The influence of acclimation on salinity and oxygen on the respiration of the brine shrimp, artemia franciscana

Camp. Biochem.

Physiol.

Vol. 98A.

No. 2. pp. 293-298,

0300-9629191

1991

$3.00 + 0.00

0 1991Pergamon Press plc

Printed in Great Britain

THE INFLUENCE OF ACCLIMATION ON SALINITY AND OXYGEN ON THE RESPIRATION OF THE BRINE SHRIMP, ARTEMIA FRANCISCANA BART DE WACHTER*and JAN VAN DEN ABBEELE* Laboratory of Biochemistry and Zoology, University of Antwerp (R.U.C.A.), Groenenborgerlaan 2020 Antwerp, Belgium. Telephone: (03) 218-03-47. Fax: (03) 218-02-17

171.

(Received 19 June 1990) Abstract-l. A multifactorial approach was used to study the influence of the acclimation on four salinities and five oxygen concentrations on the oxygen consumption of Artemiu franciscana. 2. The oxygen consumption shows a maximum at an intermediate salinity and a high oxygen concentration. 3. The dry weight of the animal being the most important factor in the respiratory response, is on its turn mainly influenced by the acclimation to salinity. 4. Multiple regression analysis indicated that the oxygen concentration is a more important factor than the oxygen pressure in determining respiration in saline waters.

INTRODUCTION The brine shrimp, Artemia sp., inhabits salt lakes which show important fluctuations in salinity and oxygen concentration. These environmental factors interact with several aspects of the biology of the brine shrimp, among which is respiration. Although several papers have been published on the single effect of oxygen concentration (Mitchell and Geddes, 1977; Decleir et al., 1980; Vos et al., 1979) or salinity (Kuenen, 1939; Gilchrist, 1956, 1959; Eliassen, 1952; Herbst and Dana, 1980) on the respiration of the adult brine shrimp, as far as we know, no attempts have been made to study or quantify their simultaneous effect. The overall metabolism of an animal can be represented by its respiration or oxygen consumption. The influence of salinity (osmo- and ion-regulation) and of oxygen (hypoxia or hyperoxia) on the metabolism could be estimated by measuring their influence on the oxygen consumption. The importance of a multifactorial approach of respiration lies in the possibility of describing interrelations between environmental factors. This way a greater insight in the influence of these interrelations on biological phenomena can be gained. Lange et al. (1972) showed that the diffusion rate of oxygen in saline waters varies proportionally to the solubility of oxygen. The solubility of oxygen decreases with increasing salinity. The oxygen availability in saline waters is thus not only dependent on the partial oxygen pressure but also on the salinity of the water. MATERIALSAND METHODS Animals For the experiments adult specimens of Artemiufianciscanu (Great Salt Lake, Northern Arm) were used. Cysts *Scholar at the Institute of Scientific Research in Industry and Agriculture (I.W.O.N.L.).

were hatched in 35% artificial seawater (HW-Wimex, Wiegandt, Krefeld, W. Germany) at 25”C, under continuous illumination and aeration. The nauplii were grown for ten days in 201 aquaria with an air-waterlift raceway according to Sorgeloos et al. (1983) (350/m,25°C). They were fed with a suspension of Yellow Mix (Artemia Systems) (2 g/day). Acclimation Five different oxygen concentrations (1, 2, 3, 4 and 5 ml/l) and four different salinities (IO, 35, 90 and 145%) were selected, resulting in twenty different combinations. Hypoxic as well as hyperoxic partial oxygen pressures were needed (Table 1). All acclimations and tests were performed at 25 + 1°C. Acclimation was carried out in oxygen acclimation jars (3 I) aerated with the appropriate N,/O, mixtures as described earlier by Vos et al. (1978). The oxygen concentration of air saturated water with a salinity of 90 and 145% were measured by the Miller method (Walker ef al., 1970; Ellis and Kanamori, 1973). Since age influences hemoglobin content and composition of the brine shrimp hemolymph (Heip et al., 1978) it was necessary to follow a strict time schedule. Ten-day-old animals were transferred to the acclimation jars. From day ten until day fourteen the animals were directly acclimated to the proper salinity at an oxygen concentration of 5 ml/l. To anticipate mortality, the animals were progressively acclimated to the highest salinity. From day ten until day twelve they were first acclimated to 90%. Thereafter they were transferred to 145%. Oxygen acclimation took place the next six days. For acclimation to the lower oxygen concentrations a stepwise decrease of the oxygen concentration was applied in order to avoid mortality by acute hypoxia. Acclimation to 1 and 2 ml 0,/l was preceded by a two-day acclimation to 2.5 ml/l. Twenty-days-old animals were fully acclimated to the required salinity and oxygen concentration. At this time the animals were adult. During acclimation the animals were fed with the same Yellow Mix suspension (100 mg suspended Yellow Mix/jar/day). Three different groups of adults were distinguished and separately tested: males, females with filled broodpouch (further called gravids) and females with empty broodpouch (further called females).

