run. After each experiment, the chamber and electrode was soaked in a dilute solution of benzalkonium chloride (0.1%) then rinsed thoroughly with distilled water to reduce microbial growth within the chamber. N’eight-specific rates of oxygen consumption (&fo,) undl:r normoxic conditions were calculated from the chart traces of changes of PO2 with time. To ensure that the rates of oxygen consumption calculated were not affected by hypoxia, tie, was calculated only for oxyl:en tensions above 100 Torr. Q,, values were calclllated for each method from the M,, values over the whole range of experimental temperature using the Van’t Hoff equation (Schmidt-Nielsen, 1990).
0.4-
0.2-
0.0
4
10
16
20 Tempsrrtur6
26
t
30
36
(“C)
Fig. 3. Relationship between oxygen consumption rate (Mo,) and temperature (“C) for nauplii of Arremiuobtained using both Gilson and oxygen electrode respirometers. Values are means + SD.
rate of oxygen consumption (ho,). Regression analysis showed that there was no significant (P 2 0.05) change in ko, with density of nauplii. In all subsequent experiments between 150 and 300 nauplii were used. The relationship between weight-specific rates of oxygen consumption (&fo,) and temperature for both nauplii and adults, determined using both methods are presented in Figs 3 and 4. The data obtained using both methods showed that the oxygen consumption rates of nauplii and adults increased linearly with temperature. The regression equations of these lines and the Q,, values calculated over the temperature range 15-35°C are given in Table 1. Covariance analysis (Sokal and Rohlf, 1981) of the data over the temperature range 15-30°C showed that there was no significant difference between the slopes of the regression lines but the elevations were significantly different (P I 0.05). The ho, values of 1.2 --CE-
ORmn
1.01
_._
10
I
lb
SO
--
56
T.ln2pO.rrtur. ('i,
RESULTS
Figure 2 shows the relationship between number of nauplii in the respirometer and the weight-specific
Fig. 4. Relationship between oxygen consumption rate (MO,) and temperature (“C) for adult Arremia obtained using both Gilson and oxygen electrode respirometers. Values are means f SD.
I. VARY et al.
554
Table I. Regression equations for the relationships between weight-specific oxygen consumption (M,, as ~nIOl0, mg ’ h ‘) for nauplii and adult Arrnnia and temperature obtained using both the Gilson and oxygen electrode respirometers. Values for the correlation coefficient (I) are also given GVXIP
Method
r
n
O,,,
Nauplii
Gilson 0, electrode
Mel = - 0.24 + 0.031 7’ ni,, = - 0.25 + 0.030 T
0.98 0.98
45 32
2.02 1.91
Adults
Gilson O> electrode
M,, = - 0.10 + 0.020 T tie,= -0.12+0.020T
0.94 0.96
134 70
1.72 1.77
Regression
the nauphi recorded using the Gilson respirometer were slightly, but significantly, higher (by approximately 10%) than those obtained using the oxygen electrode. At the highest temperature tested (35°C) however, the ho, values obtained using the Gilson were considerably higher (approximately 17%). In contrast, no significant differences in the rates of oxygen consumption of adult Artemia were recorded using the two methods. In addition, the Q,, values for adult animals obtained using the two methods were almost identical (Table 1). DISCUSSION
Early studies on fish led to the definition of three levels of metabolism in ectothermic animals namely, Standard, Routine and Active metabolism (Fry, 1947; Brett, 1964). Comparisons of metabolic rate among ectothermic animals were usually made using data on the Standard rate of metabolism. However, the rates of oxygen consumption determined during the present study were recorded in animals that were not quiescent but were showing their normal swimming behaviour. Therefore, the rates recorded were more representative of their Routine rate of oxygen consumption. It is well established that the rates of oxygen consumption of invertebrates vary with size (e.g. Zeuthen, 1953; Schmidt-Nielsen, 1984). During the present study care was therefore taken to ensure that all observations were carried out using animals of similar size. Thus, the nauplii used were always between 24 and 28 hr-old since, during this period, no significant growth was observed (Varb et al., 1991). The rates of oxygen consumption of adult Artemia were determined for animals having a limited weight range (dry wt = 0.10-0.67 mg) since preliminary experiments had shown that there was no significant difference in the values of ho, between animals within this size range. The ho, values of Artemia nauplii recorded using the Gilson respirometer were higher than those obtained using the oxygen electrode, whereas no significant difference was observed in the case of adults. Gnaigner (1983) has also shown that the oxygen consumption rates of aquatic oligochaetes were higher when measured using manometric methods than when techniques using either Winkler titrations or oxygen electrodes to record rates of oxygen consumption were used. It would appear that these
