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Thermal acclimation without heat shock, and motor responses to a sudden temperature change in Asellus aquaticus
ARTICLE IN PRESS
Journal of Thermal Biology 28 (2003) 421–427
Thermal acclimation without heat shock, and motor responses to a sudden temperature change in Asellus aquaticus Kari Y.H. Lagerspetz* Laboratory of Animal Physiology, Department of Biology, University of Turku, FIN-20014 Turku, Finland Received 25 September 2002; accepted 7 March 2003
Abstract 1. In the fresh water isopod Asellus aquaticus the critical thermal maximum (CTMax) after 2 weeks was similar in animals transferred directly from 9 C to 22 C and in animals acclimated gradually (1 C/day). 2. A sudden change of ambient temperature caused immediate changes in the locomotor behaviour of Asellus that did not persist, and especially an increase of the frequency and angle of turning (klinokinesis). 3. Asellus, acclimated to 12 C, preferred temperatures of 11–15 C in a thermal gradient, and showed klinokinetic heat avoidance reactions at effective heat shock temperature (28 C). r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Asellus aquaticus; Crustacea; Heat shock; Thermal acclimation; Avoidance; Preference; Motor activity; Klinokinesis
1. Introduction Thermal acclimation of poikilotherms can be observed in laboratory experiments but in nature acclimation is merely a part of seasonal acclimatization, and so occurs in response to slowly changing environmental temperature. Thus, thermal acclimation may occur in nature as a response to a prolonged change in the weather or in the microclimatic conditions. The changes concerned will often be relatively slow, but the organism’s responses may be made complex because they would be capable of behavioural thermoregulation, i.e. to move to a favourable temperature, and so to avoid or offset the progressive changes in ambient temperature. However, rapid changes in environmental temperature may occur in nature, e.g., when an animal crosses a thermocline in a lake, or moves from shade to sunshine, or when exposed to air on the ebb of the tide.
*Tel.: +358-2-2328566; fax: +358-2-3336590. E-mail address: kari.lagerspetz@utu.fi (K.Y.H. Lagerspetz).
In most existing experimental studies on thermal acclimation of ectotherms, animals have been transferred directly from one temperature to the other or allowed only a few hours for the temperature change to occur. The temperature difference used has been typically 5–20 C in magnitude. The effects of a heat shock on heat tolerance were generally recognized first in the 1980s (Lindquist, 1986). A shift of only 5 C (from 20 C to 25 C for 1 h) was sufficient to increase heat shock protein (HSP) production in intertidal limpets (Sanders et al., 1991). Heat shock effects occur when animals are exposed to temperatures close to the critical thermal maximum (CTMax) of the species (Tomanek and Somero, 1999). In some habitats a rapid change of environmental temperature may cause a heat shock to the animals so that occasional heat shock events would not be expected to alter the longer term effects of acclimatization, but its effect on heat tolerance would be short-term (hours). The animals are left long enough (from 5 days to several weeks) at the new temperature before the assessment of acclimation status, so that the immediate effects of a possible heat shock must be over (Sanders, 1993).
0306-4565/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4565(03)00027-5
ARTICLE IN PRESS 422
K.Y.H. Lagerspetz / Journal of Thermal Biology 28 (2003) 421–427
However, the question remains whether an initial heat shock has some effects necessary for the onset of thermal acclimation processes under laboratory conditions. As the conditions for thermal acclimation and heat shock and the additivity of their effects on thermal tolerance are well known in the freshwater isopod Asellus aquaticus (Lagerspetz and Bowler, 1993; Korhonen and Lagerspetz, 1996), the effects of a slow gradual change of environmental temperature were compared with the effects of an abrupt temperature change on CTMax and on the survival of Asellus. In addition, the initial effects of a temperature change on motor activity in Asellus were studied by a sudden transfer of the animals to a new temperature. For this, a method for the measurement of locomotor activity was developed. The thermal preference of animals was also studied using a thermal gradient.
