The body fluid osmolarity of individual rotifers was measured at 12 external concentrations ranging from 32 to 957 m-osmol/l. Brachionus plicatilis is essentially an osmoconformer, since a change in the concentration of the medium results in a corresponding change in the concentration of the body fluids. Most animals were, however, slightly hyperosmotic throughout the range tested.
The lowest body fluid osmolarity was 59 m-osmol/l at an external concentration of 32 m-osmol/l. It appears that B. plicatilis is unable to tolerate the low concentrations that are frequently associated with acid water environments and this is responsible for the restriction of this species to alkaline and brackish waters.
The Rotatoria have traditionally been divided into alkaline water, acid water and transcursion species (Harring & Myers, 1928). While many rotifers do exhibit such a distributional pattern, most workers feel that their distribution may be due to some factor or factors other than pH per se (Edmondson, 1944; Pourriot, 1965). Hutchinson (1967) 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. He suggests, among other possibilities, that the osmotically active concentration may be responsible for the observed distributional pattern in the rotifers.
Brachionus plicatilis is a common alkaline and brackish water species (Pennak, 1945) as well as a frequent inhabitant of hyperhaline ponds (Beadle, 1943; Hutchinson, 1967). Because of its restriction to waters with a high concentration of dissolved solids as well as alkaline pH values, it appears that this species might be limited by the concentration of the medium rather than, or in addition to, the pH. The purpose of this research was to measure the internal osmolarity of B. plicatilis over a range of external concentrations in order to determine (1) the nature of this species’ ability to tolerate salinities ranging from 1 to 97%o (Ito, 1956, 1960) and (2) to determine if the osmotically active concentration is a factor limiting the range of this species.
MATERIAL AND METHODS
A hyperhaline pond adjacent to Gaynor Lake in Boulder County, Colorado, was used as a source for the rotifers. The experimental animals were cultured in various osmolarities which consisted of dilutions of artifical Woods Hole sea water (Cavanaugh, 1956). All test media were maintained at pH 8·5. Rotifers could be transferred directly to salinities of up to 50% sea water, but beyond this concentration, acute exposure resulted in nearly 100% mortality. For this reason, the rotifers were acclimatized to concentrations greater than 50% s.w. by periodically transferring them to increasing salinities over a period of several months. Animals were cultured in 11 concentrations ranging from 41 to 957 m-osmol/l. One additional group was maintained at 32 m-osmol/l. As this group did not produce eggs by the seventh day, the osmotic pressure was measured on the acclimatized animals. All other measurements were made on adult pre-ovigerous females of the second generation or later. The average body length of the animals was approximately 400 μm.
A braking pipette (Claff, 1947) was used to transfer individual rotifers from the culture to a watch-glass containing mineral oil which was chilled to 4 °C on the refrigerated stage of a dissecting microscope. Excess water was removed with a fine-tipped suction pipette, thus leaving the animal completely immersed in the oil. Any water clinging to the rotifer was removed by gently swabbing it with a thin strip of filter paper that had been inserted into the oil.
The collecting pipettes were made by drawing out 0·75 mm glass tubing to form fine (50μm) tip. The tip was placed in the oil and suction applied by way of a short length of latex tubing. A small quantity of oil was thus aspirated into the pipette. The tip was placed on the corona of the animal and suction again applied until the body wall ruptured. A sample of the pseudocoelomic fluid was then drawn into the pipette, followed by an additional amount of mineral oil. The mineral oil prevented evaporation of the sample, while the chilling reduced the adsorption of water by the oil. The collecting pipette was placed in a sample holder (Laird, 1971) and the body fluids were immediately frozen in a dry ice-alcohol mixture.
The osmotic pressure was determined with the micro-cryoscope developed by Ramsay & Brown (1955) and modified by Tuft (personal communication). Further modification described by Laird (1971) provided for an accuracy of 0·007 °C (standard deviation) using samples of approximately 5 nanolitres.
Fifty-five animals were examined with the freezing point depressions of the media being determined concurrently with each body fluid sample. All results have been converted to m-osmol. The mean internal osmolarity for each of the 12 groups is shown in Fig. 1. These results indicate that Brachionus plicatilis is essentially an osmoconformer, since a change in the concentration of the medium results in a corresponding change in the concentration of the body fluids. A linear regression line was fitted to the data by the method of least squares. This line can be described by the equation: [m-osmol/l]1 × 0·9574 [m-osmol/l]0 + 36; r × 0·9982.
Despite the direct relationship between the concentration of the medium and that of the body fluids, all of the animals from 32 to 867 m-osmol were slightly hyperosmotic to their respective media. Additionally, three of the five animals examined at 957 m-osmol/l were also hyperosmotic.