eauation
differences can be attributed, at least in part, to the disturbance caused by shaking the respirometer flasks which may have significant effects on the animals’ metabolic rate. It is not clear, however, why the nauphi should be affected more than the adults. The results of the present study have shown that both the Gilson and the Strathkelvin respirometers are capable of accurately determining the ho, of small aquatic organisms. The Strathkelvin system, however, has a number of advantages over the Gilson respirometer. Firstly, the micro-cathode electrode is able to record very small changes in partial pressure so that accurate recordings can be obtained with far fewer animals than with the Gilson method. Secondly, the water in the chamber does not need to be mixed by stirring (or by shaking as with the Gilson). As a result, the animals are far less likely to be disturbed enabling measurements to be made of routine rates of oxygen consumption. In addition, the behaviour of the animals can be observed during the experiment which allows assessment of their level of activity. It is also possible to obtain continuous records of changes in PO2 within the chamber by connecting the meter to a chart recorder or to a computer. The disadvantage of this system compared with the Gilson respirometer is that it is not possible to simultaneously measure a large number of replicates unless more than one respirometer is used. In summary, the present study has shown that, although there are slight differences in the ho, values recorded using the Gilson and Strathkelvin respirometers, the data obtained using both methods are comparable. The Strathkelvin system has a number of advantages, however, which make it highly suitable for recording rates of oxygen consumption of very small aquatic animals. Acknowledgements-We should like to thank Professor G. H. Coombs for providing research facilities in the Department of Zoology, University of Glasgow. I. Varo was supported by the Generalitat Valenciana.
REFERENCES
Amat F. (1985) Utilization de Artemia en acuicultura. T&K. I&t. Ina. Pesq. 128-129, 60~. Bernaerts F.. Blust R.. Thoeve C. and Van Der Linden A. (1987) Comparison ‘of the aerobic metabolism for four strains of Ariemia. In Artemia Research and its Applications Vol. 2 Physiology, Biochemistry, Molecular Biology (Edited by Decleir W., Moens L., Sleger H..
Respiration of Artemia Jaspers E. and Sorgeloos P.), pp. 3-9. Universal Press, Wetteren, Belgium. Brett J. R. (1964) The respiratory metabolism and swimming performance of young sockeye salmon. J. Fish Res. Bd. Can. 21, 1183-1226. Browne R. A.. Sallee S. E., Grosh D. S., Segretti W. 0. and Purser S. M. (1984) Partitioning genetic and environmental components of reproduction and lifespan in Artemia. Ecology 65, 949-960. Conte F. P., Lowry J., Carpenter J., Edwards A., Smith R. ;.nd Ewing R. D. (1980) Aerobic and anaerobic metabolism of Artemiu nauplii as a function of salinity. In . Lrtemia Research and its Applications Vol. 2 Physiology, Biochemistry, Molecular Bio/ogy (Edited by Decleir W., Moens L., Sleger H., Jaspers E. and Sorgeloos P.), pp. 125-136. Universal Press, Wetteren, Belgium. De Wachter B. and Van Den Abbeele J. (1991) The influence of acclimation on salinity and oxygen on the respiration IIf brine shrimp. Artemia franciscana. Comp. Biochem. .Dhysiol. 98, 293-298.
Engel D. W. and Angelovic J. W. (1968) The influence of salinity and temperature on brine shrimp nauplii. Comp. ,%ochem. Physiol. 26, 149-752.
Fr:r F. E. J. (1947) Effects of environment on animal activity. Univ. Toronto Stud. Biol. Ser. 55., Publs. Ont. .Gh Res. Lab. 68, 162.
Graiger E. (1983) The twin-flow microrespirometer and : imultaneous calorimetry. In Polurogruphic Oxygen Sen , ors (Edited by Gnaiger E. and Forstner H.), pp. 134155. !;pringer, Berlin. Hale J. M. (1983) Factors influencing the stability of polarographic oxygen sensors. In Polurogruphic Oxygen .Lnsors (Edited by Gnaiger E. and Forstner H.), pp. 3-17. !;pringer, Berlin. Kaufmann R., Forstner H. and Wieser W. (1989)
555
Respirometry-methods and approaches. In Techniques in Comparative Resoirarorv Phvsioloav (Edited by Bridges C. R. and Butler P: J.), Society for l&&imentaiBiology, Seminar Series No. 37, pp. 51-76. Cambridge University Press, Cambridge. Klekowski R. Z. (1971) Cartesian diver microrespirometer for aquatic animals. Polish Arch. Hydrobiol. 18, 93-114. Linderstrom-Lang K. (1943) On the theory of Cartesian diver micro respirometer. C.r. Lab. Curlsberg. Ser Chim. 24, 333-398.