293

BARTDE WACHTER and

294

JAN VAN DEN ABBEELE

I. Partial oxygen pressures (kPa 0, in the 0,/N,-gas mixture) used in the acclimation experiments in function of salinity (L) and oxveen concentration (mlill

Table

Oxygen concentration

I

2

10 35 90

3.89 4.49

.37 8.98

6.68

145

13.36

8.89

17.19

Salinity

significant influence on the respiration of the brine shrimp. About half of the variation can be explained by the acclimation factors and the effect of the weight of the animal. Table 3 shows the results of the analysis of variance of the dry weight data in function of the acclimation (regression approach). A significant effect was found for salinity, oxygen concentration. group (males, females or gravids) and all interactions between these factors on the dry weight of the animals. Approximately one-third of the variation cannot be explained by the acclimation.

3 11.67 13.44 20.04 24.68

4

5

15.56 17.95

19.45 22.44

26.11 35.59

33.39 44.48

Respirometry Respiration was measured in Warburg constant volume respirometers. Series (n 2 18) of 15-m] &sks each containing five individuals. incubated in 3 ml water at the salinity and oxygen concentration of acclimation, were used. Animals were starved for 24 hr before measurement and kept in clear water. An aliquot of 0.3 ml 20% KOH together with a small filter-paper were placed in the central vial to absorb the produced CO,. To obtain the same oxygen concentrations in the incubating medium as during acclimation, the flasks were flushed with the acclimation NJO, mixture according to Umbreit et al. (1972). After 45min equilibration, the oxygen consumption was measured every 45 min over a period of 225 min. Afterwards the dry weight (24 hr at 85°C) of the animals was determined. If mortality occurred during measurement the oxygen consumption rate was always corrected accordingly.

Table analysis

4 shows the results of the regression of the respiratory data. The summary of

the model edification gives the information about the changes that occur after entering each of the variables in the model. The greatest changes (27% and 5% respectively) appear when the weight and oxygen concentration are entered. During the building of the regression model both logarithmic and linear relationships between the oxygen consumption and the dry weight were examined. The best model was obtained when the logarithm of the oxygen consumption was predicted by the dry weight. This relationship was transformed to oxygen consumption versus the exponent of the dry weight, because oxygen consumption was better predicted by the other factors in a simple linear model of the second order. The introduction of the other variables represents a smaller but still significant contribution to the explanation of the variance. The presented model explains about one-third of the total variance of the oxygen consumption. There is also no significant lack of fit, indicating that the model suits the experimental data. “Partial” values the correlation between the dependent and independent variable without interference of other variables. “Tolerance” estimates the variability of the entered independent variable that cannot be explained by other variables entered in the model. It also gives an idea of the degree of multicollinearity. The influence of the dry weight on the oxygen consumption is smaller for females than for males and smallest for gravids. Females carrying eggs respond stronger to a higher oxygen concentration

Slatistics

The statistical package SPSS PC + was used for both analysis of variance (ANOVA) and multiple regression approach to analyse the multifactorial data. Models for multiple regression were built with stepwise selection of the independent variables with P,~= 0.05, P,,~ = 0. I and a tolerance of 0.01. Also direct entering techniques were used. based on visual inspection of the data. Throughout the whole analysis oxygen consumption (in ~1 O,/i/hr) represents the respiration rate of the individual animal because of the possibility of a statistically incorrect interpretation when weight specific oxygen consumption (Packard and Boardman, 1988) is used. RESULT’S

Table 2 gives the results of the analysis of variance

of the oxygen consumption. Main factors, interactions and the covariate are assessed simultaneously and adjusted for all other effects in the model (regression approach). All factors considered have a

Table 2. Analysis of variance of the respiratory data; the dependent variable being the individual oxygen consumption. Main factors, interactions and the covariate arc assessed simultaneously and adjusted for all other effects m the model. SS: sum of squares; dfi degrees of freedom: MS: mean square Source of variation Covariate Dry weight

