The Effects of Cold Acclimation upon the Oxygen Consumption of Two Species of Free-Living Nematodes l SUSAN C. COOPER and J. HOMER FERGUSON2 Abstract: Two species of free-living nematodes, Panagrellus redivivus and Turbatrix aceti, were cultured axenicaUy at control (20 C) and cold (10 C) temperatures. Oxygen consumption of worms from each population was measured manometrically on days 2 through 8 after exposure to these temperatures. In both species, the slope of the oxygen consumption curve for the controls was greater than that of the worms exposed to the cold on day 2. The slope of the curve of the cold-exposed worms gradually increased until day 7. At this time, the slope of the oxygen consumption curve from the cold-exposed worms exceeded or equaled that of the controls. This is taken as an indication of the onset of the cold-acclimated state in both species of worms by day 7. For many years, a number of parameters stoppered with cotton balls wrapped in cheese have been used to denote the onset of the cloth (6). At weekly intervals, the worms were acclimation of a poikilothermal organism to subcultured aseptically by draining the worms various environmental conditions, particularly and the old medium from each funnel and to temperature (1, 9, 10). Among the criteria adding them to another funnel containing fresh utilized have been such rate functions as heart medium. beat (20), ciliary gill movements (19) and The worms were exposed to a temperature metabolic rate; e.g., rate of an enzymatic of 20 C until use in this experiment. They were reaction (7, 10, 11). subjected to a photoperiod of 12 hr of light and In most species of poikilothermic animals, 12 hr of dark throughout the study. exposure to a cold but nonlethal temperature The basic medium consisted of an will result in an initial decrease in metabolic autoclaved aqueous solution of 4% soya rate according to Van't Hoff's principle (1). peptone and 3% yeast extract (2) to which However, after a certain period of time during heated liver extract (17) was added to make up which the organism has been exposed to cold, it 10% of the volume of the medium (2). This often responds by acclimating to the new medium was used for P. redivivus. For T. aceti, temperature. This is frequently manifested as glacial acetic acid was added to make up 3% of an increased metabolic rate (10). the basic medium (14). In the present study, two species of The cultures were checked periodically for free-living nematodes were acclimated to cold. signs of bacterial or fungal contamination by Metabolic rates of acclimated and control using agar plates of brain-heart infusion or worms were compared, and rates of acclimation thioglycollate medium incubated at room were determined in both species. temperature for 1 week. Contamination in a culture was readily apparent, however, because MATERIALS AND METHODS it caused a subsequent discoloration of the Culture methods: Axenic cultures of medium. Panagrellus redivivus and Turbatrix aceti were Cold acclimation: Each population of maintained continuously in 50-ml cylindrical worms harvested from the funnels received separatory funnels lined with glass wool and approximately 5 ml of fresh medium. The medium of each species was divided evenly between two sterile 125-ml Erlenmeyer flasks, Received for publication 13 April 1972. which then were covered with aluminum foil. 1 This work is part of a doctoral dissertation by the One flask was .returned to the control (20 C) senior author supported in part from a NASA predoctoral fellowship during part of the temperature, whereas the second was investigation. introduced to'the cold (10 C). 2 Susan C. Cooper, Department of Biological Sciences, On days 2-8 following the initiation of the University of Idaho, Moscow 83843; and J. Homer treatment, two 0.5-ml aliquots of each Ferguson, Professor of Biology, Division of Earth and Life Sciences, The University of Texas at San worm-medium mixture were removed, each of Antonio, 4242 Piedras Drive, East, San Antonio which was pipetted into the main compartment 78284. The authors thank Morton Rothsteln (State University of New York at Buffalo) who g~aciously of a Warburg reaction vessel for manometric supplied cultures of axenic Panagrellus redivivus and studies. From time to time, aliquots were Turhatrix aceti. extracted and counted to verify accuracy of the 241 242 Journal ofNematology, Vol. 5, No. 4, October 1973 procedure. There were no significant changes in oxygen per unit time by day 4 and much more number of worms over the time period studied by days 5 and 6; oxygen consumption by when compared on a statistical basis. control worms decreased again by day 7. Each reaction flask also contained 1.5 ml of Enzyme accumulation