The ability of B. plicatilis to withstand large changes in the concentration of the medium is clearly due to its ability to tolerate considerable variation in the concentration of its body fluids. This faculty of tolerance is quite common in brackish-water animals, more so than that of osmoregulation (Potts & Parry, 1964), but what is unusual is the range of tolerance in B. plicatilis. The internal concentration of this animal was found to vary from approximately 6% to nearly full strength sea water (59–936 m-osmol/l). The only metazoan known to have a greater tolerance to internal changes is the serpulid polychaete, Mercierella enigmatica. The blood concentration of this animal was found to vary from 94 to 1788 m-osmol/l and it can withstand prolonged exposure to glass distilled water (Skaer, 1974). Above an external concentration of 1620 m-osmol/l, the blood concentration of M. enigmatica rises rapidly and the animal begins to die. Ito (1960) reports that Brachionus plicatilis is able to tolerate external concentrations as high as 2860 m-osmol/l (54‰ Cl) and it would, therefore, appear that B. plicatilis may have an internal range equivalent to or greater than even M. enigmatica. In the present study, however, we were unable to acclimatize B. plicatilis to concentrations greater than 957 m-osmol/l.
At an external concentration of 32 m-osmol/l, the [m-osmol/l]1 of B. plicatilis fell to 59 m-osmol/l. Correspondingly low values have been recorded for a number of freshwater animals (Kitching, 1938; Potts, 1954; Lilly, 1955; Potts & Parry, 1964). Anodonta cygnaea, with a [m-osmol/l]1 of 44, has the lowest internal osmolarity of any animal (Hutchinson, 1967). Potts & Parry (1964) have suggested that the low concentration of these animals may be associated with the low metabolic rates of such sedentary or sessile animals. In contrast, they mention the relatively high internal osmolarities of a number of active protozoans. The osmotic pressure of the large rhizopod, Pelomyxa carolinensis, is estimated at between 94 and 103 m-osmol/l (Lovtrupt & Pigon, 1951; Prosser, 1973) and that of the ciliate, Spirostomum, at 89 m-osmol (Prosser, 1973). Tetrahymena has a recorded [m-osmol/l]1 of approximately no in a dilute medium (Stoner & Dunham, 1970). The low [m-osmol/l]1 recorded for B. plicatilis (59 m-osmol/l) may then be viewed as an anomaly when considering the rather high metabolic rate of such an active planktonic animal. Anodonta, for instance, has a VO2 of 0·002 ml O2/g.h (Prosser, 1973). Using the 17% dry matter figure given by Picken (1931), the Q02 of Anodonta is calculated to be approximately o·008 ml O2/g.h as compared to 12–16 ml O2/g.h for B. plicatilis (Doohan, 1973; Epp & Winston, in manuscript).
Under controlled laboratory conditions with an abundance of food and a lack of competition, B. plicatilis can live for some time at a concentration as low as 3 2 m-osmol/l, but it has never been recorded in nature at such a low concentration. With a dissolved solids content of 1100 mg/1, this water represents oligohaline brackish water (Venice Symposium, 1959). The lower osmolarities encountered in soft fresh water would be expected to cause a further dilution in the body fluids of B. plicatilis to the point at which even normal metabolic functions would be impossible. It therefore appears that B. plicatilis is limited to hard alkaline waters because of its inability to tolerate the low osmolarities normally associated with soft water environments. Whether this factor of osmolarity is responsible for the distributional pattern of the Rotatoria as a whole is uncertain, but it appears as a possibility in the numerous groups with both marine and freshwater representatives.
The Rotatoria is predominately a freshwater group and is considered to have arisen from some freshwater ancestor. While the results of this study do not necessarily disprove this idea, they do illustrate a significant difference between the osmotic regulating process in B. plicatilis and that of other hyperhaline animals of freshwater origin. Most hyperhaline animals have well developed powers of hyposmotic regulation (Beadle, 1943), for example, the brine shrimp, Artemia salina (Croghan, 1958) and the saline mosquito, Aedes detritis (Wigglesworth, 1933,1938; Beadle, 1939). This capacity for hyposmotic regulation has, then, been given as evidence of the freshwater origin of these animals (Beadle, 1943; Croghan, 1958; Potts & Parry, 1964). Furthermore, numerous brackish and marine animals are capable of regulating their internal concentration below that of the medium. Panikkar (1941) suggests that the high degree of osmotic independence in Palaemontes varians (Panikkar, 1939), Leande serratus (Panikkar, 1940) and L. squilla (Panikkar, 1941) indicates that these animals must have inhabited fresh water at some time and are possibly of freshwater origin. The marine teleosts are also capable of hyposmotic regulation (Smith, 1932; Keys, 1933; Baldwin, 1937) and this has been correlated with their freshwater origin (Beadle, 1943; Croghan, 1958).
In contrast to all of these animals, B. plicatilis is hyperosmotic to concentrations of 867 m-osmol/l and below. Additionally, three of the five animals studied at 957 m-osmol were hyperosmotic. The lack of the ability to regulate hyposmotically at concentrations approaching full strength sea water suggests a marine ancestry for this animal.