2. Materials and methods 2.1. Animals and acclimation Adults of A. aquaticus (6–12 mm long) were collected from a shallow pond near Turku. They were kept in laboratory in charcoal-filtered tap water at 9–10 C or 12–13 C with decaying alder leaves for food. For thermal acclimation, the animals were kept either in groups of 5–40 in containers with 2–5 l of water, or for CTMax experiments individually in clear plastic vials (diameter 4.5 cm) with 40 cm3 of water at the acclimation temperature (Lagerspetz and Bowler, 1993). In these vials a thin layer of fine sand and pieces of decaying alder leaves were present for attachment and food. A total of 5 cm3 of water was changed daily. 2.2. CTMax determination Individuals from the group of animals were subjected to an increase of environmental temperature from 9 C to 22 C (1 C/day), which was slow enough as not to induce a heat shock responds. Individuals from another group were transferred abruptly from 9 C to vials at 22 C for the same period of time (13–14 days). The control animals remained at 9 C in individual containers. CTMax was determined by subjecting each animal in a plastic vial with 40 cm3 of water, with a little sand but no food to a temperature rise of 12 C/h in a Lauda RC 20-RCS thermocontrolled bath, until they could not right themselves within 60 s after being turned on their backs using a small paint brush (Lagerspetz and Bowler, 1993). Temperature close to the animal was measured by a 0.5 mm thermocouple and a calibrated Ellab thermogalvanometer with an accuracy of 0.1 C. This was done at intervals of 5 min and more often close to the CTMax.
2.3. Measurement of motor activity For the measurement of motor activity one animal at time was transferred to a clear plastic Petri dish of 14 cm in diameter with water at the desired temperature, which was monitored with a thermocouple. The water depth of 6–9 mm allowed for some vertical swimming, but this did not occur in our experiments. The animals walked often along the perimeter of the dish keeping one of the antennae in contact with the wall. The illumination of the dishes was measured by a quantum counter (Li-Cor, model Li-189) and adjusted to be in the same range in all experiments. Pilot experiments showed that a change from 0.12 to 3.5 mmol of quanta s1 m2 did not affect the behaviour of the animals. A paper marked with eight equal sectors and a circle half of the diameter of the dish was placed beneath the dish. This allowed the tracing of the movements of animals on similar charts (Fig. 1). Using a stopwatch, each minute was marked on the chart, as were all points where animals stopped moving. These charts were later measured with a map-measurer. The speed of the animals could be analysed for each minute, and the number of times they stopped as well as the number and angle of turnings they made could be estimated. These data were obtained from 6 min sample recordings of motor activity made at different intervals. Another method for retrieving data from the track charts for 6 min periods was to measure linear movement and turning angles separately for each sector of the chart and to add them up. In this study, two measures of locomotor activity were used: (1) linear velocity (LV, cm/min) which was calculated from the length of the track, and (2) the rate of change of direction (angular velocity (AV) of the movement, /min). In calculating this, the changes of direction caused by animals following the perimeter of the dish were not taken into account. After the animal had been accustomed to the experimental conditions for half an hour, it could be transferred to another similar dish with water at the same temperature without a change in its LV and with a relatively small change in AV (Table 2). In experiments on the effects of temperature change, one animal at time was transferred in 1 min to a similar dish with water of a different temperature. Its subsequent motor activity was measured immediately and later for periods of 6 min. After the experiment, the animal was transferred to 18 C, where all animals recovered. 2.4. Thermal preference and avoidance A gradient apparatus similar to that of Hodgson (1951) was used, but with two sources of flowing water
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of different temperatures. A thermal gradient from 9 C to 33 C was established in the glass tube. The water phase in the tube was 33 cm long and 1.5 cm deep. Temperatures in the different parts of the tube were measured by thermocouples inserted through holes on the upper surface of the tube. The position of the animals was recorded at 5 min intervals. The direction of the gradient was changed between the experiments. The total water flow through the gradient tube was 50 cm3/min, at which rate the flow did not disturb the animals. Six experiments of 1 h with ten 12 C-acclimated animals in each gave 720 position records. Four experiments with each of four animals were done in order to decide, if the heat avoidance responses of Asellus depended on klinokinetic or klinotactic orientation mechanism (see Section 4). A 13 C-acclimated animal was held for 30 min in the conditions used in measuring motor activity, before a plastic bag containing 10 cm3 water of 30 C was introduced and punctured, allowing the warmer water to reach the animal. The subsequent movements of the animal were recorded.