Main effects Salinity 0, cont. Group Interactions &Way Sal*O, Sal’group o,*group 3.way Sal*O,*group Explained Residual Total

MS

F-Value

Sign F

90,527 12.558 Il.955 15.577 3.188

0.000

4 2

24.053 3.337 3.176 4.139 0.847

41.866 27.042 10.844 4.674

26 12 6 8

I.610 2.254 1.802 0.584

6.060 8.482 6.802 2.199

0.000 0.000 0.000 0.026

11.053 173.516 169.514 343.030

24 60 638 698

0.416 2.892 0.266 0.491

1.733 10.844

0.017 0.000

ss 24.053 30.030 9.529 16.555 I.694

df

9

0.000

0.000 0.000

0.042

Salinity, oxygen and artemia respiration

295

Table 3. Analysis of variance of the weight data; the dependent variable being the individual dry weight. Main factors and interactions are assessed simultaneously and adjusted for all other effects in the model. SS: sum of squares; df: degrees of freedom; MS: mean square

ss

Source of variation Main effects Salinity 0, cont. Group Interactions 2.Way Sal’O, sa1*group 0,‘group 3-way Sal*O,*group Explained Residual Total

MS

F-Value

Sign F

I .092 1.066 0.167 2.804

112.646 109.967 17.219 289.404

0.000 0.000 0.000 0.000

26 12 6 8

0.059 0.064 0.078 0.033

6.083 6.655 8.043 3.430

0.000 0.000 0.000 0.00 I

24 59 646 705

0.020 0.201 0.010 0.026

2.098 20.745

0.002 0.000

df

9.824 3.197 0.667 5.609

9 3 4 2

I.532 0.774 0.468 0.266 0.488 11.860 6.260 18.120

salinity and the squared salinity are the major factors in this model. Gravids are heavier than the other two groups. An increased salinity has a greater decreasing effect on the dry weight than an increase in the oxygen concentration. Figure 3a-d gives the dry weight in function of the oxygen concentration for each of the four salinities and for each group. Means and standard deviation of the observed values (open symbols) and the predicted values (filled symbols) are presented on the same plot. Figure 4 shows the regression equation for males and females and for gravids as a contour plot in function of salinity and oxygen concentration. Here the maximum response is located in the salinity range around 60%0 and in the lower oxygen concentrations.

and are less influenced by salinity. Generally oxygen consumption rises with oxygen concentration. Figure la-d gives the oxygen consumption in function of the oxygen concentration for each salinity and for the three distinct groups. Means and standard deviations are given for the observed (open symbols) and predicted values (filled symbols). The regression equation is plotted as a contour plot in Fig. 2, for males and gravids for the mean weight of 0.48 mg. The oxygen consumption is maximum for males and females at an oxygen concentration of about 4.5 ml/l and a salinity of about 90X. For gravids this maximum is situated outside the test area. Table 5 gives the results of the regression analysis of the weight data. As can be seen the variable gravid, Table 4. Results A. Summary of model edification Variable entered Rsq EXP (Wd) 0.2697 Oxygen 0.3185 Exp (gravid’wd) 0.3261 Exp (female*Wd) 0.3353 Gravid’oxygen 0.3406 Oxygen’ 0.3451 Gravid*salinity 0.3488 Salinity 0.3530 Salinity* 0.3607 B. Analysis

of variance

Regression Residual Lack of Fit Pure Error

of the model df 9 689 50 639

C. Summary of the model Variable B Constant -0.86390 Salinity 0.006583 Gravid’salinity -0.002268 Salinity’ - 0.00003698 Oxygen 0.25069 Gravid*oxygen 0.09429 Oxygen’ -0.02787 I.74138 EXP (Wd) Exp (gravid*Wd) -0.46793 Exp (female*Wd) -0.26458

adjRsq 0.2686 0.3166 0.3232 0.3314 0.3359 0.3394 0.3422 0.3455 0.3524

of the renression F (Eq”) 257.395 162.652 112.089 87.508 71.596 60.774 52.863 47.055 43.203

ss 123.74780 219.28240 25.71528 193.56712 SE B 0.21396 0.001972 0.008342 0.00001278 0.07857 0.02745 0.01278 0.15700 0.13321 0.08950

-

Beta 16.302 0.48152 -0.14053 - 0.42905 0.51780 0.20838 -0.35341 0.69115 -0.28932 -0.11121

analvsis

on the resoiratorv

Sig F 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

RsqCh 0.2697 0.0488 0.0076 0.0092 0.0053 0.0045 0.0037 0.0042 0.0078 MS 13.74976 0.31826 0.51431 0.30292

