The effects of salinity and pH on the activity and oxygen consumption of Brachionus plicatilis (rotatoria)

Comp. Biochem. Physiol., 1978,Vol. 59A,pp. 9 to 12. Pergamon Press. Printed in Great Britain

THE EFFECTS OF SALINITY AND pH ON THE ACTIVITY AND OXYGEN CONSUMPTION OF B R A C H I O N U S P L I C A T I L I S (ROTATORIA) R. W. EPP and P. W. WINSTON Hygrobiology Laboratory, Department of Environmental, Population and Organismic Biology, University of Colorado, Boulder, CO 80309, U.S.A. (Received 28 March 1977)

Al~tmet--1. Activity and respiratory rates of the rotifer, Brachionus plicatilis, were determined following exposure to pH values of 6.5, 7.5 and 8.5 and to concentrations of 10, 50 and 100 mOsm. 2. Changes in the hydrogen-ion concentration had no detectable effect on either activity or metabolism. 3. Acute reduction in osmolarity of the medium resulted in a reduction in oxygen consumption and activity. 4. Both activity and oxygen consumption increased upon acclimatization to osmolarities of 50 and 100 mOsm.

INTRODUCTION

Throughout the nineteenth century, the rotifers were considered to be a truly cosmopolitan group of animals (Ehrenberg, 1838). Jennings (1900) revised this view somewhat when he referred to them as "potentially cosmopolitan" with respect to their means of distribution (i.e. the resting eggs and desiccation cysts). He went on to say "The problem of the distribution of the Rotifera is, then, a problem of the conditions of existence, not a problem of the means of distribution". As a result of their extensive collections in Wisconsin and the Washington, D.C. area, Harring & Myers (1928) were able to divide the ploimate rotifers into three ecological categories (acid water, alkaline water and transcursion species) based on the "conditions of existence". While this classification is an accurate representation of the distributional pattern of several hundred rotifer species (Myers, 1931 ; Edmondson, 1944), many workers feel that the actual cause may be some related variable rather than pH, per se. Pourriot (1965) notes that "Dans les eaux naturelles, le pH est la la rtsultante des actions combintes des difftrents constituants chimiques. Tousles limnolognes s'accordent actuellement pour reconna~tre que tr~s souvent le facteur limitant n'est pas le pH lui-m&ne mais d'autres facteurs dont le pH n'est que le reflect". Hutchison (1967) also points out that numerous factors may vary in a correlative fashion with pH, so that an apparent limitation to pH may be due to one of the related variables. Among other possibilities, he suggests the osmotically active concentration as a potential limiting factor. Osmolarity has, indeed, been shown to be limiting in the distribution of B r a c h i o n u s plicatilis (Ito, 1956; Epp & Winston, 19771 This species is a common alkaline and brackish water rotifer (Pennak, 1945) as well as a frequent inhabitant of hyperhaline ponds (Beadle, 1943; Hutchison, 1967)~ The lower osmolarities usually associated with soft, acid-water corn-

munities cause a reduction in the hatching of the resting eggs (Epp, in press). Furthermore, B. plicatilis has been shown to be essentially an osmo-conformer with exposure to low external concentrations causing the internal concentration of this rotifer to fall to exceptionally low levels (Epp & Winston, 1977). In such osmo-conforming animals, a decrease in the concentration of the medium often results in a decrease in metabolism (Potts & Parry, 1964). The purpose of this research was to determine if this relationship is true for B. plicatilis. T h e effect of pH on metabolism was also studied. It was felt that if pH did have an effect on the distribution of this species, this effect might ultimately be due to the influence of pH on the physiology of this animal. This might manifest itself in a change in metabolism resulting from a change in pH. These two variables were studied concurrently in hopes of determining the factor with the most significant effect on metabolism and, thereby, the most likely factor affecting the distribution of B. plicatilis. Because of the relationship between activity and metabolism (Prosser, 1973), the activity of individual rotifers was measured under the same conditions as were used in the metabolic studies.