Schmidt-Nielsen K. (1984) Scaling: Why is Animal Size so Important? p. 241. Cambridge University Press, Cambridge. Schmidt-Nielsen K. (1990) Animal Physiology: Adnpration and Environment 4th edn. p. 602. Cambridge University Press, Cambridge. Sokal R. R. and Rohlf F. J. (1981) Biometry. The Principles and Practice of Statistics in Biology Research 2nd edn. W. H. Freeman and Co., San Francisco. Varo I., Taylor A. C., Navarro J. C. and Amat F. (1991) Effects of temperature and oxygen tension on oxygen consumption rates of nauplii of different Artemiu strains. Mar. Ecol. Prog. Ser. 76, 25-31.
Vos J., Bemaerts F., Gabriels I. and Decleir W. (1979) Aerobic and anaerobic respiration of adults Artemia salina L., acclimated to different oxygen concentrations. Comp. Biochem. Physiol. 62, 545548.
Zeuthen E. (1943) A Cartesian diver micro respirometer with a gas volume of 0.1 ~1 (respiration measurements with an experimental error of 2 x 10-5~1). C.r. Lob. Carlsberg, Ser. Chim. 24, 479-518.
Zeuthen E. (1950) Cartesian diver respirometer. Biol. Bull. mar. biol. Lab., Woods Hole 98, 303-318.
Zeuthen E. (1953) Oxygen uptake as related to body size in organism. Q. Rev. Biol. 28, I-12.
1993 Vol.106A.No. 3.pp.551-555, Camp. Biochem.fhysiol.
0 1993Pergamon Press Ltd
Printedin Great Britain
COMPARISON OF TWO METHODS FOR MEASURING THE RATES OF OXYGEN CONSUMPTION OF SMALL AQUATIC ANIMALS (ARTEMIA) I. VA&I*,
A.
C.
TAYLOR~$
and F.
AMAT*
*Institute de Acuicultura de Terre de la Salle (C.S.I.C.), El2695 Ribera de Cabanes, Castel16n. Spain tDepartment of Zoology, University of Glasgow, Glasgow G12 8QQ. U.K. (Fax 041-330-5971) (Received 22 December 1992; accepted 29 January 1993) Abstract-l. Two methods for measuring rates of oxygen consumption of small aquatic animals namely, the Gilson differential respirometer and a micro-cathode oxygen electrode contained within a closed micro-respirometer have been compared. 2. The rates of oxygen consumption of Artemia nauplii, but not the adults, obtained using the Gilson respirometer were slightly higher than those obtained using the oxygen electrode technique. 3. The weight-specific rates of oxygen consumption of the nauplii were not affected by the density of animals within the oxygen electrode respirometer chamber. 4. The advantages and disadvantages of the two methods are discussed.
INTRODUflION
A wide variety of methods have been used to measure oxfgen consumption rates of small (S-10 mg dry wt) aq latic organisms. These include the classical manometric or volumetric systems, such as Warburg, Gilson and Cartesian Diver respirometers, as well as numerous types of respirometers which incorporate oxvgen electrodes to measure changes in oxygen ter sion within the respirometer chamber (see reviews of Gnaiger, 1983; Kaufmann et al., 1989). e3ne of the most sensitive closed respirometers is tht. Cartesian diver which enables oxygen consumption rates of animals weighing only a few pg to be measured (Linderstrom-Lang, 1943; Zeuthen, 1943, 19iO; Klekowski, 1971). Despite its sensitivity, the tee hnical difficulties associated with the use of this type of respirometer have resulted in it being a rather unpopular technique. The Warburg and the Gilson rerpirometers, although less sensitive, are often used in studies on small aquatic animals if the oxygen uptake rates are in the order of 1 pmol O,/hr (Gnaiger, 1983). The relative ease of use of the Gilson respirometer combined with the possibility of carryinl: out concurrent replicate determinations has contriruted to the popularity of this technique. For ex.tmple, Gilson respirometers have frequently been used to measure oxygen consumption rates of nauplii anl adult Artemia (Engel and Angelovic, 1968; Vos et A., 1979; Conte et al., 1980; Bernaerts et al., 1987; De Watchtter and Van Den Abbeele, 1991). Few studies have been carried out, however, to compare the values for rates of oxygen consumption obtained using different types of respirometric techniques. fTo whom all correspondence should be addressed.