SE Beta 0.0001 0. I4420 0.05169 0.14833 0. I6229 0.06067 0. I6206 0.0623 I 0.08237 0.03762

data FCh 257.395 49.865 7.789 9.603 5.618 4.735 3.880 4.516 8.366

Sig Ch 0.000 0.000 0.005 0.002 0.018 0.030 0.049 0.034 0.004

Carrel. 0.5193 0.1172 0.3512 -0.0639 0.2760 0.0979 0.0827 -0.0840 -0.1509

F-Value

Sig F

43.20265

0.0000

I .69784

0.0025

Partial

Tolerance

F

Sig F

0. I2620 -0.10302 -0.10953 0.12066 0. I2974 -0.08279 0.38923 -0.13264 -0.11192

0.04462 0.3472 I 0.04217 0.03523 0.25205 0.03533 0.23894 0. I3676 0.65567

II.151 7.390 8.366 IO.180 I I.796 4.755 123.024 12.338 8.740

0.0009 0.0067 0.0039 0.0015 0.0006 0.0295 0.0000 0.0005 0.0032

Oxygen consumption (~1 O,/l/hr) being the dependent variable. Gravid: nominal variable for the females with filled broodpouch; female: nominal variable for the females without filled broodpouch. Oxygen: oxygen concentration in ml/l; salinity in %a; dry weight in mg. Rsq: squared correlation coefficient; AdjRsq: adjusted Rsq; F (Eqn): F value of the total equation; Sig F: significance of F value; RsqCh: change in Rsq value after entering a variable. FCh: change in F value. SigCh: significance of the change; carrel.: correlation between oxygen consumption and the independent variable; df: degrees of freedom; B: regression cofficient; SE B: standard error on regression coefficient; beta: standardized regression coefficient; SE beta: standard error on beta; partial: partial correlation with the oxygen consumption.

BART DE WACKIER

296

Oxygen

concentration

o&-t---+-

0

0

Oxygen

3

concentration

JAN VAN DEN ABBEELE

(ml/l)

Oxygen

I

: 2

1

and

5

4

6

z 0

oL----+-. 0 1

(ml/l)

concentration

2

Oxygen

: 3

concentration

(ml/l)

I 5

4

6

(ml/l)

Fig. I. Oxygen consumption in function of the oxygen concentration in the saline water for a salinity of 10% (A), 35%0(B),90% (C) and 145% (D). Each figure contains mean and standard deviation of the observed data for females (O), gravids (A) and males (O). Filled symbols are mean and standard deviation for the predicted values for each group. DlSCUSSION

Previous studies on the effect of salinity on the respiration of the brine shrimp reported contradictory results. Kuenen (1939) found an increase in respiration with increasing salinity (at 30, 60 and 120%) while Eliassen (1952) found a decrease (at 10, 35 and 50%0). Gilchrist (1956, 1959) stated that the respiration of female Artemia fkmciscana was inde-

pendent of the salinity, while the respiration of male brine shrimps was higher at 35% than at 140%0, which could be related to morphological differences. Herbst and Dana (1980) found that respiration decreased at high salinities (> 200~~) for both females and males of Artemiu ~o~~cQ, and at low salinities (25o/oo)for males. For nauplii (Great Salt Lake, Utah), Engel and Angelovic (1968) found only a significant influence of the salinity in combination with tempera-

160.0

160.0

140.0

140.0

120.0

120.0

g

100.0

g

100.0

z.G ::

80.0

g .E ;

80.0

60.0

60.0

40.0

1.0

2.0 Oxygen

3.0 Concentration

4.0 ml,/1

5.0

1.0

2.0 Oxygen

3.0 Concentration

4.0 ml/l

Fig. 2. Contour plot of the regression equation. The oxygen consumption (pI/hr) is given in function of the oxygen concentration and salinity for males (A) and gravids (B) with a weight of 0.48 mg, being the mean observed weight.