3. Results 3.1. CTMax after acclimation with or without heat shock Animals subjected to a slow rise (1 C/day, from 9 C to 22 C) in their environmental temperature had a CTMax of 34.070.2 C (n ¼ 12), which was similar to CTMax in animals directly transferred from 9 C to 22 C, after 2 weeks of individual acclimation to the latter temperature (34.570.2 C; n ¼ 12). The mean values of both groups were similar to those reported earlier for abruptly transferred Asellus, but differed significantly (t-tests, Po0:001) from the controls kept in individual vials for the same time at 9 C (CTMax 30.470.4 C; n ¼ 16; Lagerspetz and Bowler, 1993). 3.2. Motor activity
Fig. 1. Three examples of records of 6 min tracks of Asellus in the Petri dish arena (diameter 14 cm). Arrowheads mark stops, transverse bars minutes 1–6. (A) 13 C, 30–36 min after the start of the experiment (Exp. A10:2, LV 30 cm/min, AV 20 /min). (B) The same animal 0–6 min after transfer to 25 C (Exp. A10:3, LV 27 cm/min, AV 173 /min). (C) Another animal at 5 C, 6–12 min after transfer from 13 C (Exp. A18:4, LV 1 cm/min, AV 30 /min).
3.2.1. Control experiments The transfer of an animal from the acclimation aquarium to the Petri dish used for the measurement of motor activity caused at first a high explorative activity of the animals. Therefore, each experiment was preceded by 36 min to allow animals to adapt to the new measurement conditions. This adaptation to the new measurement conditions was seen as a decrease of the initially frequent and large turnings by the animals (Table 1), which occurred in about 6 min in the experimental dish (data not shown). The motor activity of individual animals measured at 0–6 min and at 30–36 min in the experiment conditions (Table 1) did not show statistically significant
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Table 1 Adaptation of individual Asellus aquaticus to the conditions for measurement of motor activity at 13 C (n ¼ 21)
Table 3 Effect of a sudden change of temperature on the linear velocity (LV, cm/min) in Asellus (n ¼ 4; for 24 C n ¼ 5)
Time (min)
LV
AV
LV at 13 C
24719
3673b
2875
2479
0–6 30–36
35.072.6 30.574.7
191.4726.9a 46.9712.2a
New temp.
5 C
18 C
24 C
28 C
0–6 min 6–12 min 30–36 min
874 775 14713
4871b 4678 46710
31710 4578 2377
2778 3574 23711n
Note: LV—linear velocity (cm/min), AV—angular velocity ( /min). Mean7S.E.M. Statistically significant differences (t-test) between data in each column are marked by superscripts: a Po0:001; bPo0:01; cPo0:02; dPo0:05:
Table 2 Immediate effects on motor activity of the transfer of Asellus aquaticus from one recording arena to another at the same temperature (13 C)
Before Transfer After
Note: Other explanations as in Table 1. n At 60–66 min.
Table 4 Effect of a sudden change of temperature on the angular velocity (AV, /min) in Asellus (n ¼ 4; for 24 C n ¼ 5) AV at 13 C
19711c,c
38714b
29713d
109747c
LV
AV
New temp.
5 C
18 C
24 C
28 C
3672.8
34719.9d
3874.4
74727.5d
0–6 min 6–12 min 30–36 min
58720c 105734c 38728
206740b 74774 39728
274785d 162771 146762
355774c 1877135 86731n
Note: Data from the last 6 min before and the first 6 min after the transfer (n ¼ 5). Other explanations as in Table 1.
Note: Other explanations as in Table 1. n At 60–66 min.
correlations: for LV measurements r ¼ 0:39; for AV measurements r ¼ 0:13; respectively. Therefore nonpaired t-tests were used throughout this study. As an abrupt change of temperature in this study involved also the transfer of the animal to another similar experiment arena, the changes of motor activity must be compared to the changes observed when an animal is simply transferred from one dish to another at the same water temperature. In these control experiments (Table 2) only a slight transient increase in AV was observed. The change of LV was not significant.
keep an upright position was disturbed by muscle spasms for about 4 (1–6) min. During this period the animals moved by lateral crawling with frequent apparently random turnings. The AV of motor activity was increased. After a transfer of animals from 13 C to 28 C, AV increased for at least 12 min (Table 4). Some motor disturbances occurred at least for half an hour, even for the whole recording period. The duration of the time at 28 C was extended to 60–66 min, as such heat shocks had been previously found to be effective in Asellus (Korhonen and Lagerspetz, 1996).