The introduction of oxygen electrodes, especially micro-cathode electrodes having low rates of O2 consumption (Hale, 1983), has led to their use in both open and closed respirometers (Kaufmann et al., 1989). Although the sensitivity achieved using oxygen electrodes is considerably less than can be achieved with the Cartesian diver, their ease of use offers an attractive alternative for measuring oxygen consump tion rates of even very small aquatic animals. The present study was carried out to compare the respiration rates of both nauplii and adult Artemia obtained with the Gilson differential respirometer and with a micro-cathode oxygen electrode contained within a closed micro-respirometer. MATERIALS
Experimental
ANDMETHODS
animals
Artemia nauplii were obtained from dried cysts of
the parthenogenetic diploid strain collected from La Mata, Alicante, Spain. Cysts were hatched in 30% artificial sea-water (Tropic Marine) at 28°C under conditions of continuous illumination and aeration. After hatching, the nauplii were separated from their shells and any remaining unhatched cysts discarded (Amat, 1985). Culture conditions
Each batch of newly hatched nauplii was washed in thoroughly filtered (0.2 pm, Whatman WCN/filters) artifical sea water (salinity = 30%), transferred to clean filtered sea water and then acclimatized to 20 + 0.5”C for 24 hr in an incubator under constant aeration and illumination before oxygen consumption rates were determined. Additional batches of nauplii were transferred to 551
552
1. VARb
water having a salinity of 90% and reared in 2 1 glass flasks at 25 f 05°C under 12: 12 1ight:dark regime. Previous work has shown that the adult stage is reached quickly at this temperature with very low mortality rates (Browne et al., 1984). The animals were fed regularly on the alga, Dunaliella sp., and the medium changed every 2-3 days. Animals reached the adult stage after 8-10 days of culture when they had attained a length of 5.6-6 mm. The females became sexually mature between 1416 days of culture (oocytes were present in the ovarium), having reached a size of 8-9 mm. Animals above this size were not used because the determination of oxygen consumption rates would be complicated by the presence of cysts or embryos in the brood pouch. Prior to measuring the rates of oxygen consumption, the animals were washed thoroughly in water (salinity = 90%) and transferred to clean filtered water and acclimated to the same conditions for 24 hr without being fed. Measurements of oxygen consumption rates Two methods for measuring rates of oxygen consumption rates were used, namely the Gilson differential respirometer and an oxygen electrode contained within a micro-respirometer. Gilson (Gilson Medical Electronics, Wisconsin, U.S.A.). Before they were transferred to the flasks the animals were again washed with filtered sea-water at the appropriate salinity to ensure that bacterial contamination was kept to a minimum. A 3 ml suspension of nauplii or adults was placed into a series of 15 ml flasks and left ,at the experimental temperature for 30 min during which time the flasks were continuously shaken at the minimum speed available (45 oscillations per min). The number of nauplii added varied between a minimum of 520 and a maximum of 1450 depending on the experimental temperature. It was found to be essential to use higher numbers of nauplii at low temperatures to obtain a clear change in the level of the manometric fluid during the period of observation. Similarly, between two and six adults were used depending on the experimental temperature, although in most experiments only three individuals were used. Two control flasks containing no animals were used for each experimental run. After equilibration, oxygen consumption was measured every 30-45 min over a period of 24 hr. At the end of each experiment individuals were removed from the flasks, the nauplii counted and the total body lengths of the adults measured. Flasks were washed with concentrated detergent (PCC-54, Pierce Chemical Company, U.S.A.) to minimize microbial contamination. Rates of oxygen consumption of the nauplii and adult Artemia were determined at temperatures of 15, 20, 25, 30 and 35°C. Weight specific rates of oxygen consumption (~1 Or mg-’ h-i) were calculated by dividing the total oxygen consumption of each batch of animals by the number of individuals in the flask
et a/.