5.0

Salinity, oxygen and artemia respiration Table 5. Results

of the regression

A. Summary of model edification Variable entered Rsq Gravid 0.3028 Salinity2 0.4192 Salinity 0.4952 Oxygen’ 0.5222 Gravid*salinity* 0.5356 B. Analysis

of variance

Regression Residual Lack of Fit Pure Error

analysis

adjRsq 0.3018 0.4175 0.4930 0.5195 0.5323

on the dry weight data. Abbreviations Sig F 0.000 0.000 0.000 0.000 0.000

F (Eq”) 305.720 253.685 229.526 191.566 161.473

of the model df 5 700 54 646

and symbols as in Table 4

RsqCh 0.3028 0.1164 0.0760 0.0271 0.0134

Beta 0.43299 0.67297 -0.16559 I.15081 - I .37476 -0.18065

SE B 0.01148 0.01270 0.0004665 0.0003502 2.254.10-6 1.119.10~~

ture (P < 0.10). An increased salinity resulted in a decreased oxygen consumption rate. The papers of Mitchell and Geddes (1977), Vos et al. (1978) and Decleir et al. (1980) showed that Artemia is a respiratory regulator up until about 2 ml 0*/l and that the regulatory capacity increased with acclimation to lower oxygen concentrations. They did not report any influence of the oxygen concentration in the regulated part of the oxygen concentration range on the oxygen consumption.

Sig Ch 0.000 0.000 0.000 0.000 0.000

FCh 305.720 140.899 105.666 39.713 20. I59

MS 1.94104 0.01202 0.0399 I 0.00969

ss 9.70521 8.41460 2. I5488 6.25972

r Summary of the model B Variable Constant 0.41045 Gravid 0.24000 Oxygen’ -0.002992 Salinity 0.003610 Salinity’ -0.00002722 Gravid*salinity* -5.026.10-”

291

Carrel. 0.5503 -0.2915 -0.2140 -0.2036 0.1266

F-Value

Sig F

161.47276

0.0000

4.11861

0.0000

SE Beta

Partial

Tolerance

0.03560 0.02582 0.11166 0.11385 0.04024

0.58137 -0.23560 0.36299 -0.41521 -0.16731

0.52350 0.99525 0.05321 0.051 I9 0.40978

F 1278.100 357.382 41.137 106.228 145.821 20. I59

Sig F 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000

A factor that has been neglected during all previous experiments dealing with the effect of salinity on the respiration is the impact of oxygen. Since the oxygen concentration in saline waters is dependent on oxygen pressure and on salinity, they should be both taken into account when examining the effect of the salinity on respiration. Respiration is a diffusion process with the oxygen pressure as the driving force. In saline environments the oxygen concentration can be primal in determining the oxygen availability, since its

0.6

0.04 0

I

Oxygen

0.04

0

1 Oxygen

2

3

4

concentratrion

2

3

concentratrion

5

b

0

4

5 (ml/l)

1 Oxygen

(ml/l)

I

6

o.oL--0

1 Oxygen

2

3

4

concenfratrion

2

3

concentratrion

5 (ml/l)

4

5 (ml/l)

Fig. 3. Dry weight in function of the oxygen concentration in the saline waer for a salinity of 10% (A), 35% (B), 90% (C) and 145L (D). Each figure contains mean and standard deviation of the observed data for females (O), gravids (A) and males (0). Filled symbols are mean and standard deviation for the predicted values for each group.

I

6

298

BART DE WACHTER and JAN VAN DEN

ABBEELE REFERENCES

160.0 140.0 120.0 g

100.0

.&- 80.0 .E 2

60.0 40.0

.O

I 2.0 Oxygen

/

I

I u 3.0 Concentration

2.0 Oxygen

3.0 Concentration

, uLLUIL._I 4.0 5.0 ml/l

160.0

g

100.0

.z.c

80.0

2

60.0 40.0 20.0 .O 1 .o

5.0

4.0 ml/l

Fig. 4. Contour plot of the regression equation. The dry weight (mg) is given in function of the oxygen concentration and salinity for males (A) and gravids (B).

diffusion rate is concentration dependent. It can therefore be more important than the partial oxygen pressure. We offered both oxygen pressure and oxygen concentration as possible variables in the regression model. In both cases, when determining the effect on weight and on oxygen consumption, this partial oxygen pressure has been removed out of the model in favour of the oxygen concentration which was selected. This indicates that weight and oxygen consumption fits better with oxygen concentration than with oxygen partial pressure. These findings support the suggestion of Lange et al. (1972) that in saline waters oxygen concentration is more important than the oxygen partial pressure in controlling metabolic responses. The importance of the salinity with regard to the dry weight is probably related with the osmotic properties of the environment. Croghan (1958) showed that the brine shrimp is a hyperosmotic regulator. The energy this regulation demands may be responsible for the smaller dry weight after acclimation to higher salinities.

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