3.2.2. Motor activity after a sudden change of temperature When Asellus were transferred from 13 C to 5 C, their LV decreased and stayed at a low level (Table 3), while AV showed a transient increase for at least 12 min (Table 4). The animals assumed a special stance: legs were stretched and telson and caudal abdomen lifted up, so they stood and walked in a stilted fashion. Earlier, Kivivuori (1980) found the same phenomenon in the crayfish Astacus astacus at low temperatures. When the animals were transferred from 13 C to 18 C (Tables 3 and 4), there was a permanent increase in LV and a transient increase (for about 6 min) in AV. When the temperature difference was larger, from 13 C to 24 C (Tables 3 and 4), the ability of animals to
3.2.3. Thermal preference and avoidance The distribution of 12 C-acclimated Asellus in the thermal gradient used is shown in Fig. 2. The animals preferred temperatures of 11–15 C. There is a secondary maximum of position records at 26–31 C. This may depend on heat avoidance reactions which were clear at 28 C. As the low temperature end of the gradient was as high as 9 C, cold avoidance reactions could not be observed. Heat avoidance responses were studied in separate experiments. The animals did not make 180 turns away from water of 30 C, but several smaller random turnings and short stops.
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survived for 52 days at 13 C in laboratory. As these animals (later used in behaviour experiments) were kept at their original temperature, a selection by temperature could not occur among them. The animals fed on the alder leaves, and when housed in groups even reproduced. Some cannibalism probably occurred, as pointed out by Sprague (1963). An addition of animal food would perhaps be necessary to avoid this in laboratory populations of Asellus. 4.3. Motor activity and thermal behaviour
Fig. 2. Thermal preference of Asellus acclimated to 12 C. The distribution of animals in a thermal gradient extending from 9 C to 33 C. Based on position records (PR) taken at 5 min intervals in six 1 h experiments, each with 10 animals. Redrawn from Lehtonen (1960).
4. Discussion 4.1. Thermal acclimation without a heat shock The first experiment confirms that a heat shock is not a necessary prerequisite for thermal acclimation. This result agrees with the earlier studies on fish cell cultures (Merz and Laudien, 1987) and on cellular functions in isolated mussel gills (Lagerspetz et al., 1995) and on CTMax in A. aquaticus (Korhonen and Lagerspetz, 1996), in which the effects of heat shock and thermal acclimation have been shown to be separate because of their different time courses. 4.2. Survival The survival of Asellus in laboratory, especially in acclimation conditions is of interest, because a selection among the original sample of animals taken from a natural population may occur. Sprague (1963) found an average mortality of 28% for Asellus intermedius during 4 weeks of acclimation in groups at 20 C and 30 C. The mortality was higher at more crowded laboratory populations. In the experiments of Lagerspetz and Bowler (1993) with separately housed individuals of A. aquaticus, a loss of about 50% of animals was found during the acclimation period of 15–23 days. However, these animals were subjected at times to an assessment of their CTMax which involved a heat shock. When in this study the animals were acclimated separately, and subjected to either a sudden or gradual transfer from one temperature to another, a loss of 10–20% was observed. Of a group of 40 animals, 20
The possible changes of motor activity caused by a sudden transfer of animals to another temperature have seldom been studied in any invertebrate (Kerkut and Taylor, 1958; Perttunen and Paloheimo, 1964; Marsden, 1976). Also the initial changes in metabolism after temperature change have received little attention (Grainger, 1956, 1958). This is astonishing because of the growing number of heat shock and thermal acclimation studies. The work done by Marsden (1976) on another isopod, Sphaeroma rugicauda, is especially relevant. This work recorded the percentage of animals walking, swimming, inactive, or rolled (total inactivity) at 1 min intervals during the first 10 min and at 30–40 min after the transfer of 10 animals in each group from 10 C to temperatures ranging between 2.5 C and 35 C. At temperatures below 4 C and above 30 C most isopods were inactive, but between 14.5 C and 30 C all isopods showed either walking or swimming activity. The response of isopods following 30 min exposure to a new thermal regime was similar to that found immediately after the transfer. When the temperature was increased at a rate of 1 C/min from 2 C to 34 C, and then decreased at a similar rate, the percentage of isopods displaying the various behaviour patterns was generally similar to the results obtained at the same temperature on direct transfer (Marsden, 1976). In that work only walking and swimming were discerned, and the changes of direction were not recorded. These results on Sphaeroma are comparable to those given for Asellus in Table 3 in the present study. A stay for 30 min at the new temperature did not alter the motor activity (LV) from that recorded immediately after the transfer. Mechanical registration and photocell response counting are not useful for smaller animals, and these methods do not differentiate between different types of motor activity. Videotaping with subsequent observation (or suitably programmed imaging) would be as good as the simple chart tracing method used here. It can distinguish between linear locomotory movement, stops, and the frequency and angle of turning. The two first are in this study summated as LV, the two latter as AV.