apparently peaks after 1 M phosphate buffer (pH 8) in the main day 4, hence the notable increase in oxygen compartment, 1 ml of 10% glucose in the consumption on day 5. sidearm, and a piece of fluted filter paper Whereas little oxygen was consumed by soaked with 0.2 ml of 20% KOH in the center cold-exposed T. aceti (Fig. I-B) on days 2-4, well (22). After equilibration of the reaction uptake increased slightly on day 5 and vessel in the Warburg apparatus for 15 min, the considerably more on days 6 and 7. By day 7, substrate was tipped into the main the rate of oxygen consumption by worms compartment, and oxygen consumption was exposed to the cold was appreciably greater measured for 105 min. than that of the controls. Again, no further Originally, the rate of oxygen uptake was difference was detected on day 8. measured at 37 C. However, T. aceti failed to Unlike the linear curves obtained for P. consume detectable quantities of oxygen at this redivivus (Fig. l-A) which are typical of oxygen temperature. Therefore, all subsequent oxygen consumption curves, those obtained by plotting consumption measurements were made at 30 C oxygen uptake of T. aceti versus time often for both species. showed a distinct plateau in the first few days The experiment subsequently was repeated after the initiation of the experiment (control: using worms obtained from a second day 2; cold: days 2-5). Subsequently, however, subculture. the more conventional, linear pattern gradually replaced the horizontal nature of the lines, the RESULTS slopes becoming greater and greater with time (with the exception of that of the day 7 The control of P. redivivus consumed control, where the slope decreased with respect considerably greater amounts of oxygen than to that of day 6). did the worms exposed to the cold (Fig. l-A). The difference between oxygen consumption of the two groups decreased gradually until very DISCUSSION little difference appeared by day 5. On day 6, Typically, the amount of oxygen although the difference was very slight between consumption increased in the control worms of the two groups, the cold-exposed worms both species shortly after the initiation of the consumed more oxygen per unit time than did experiment (P. redivivus: days 2-4; T. aceti: the controls. This trend was accentuated by day days 2-5). Gradually, the slopes obtained from 7. No further difference was detected by day 8. the worms subjected to the cold increased with A lag in oxygen consumption following the time until they clearly exceeded those of the addition of exogenous glucose was evident in control worms by day 7. This is the classic some of the curves (Fig. I-A). In the control picture of cold acclimation as presented by worms, this lag either was not apparent (days many authors, in which some rate function 2-4) or persisted for only 5-10 min after glucose (usually rate of oxygen uptake) increases at a supplementation (days 5-7). On the other hand, given temperature in a cold-acclimated animal relatively long lag periods of 45 rain (day 2), with respect to that of a control. Precht (9) approximately 30 rain (day 3-4) and 10 min described five patterns of temperature (day 5) were revealed in the curves obtained acclimation, ranging from supra-optimal from the cold-exposed worms. No lag period compensation (type 1) to inverse compensation appeared after 6 or 7 days of cold exposure. (type 5). The most common pattern is one in The results obtained from T. aceti are not as which the normal rate is greater in cold- than in clear-cut as are the data from P. redivivus. At warm-acclimated animals (9). This pattern was days 2 and 3 (Fig. I-B) little difference was observed in this experiment for both P. observed between the oxygen consumption of redivivus and T. aceti. Most frequently, partial the control and that of the cold-exposed compensation (type 3) is observed (9, 10), but worms. In both cold-acclimated and control often the rate function approaches perfect groups, oxygen uptake, although greater after the compensation (type 2) (10). Bullock (1) addition of exogenous glucose, was very low. pointed out that this type of compensation to The control worms consumed slightly more temperature by various rate functions has the Nematode Cold Acclimation: Cooper, Ferguson 243 over-all effect of conferring relative homeostasis from the control and the cold-exposed worms. to poikilotherms that can acclimate to changing In both species, the curve obtained from the temperatures over a nonlethal temperature cold-exposed worms increased with time until range. day 7, when the rate of oxygen consumption In the oxygen consumption-time plots for was appreciably greater than that of the both P. redivivus and T. aceti, a change controls.