MATERIALSAND METI4.ODS Activity was measured by placing individual rotifers in a fiat-bottomed watch-glass containing the desired medium. After an equilibration period of 1 hr, the watchglass was placed under a dissecting microscope and on a piece of graph paper with 1 mm grids. The animal was observed for a 5-rain period and the humber of grids entered was recorded. Oxygen consumption of individual, pre-ovigerous female rotifers was measured with a Cartesian diver mt_fr'o-respirometer (Linderstr~m-Lang, 1943; Holter & Zeuthen, 1966; Klekowski, 1971). The divers had gas volumes ranging from 0.75 to 1.25#1 and were of the straight-necked variety (Glick, 1947). Loading procedures were modified from

10

R.W. EPP AND P. W. WINSTON

Holter & Zeuthen (1966). A fine-tipped pipette was used to completely fill the diver with the desired test medium. The test animal was then placed in the diver neck with a "braking pipette" (Claff, 1947). As the rotifer swam toward the bottom of the diver, the excess medium was removed with a suction pipette. The inside of the diver neck was then dried with thin strips of lintless filter paper. NaOH and oil seals were added in the standard manner. The diver was then placed in a small beaker containing 50ml of Holter's flotation medium (Glick, 1947). Small amounts of air were aspirated from the diver neck with a micro-pipette until the diver slowly sank in the flotation medium. This procedure provided an initial flotation pressure of less than 25 mm of Brodie's solution (Umbreit, 1964). The volume of the oil was determined with an ocular micrometer. Following each experimental run, the mouth seal was removed and the diver was carefully rinsed with distilled water. The weight of the diver, oil, NaOH and test medium was determined with a Cahn electro-balance. The volume of the medium and NaOH could then be calculated by subtracting the weight of the oil and the empty diver. The gas volume of the diver was then calculated according to the standard formula (Holter & Zeuthen, 1966). The divers were allowed to equilibrate for 1 hr prior to the initial measurement and each trial lasted approximately 1.5hr following the initial measurement. All measurements were performed at 20°C. A hyperhaline pond adjacent to Gaynor Lake in Boulder County, Colorado was used as a source for the rotifers. The experimental media consisted of dilutions of artificial Woods Hole sea water (Cavanaugh, 1956). Three osmolarities of 10, 50 and 100 mOsm were used which correspond to approximately 350, 1750 and 3500mg/l total dissolved salts. Three pH values of 6.5, 7.5 and 8.5 were prepared by the addition of small amounts of 0.1 N NaOH. In the initial experiments, the animals were cultured in calcareous pond water from the source with a pH of 8.7 and an osmolarity of 487 mOsm. The activity and oxygen consumption of these animals was measured following acute changes in pH and osmolarity. Additional animals were cultured in the test media at osmolarities of 50 and 100 mOsm and pH values of 6.5, 7.5 and 8.5. The metabolic and activity studies were then repeated on these acclimatized animals. Since the animals could not be cultured at an osmolarity of 10mOsm, the effect of acclimatization could not be determined at this concentration.

Table 1. Activity of Brachionus plicatilis in mm 2 entered during a 5-min period (±S.E.)

RESULTS

Acute exposure to the test media resulted in a reduction in b o t h activity and oxygen c o n s u m p t i o n as evidenced by the subsequent increase following acclimatization. There appears to be little d o u b t that these reductions were due to the reduced osmolarity

Activity measurements The effects of acute changes in pH and osmolarity were measured On the activity of 15 animals from each of the nine test media. Immediately following transfer from the culture to the test medium, nearly all animals showed a m a r k e d decrease in movement. In all of the media except those at 10 mOsm, the activity began to increase shortly after the transfer. After 1 hr equilibration, the m o v e m e n t of the animals at 1 0 m O s m was negligible, while the rotifers from the other six media were all actively swimming (Table 1). The m o v e m e n t of the animals at 10 m O s m was significantly lower t h a n that at the other osmolarities (P < 0.001) while there were no significant differences between the other six groups (t-test). Ten animals were examined from each of the six cultures (Table 1). Again there were n o significant differences between any of the six groups, but the activity was greater t h a n that of the animals subjected to acute changes in pH and osmolarity (P < 0.1301).

Osmolarity

6.5

pH 7.5

8.5

10 50 100

3.3 ± 1.6 38.8 ± 6.9 47.4 ± 7.3

1.6 ± 0.6 53.9 ± 9.1 47.4 ± 6.6

2.5 + 0.7 44.9 ± 5.8 51.9 ± 2.7

50 100

137 ± 21 165 ± 20

144 ± 10 153 ± 23

135 ± 23 147 ± 31

Upper portion represents activity following acute exposure to the nine test media, while the lower portion represents the results following acclimatization. Concentrations are expressed as mOsm.