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The idea of tracing the movements of animals on charts comes from the original work of Fraenkel and Gunn (1961) and their associates, done in 1930s. The use of the rate of change of direction (or AV without regard of the direction of the angle) as a measure of klinokinesis is due to Ullyott (1936) and Amos (1965). The method used here employs a circular arena, in which the animal is moving. For swimming animals, like Daphnia, long tubes have been used, relying on their positive phototaxis (Kobayashi and Gonoi, 1985), and for Daphnia, also counting of the number of strokes of the antennae using a microscope (McKenzie et al., 1992). These methods do not easily allow the measurements of turning, which crawling animals may do frequently. ‘‘Linear movement’’ in the present case was often movement around the circular periphery of the arena. According to Fraenkel and Gunn (1961), ‘‘The term taxis is to-day used for directed orientation reactions. yWe use the word only for reactions in which the movement is straight towards or away from the source of stimulation. yUndirected locomotory reactions, in which the speed of movement or the frequency of turning depend on the intensity of stimulation, we call kineses’’. These commonly adopted definitions have been followed here. The present results show that a sudden transfer of Asellus to another temperature changes its motor behaviour, usually immediately increasing its turning frequency and angles. At temperatures more distant from the holding temperature indicators of increased muscle tonus (the stance of the animals, muscle spasms) appear. The transfer of Asellus to 28 C for 1 h causes a heat shock (Korhonen and Lagerspetz, 1996), but also disturbs at least temporarily its motor behaviour, and so probably the function of its nervous system. This effect is rapid, as shown also by the avoidance responses at 28 C in the thermal gradient. The observed heat avoidance of Asellus seems to be based on klinokinetic behaviour as in Daphnia (Lagerspetz, 2000), and not on klinotactic, stimulus oriented responses. The interindividual variation in the motor activity of Asellus is large. It seems not depend on the size or sex of the individuals. The feeding and molting cycles which were not controlled in the present study are probably important sources of variation. In conclusion, A. aquaticus uses three defence mechanisms against high temperatures: (1) behavioural avoidance reactions which occur by increase of turnings (klinokinesis), (2) induced or increased heat shock protein production, which is associated with an increase of thermal tolerance, and (3) thermal acclimation which is slower but longer lasting and probably depends on CNS and/or endocrine effects. These behavioural, molecular and systemic mechanisms are activated and functioning independently of each other.
Acknowledgements I thank Dr. Liisa A. Vainio for help in collecting the animals and for many discussions, Risto Heikkinen (M.Sc.) and Tapio Haavisto (M.Sc.) for help in statistical treatment of the data, and Aila Martikkala (M.Sc.) (b. Lehtonen) for allowing to use parts of her original M.Sc. thesis (Lehtonen, 1960) material.