and by their mean dry weight. The total dry weight of the adults in each flask was obtained after ovendrying (24 hr at 60°C). For the nauplii, the mean dry weight of a 24 hr nauplius (2.18 f 0.07 p(p) obtained by Varo et al. (1991) was used in these calculations. Finally, the weight specific rates of oxygen consumption were expressed as pmol Or mg-’ h-’ (ko,). Oxygen electrode. A micro-cathode oxygen electrode contained within a closed glass micro-respirometer (RC-300 respiration cell and 1302 electrode, Strathkelvin Instruments, Glasgow, U.K.) was used to determine the rates of oxygen consumption of both nauplii and adult Artemia (Fig. 1). The oxygen electrode was connected to an oxygen meter (Model 781, Strathkelvin Instruments) whose output was displayed on a pen recorder. The advantage of using a micro-cathode oxygen electrode is that it has an extremely low. rate of oxygen consumption (approximately 3 x 10-6~mol O,/hr, at 20°C with 20pm polypropylene membranes) due to the small surface area of the cathode. Since, with these membranes, the micro-electrode does not cause significant local depletion of oxygen, it was not necessary to stir the water in the respirometer chamber. This was very important because preliminary experiments had shown that even gentle stirring had an adverse effect on the behaviour of the animals in the respirometer and, in the case of the nauplii, may cause very high mortalities. During these experiments we also observed that the swimming movements of the animals kept the water in the chamber well mixed making any additional stirring unnecessary. The oxygen electrode was calibrated before each experiment using a solution of sodium sulphite and 0.01 M sodium tetraborate (Po, = 0 Torr), and aer-
Oxygen electrode
1
1cm
4
Fig. 1. Diagram of the RC-300 respirometer cell. The oxygen electrode is contained within the central holder.
Respiration of Arkmiu 1.0
1.2-
0.8
1.0-
0.8
L
553
0.8-
';a E 0"
0.6.
I. $
0.0
4 0
100
200
300
Number
400
500
600
of ncluplll
Fig. 2. Relationship between number of nauplii in the resp rometer chamber and the rate of weight-specific oxygen consumption (lir,,) of an individual nauplius. Recordings wen carried out at a salinity of 30% and a temperature of 20°C.
atec , filtered water at the salinity of the culture and at tie experimental temperature used. Although the volume of the respirometer chamber can he varied between 0.3 and 1 ml, in all experiments a chamber volume of 1 ml was used.
The nauplii were maintained for 30 min in clean filtered sea-water at the experimental temperature befclre a 1 ml suspension was transferred to the respirometer chamber. The number of nauplii added varied between 100 and 500 although, in most of the experiments, between 150 and 300 nauplii were used. Preliminary experiments were carried out to study the effec:t of the density of the nauplii within the chamber on oxygen consumption rate. The number of nauplii usetl varied from 40 to approximately 600 in 1 ml of watl:r. A similar protocol was used for the adults, but onl) one or two animals were placed in the chamber. At the end of each experimental run, the nauplii were: counted and the dry weights determined as abo r’e.Control experiments in which no animals were present in the chamber were run at the end of each expcsrimental run. After each experiment, the chamber and electrode was soaked in a dilute solution of benzalkonium chloride (0.1%) then rinsed thoroughly with distilled water to reduce microbial growth within the chamber. N’eight-specific rates of oxygen consumption (&fo,) undl:r normoxic conditions were calculated from the chart traces of changes of PO2 with time. To ensure that the rates of oxygen consumption calculated were not affected by hypoxia, tie, was calculated only for oxyl:en tensions above 100 Torr. Q,, values were calclllated for each method from the M,, values over the whole range of experimental temperature using the Van’t Hoff equation (Schmidt-Nielsen, 1990).
0.4-
0.2-
0.0
4
10
16
20 Tempsrrtur6
26
t
30
36
(“C)
Fig. 3. Relationship between oxygen consumption rate (Mo,) and temperature (“C) for nauplii of Arremiuobtained using both Gilson and oxygen electrode respirometers. Values are means + SD.
rate of oxygen consumption (ho,). Regression analysis showed that there was no significant (P 2 0.05) change in ko, with density of nauplii. In all subsequent experiments between 150 and 300 nauplii were used. The relationship between weight-specific rates of oxygen consumption (&fo,) and temperature for both nauplii and adults, determined using both methods are presented in Figs 3 and 4. The data obtained using both methods showed that the oxygen consumption rates of nauplii and adults increased linearly with temperature. The regression equations of these lines and the Q,, values calculated over the temperature range 15-35°C are given in Table 1. Covariance analysis (Sokal and Rohlf, 1981) of the data over the temperature range 15-30°C showed that there was no significant difference between the slopes of the regression lines but the elevations were significantly different (P I 0.05). The ho, values of 1.2 --CE-
ORmn
1.01
_._
10
I
lb
SO
--
56
T.ln2pO.rrtur. ('i,
RESULTS
Figure 2 shows the relationship between number of nauplii in the respirometer and the weight-specific
Fig. 4. Relationship between oxygen consumption rate (MO,) and temperature (“C) for adult Arremia obtained using both Gilson and oxygen electrode respirometers. Values are means f SD.