Respiratory studies An average of 16 rotifers were used from each of the nine test media for a total of 145 animals. Despite the small size of the divers, the activity of the rotifers did not appear to be hampered. Acute exposure. The effects of acute changes in pH and osmolarity are shown in Table 2. Two-way analysis of variance (ANOVA) indicated that the hydrogen ion concentration had n o effect on metabolism and there was no interaction between pH and osmolarity. Osmolarity had no significant effect o n metabolism within the 50 and 100 m O s m media, but oxygen consumption was considerably reduced at 10mOsm. Overall, the effect of osmolarity was significant at the 0.001 level ( A N O V A ; F = 38, df = 2/136). Acclimatization. Measurements identical to those described above were performed o n 60 animals that were acclimatized to the six-test media. The results of this portion of the experiment are shown in Table 2. There was no significant difference with respect to pH ( A N O V A and linear regression) or osmolarity (ANOVA). All values were, however, higher than those for the animals subjected to acute changes in pH and osmolarity (t-test; P < 0.001). DISCUSSION

Table 2. Oxygen consumption of Brachionus plicatilis in /al 0 2 x t0 -4 per animal per hr (__S.E.) Osmolaxity

6.5

pH 7.5

8.5

10 50 100

11.52 -I- 1.06 8.51 __ 1.44 10.04 + 0.53 17.13 ± 1.15 15.97 + 0.61 16,42 __ 1.24 16.21 ± 0.81 15.18 _____0.69 16.98 + 0.89

50 100

18.31 + 0.67 19.39 __ 0.71 19.13 _____0.57 20.06 + 0.52 20.13 + 0.92 19.75 ± 1.15

Upper portion represents oxygen consumption following acute exposure to the test media, while the lower portion represents the results following acclimatization. Concentrations are expressed as mOsm.

Salinity and pH effects on Brachionus plicatilis rather than the change in pH. The water from the source had an osmolarity of 485 mOsm and a pH of 8.7. Transfer to the test medium of 100mOsm and pH of 8.5 resulted in only a minor change in pH, even though the osmolarity had been reduced fivefold. Furthermore, there were no significant differences between pH groups in any of the experiments, but both activity and oxygen consumption were lower at 10 mOsm than at either 50 or 100. Our findings concerning activity are consistent with the observations of Worley (1928), who noted a temporary sluggishness in B. mulleri (=plicatilis) following transfer from a salinity of 45 to 329oo. A similar reduction in the activity of Maja verrueosa following a decrease in salinity was shown by King (1965). The existence of a correlation between 02 consumption and salinity has been known for some time (Remane & Schlieper, 1971). Roch (1924) noted that the coelenterate Cordylophora lacustris could be found in stagnant or well-oxygenated water provided it was brackish, but it occurred in only well-oxygenated fresh water. Thienemann (1928) found a similar situation with M y s i s relica. Schlieper (1929), who was the first to measure changes in 02 consumption following changes in the concentration of the medium, found a permanent increase in 02 consumption in C a r c i n u s m a e n a s and a temporary increase in Nereis diversicolor following a reduction in salinity. Similar interpretations can be made about the studies of Beadle (1931), Schwabe (1953), Rao (1958) and Lofts (1956). On the other hand, animals such as Asterias rubens (Meyer, 1935), Mytilis edulis (Bouxin, 1931), Metridium marginatum (Schoup, 1932), Maja verrucosa and Hyas araneus (Schwabe, 1953; King, 1965)all show a reduction in 02 consumption following any change in salinity from the normal acclimatized concentration. From this information, Potts & Parry (1964) concluded that osmoregulating animals respond to a decrease in concentration by increasing oxygen consumption, while osmo-conformers respond to any change in the osmotic concentration by decreasing metabolism. This appears to be the case with Brachionus plicatilis. In this species, which has already been shown to be an osmo-conformer (Epp & Winston, 1977), a reduction in the external concentration resulted in a decrease in oxygen consumption. The relationship between activity and oxygen consumption in B. plicatilis is unclear. It has been suggested that the increase in oxygen consumption following a dilution of the medium is due to random or escape movements (Gross, 1957). King (1965) suggests that the decrease in oxygen consumption in M a j a verrucoa is directly due to the decrease in movement and the lower 02 availability resulting from the reduction in ventilation movement. In the present study, it seems possible that the decrease in oxygen consumption results from a decrease in the availability of metabolically active ions and a reduction in the concentration of enzymes resulting from tissue hydration. The reduction in activity may, then, be a result of the overall reduction in metabolism, rather than its cause. The results of this and previous studies on B. plicatills (Ito, 1956; Epp & Winston, 1977; Epp, in press) indicate that the low osmolarities of soft, acid-

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waters are restricting the distribution of this species, rather than the acid pH values. Osmolarity may, in fag, be responsible for the distributional pattern of many of the rotifer groups with both marine and fresh water representatives, but further study is necessary before making any generalizations. REFERENCES BEADLE L. C. (1931) The effects of salinity changes on the water content and respiration of marine invertebrates. J. exp. Biol. 8, 211-227.

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