I. VARY et al.
554
Table I. Regression equations for the relationships between weight-specific oxygen consumption (M,, as ~nIOl0, mg ’ h ‘) for nauplii and adult Arrnnia and temperature obtained using both the Gilson and oxygen electrode respirometers. Values for the correlation coefficient (I) are also given GVXIP
Method
r
n
O,,,
Nauplii
Gilson 0, electrode
Mel = - 0.24 + 0.031 7’ ni,, = - 0.25 + 0.030 T
0.98 0.98
45 32
2.02 1.91
Adults
Gilson O> electrode
M,, = - 0.10 + 0.020 T tie,= -0.12+0.020T
0.94 0.96
134 70
1.72 1.77
Regression
the nauphi recorded using the Gilson respirometer were slightly, but significantly, higher (by approximately 10%) than those obtained using the oxygen electrode. At the highest temperature tested (35°C) however, the ho, values obtained using the Gilson were considerably higher (approximately 17%). In contrast, no significant differences in the rates of oxygen consumption of adult Artemia were recorded using the two methods. In addition, the Q,, values for adult animals obtained using the two methods were almost identical (Table 1). DISCUSSION
Early studies on fish led to the definition of three levels of metabolism in ectothermic animals namely, Standard, Routine and Active metabolism (Fry, 1947; Brett, 1964). Comparisons of metabolic rate among ectothermic animals were usually made using data on the Standard rate of metabolism. However, the rates of oxygen consumption determined during the present study were recorded in animals that were not quiescent but were showing their normal swimming behaviour. Therefore, the rates recorded were more representative of their Routine rate of oxygen consumption. It is well established that the rates of oxygen consumption of invertebrates vary with size (e.g. Zeuthen, 1953; Schmidt-Nielsen, 1984). During the present study care was therefore taken to ensure that all observations were carried out using animals of similar size. Thus, the nauplii used were always between 24 and 28 hr-old since, during this period, no significant growth was observed (Varb et al., 1991). The rates of oxygen consumption of adult Artemia were determined for animals having a limited weight range (dry wt = 0.10-0.67 mg) since preliminary experiments had shown that there was no significant difference in the values of ho, between animals within this size range. The ho, values of Artemia nauplii recorded using the Gilson respirometer were higher than those obtained using the oxygen electrode, whereas no significant difference was observed in the case of adults. Gnaigner (1983) has also shown that the oxygen consumption rates of aquatic oligochaetes were higher when measured using manometric methods than when techniques using either Winkler titrations or oxygen electrodes to record rates of oxygen consumption were used. It would appear that these
eauation
differences can be attributed, at least in part, to the disturbance caused by shaking the respirometer flasks which may have significant effects on the animals’ metabolic rate. It is not clear, however, why the nauphi should be affected more than the adults. The results of the present study have shown that both the Gilson and the Strathkelvin respirometers are capable of accurately determining the ho, of small aquatic organisms. The Strathkelvin system, however, has a number of advantages over the Gilson respirometer. Firstly, the micro-cathode electrode is able to record very small changes in partial pressure so that accurate recordings can be obtained with far fewer animals than with the Gilson method. Secondly, the water in the chamber does not need to be mixed by stirring (or by shaking as with the Gilson). As a result, the animals are far less likely to be disturbed enabling measurements to be made of routine rates of oxygen consumption. In addition, the behaviour of the animals can be observed during the experiment which allows assessment of their level of activity. It is also possible to obtain continuous records of changes in PO2 within the chamber by connecting the meter to a chart recorder or to a computer. The disadvantage of this system compared with the Gilson respirometer is that it is not possible to simultaneously measure a large number of replicates unless more than one respirometer is used. In summary, the present study has shown that, although there are slight differences in the ho, values recorded using the Gilson and Strathkelvin respirometers, the data obtained using both methods are comparable. The Strathkelvin system has a number of advantages, however, which make it highly suitable for recording rates of oxygen consumption of very small aquatic animals. Acknowledgements-We should like to thank Professor G. H. Coombs for providing research facilities in the Department of Zoology, University of Glasgow. I. Varo was supported by the Generalitat Valenciana.
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