References Amos, T.G., 1965. Kinesis. Nature 208, 908–909. Fraenkel, G.S., Gunn, D.L., 1961. The Orientation of Animals, 2nd Edition. Dover Publications, New York, 376pp. Grainger, J.N.R., 1956. Effects of changes of temperature on the respiration of certain Crustacea. Nature 178, 930–931. Grainger, J.N.R., 1958. First stages in the adaptation of poikilotherms to temperature change. In: Prosser, C.L. (Ed.), Physiological Adaptation. American Physiological Society, Washington, DC, pp. 79–91. Hodgson, E.S., 1951. Reaction thresholds of an aquatic beetle, Laccophilus maculosus Germ., to salts and alcohols. Physiol. . 24, 131–140. Zool. Kerkut, G.A., Taylor, B.J.R., 1958. The effect of temperature changes on the activity of poikilotherms. Behaviour 13, 259–279. Kivivuori, L., 1980. Effects of temperature and temperature acclimation on the motor and neural functions in the crayfish Astacus astacus L. Comp. Biochem. Physiol. 65A, 297–304. Kobayashi, M., Gonoi, H., 1985. Horizontal movement of pale and red Daphnia magna in low oxygen concentration. Physiol. Zool. 58 (2), 190–196. Korhonen, I.A., Lagerspetz, K.Y.H., 1996. Heat shock response and thermal acclimation in Asellus aquaticus. J. Therm. Biol. 21, 49–56. Lagerspetz, K.Y.H., 2000. Thermal avoidance and preference in Daphnia magna. J. Therm. Biol. 25, 405–410. Lagerspetz, K.Y.H., Bowler, K., 1993. Variation in heat tolerance in individual Asellus aquaticus during thermal acclimation. J. Therm. Biol. 18, 137–143. Lagerspetz, K.Y.H., Korhonen, I.A., Tiiska, A.J., 1995. Heat shock response and thermal acclimation effects in the gills of Anodonta cygnea: ciliary activity, stress proteins and membrane fluidity. J. Therm. Biol. 20, 43–48. Lehtonen, A., 1960. Unpublished Master’s Thesis, Department of Biology, University of Turku, Finland. Lindquist, S., 1986. The heat-shock response. Ann. Rev. Biochem. 55, 1151–1191. Marsden, I.D., 1976. Effect of temperature on the microdistribution of the isopod Sphaeroma rugicauda from a saltmarsh habitat. Mar. Biol. 38, 117–128. McKenzie, J.D., Calow, P., Clyde, J., Miles, A., Dickinson, R., Lieb, W.R., Franks, N.P., 1992. Effects of temperature on the anaesthetic potency of halothane, enflurane and ethanol in Daphnia magna (Cladocera: Crustacea). Comp. Biochem. Physiol. 101C, 15–19.
ARTICLE IN PRESS K.Y.H. Lagerspetz / Journal of Thermal Biology 28 (2003) 421–427 Merz, R., Laudien, H., 1987. Two types of heat tolerance in FHM-cells. Induction by heat-shock versus elevated culturing temperature. J. Therm. Biol. 12, 281–288. Perttunen, V., Paloheimo, L., 1964. Locomotory activity of Tenebrio molitor L. (Col., Tenebrionidae) at different temperatures and light intensities. Ann. Entomol. Fenn. 30, 156–172. Sanders, B.M., 1993. Stress proteins in aquatic organisms: an environmental perspective. Crit. Rev. Toxicol. 23, 49–75. Sanders, B.M., Hope, C., Pascoe, V.M., Martin, L.S., 1991. Characterization of the stress protein response in two
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species of Collisella limpets with different temperature tolerance. Physiol. Zool. 64 (6), 1471–1489. Sprague, J.B., 1963. Resistance of four freshwater crustaceans to lethal high temperature and low oxygen. J. Fish. Res. Board Can. 20 (2), 387–415. Tomanek, L., Somero, G.N., 1999. Evolutionary and acclimation-induced variation in the heat-shock responses of congeneric marine snails (genus Tegula) from different thermal habitats: implications for limits of thermotolerance and biogeography. J. Exp. Biol. 202, 2924–2936. Ullyott, P., 1936. The behaviour of Dendrocoelum lacteum. I–II. J. Exp. Biol. 13, 253–278.
Journal of Thermal Biology 28 (2003) 421–427
Thermal acclimation without heat shock, and motor responses to a sudden temperature change in Asellus aquaticus Kari Y.H. Lagerspetz* Laboratory of Animal Physiology, Department of Biology, University of Turku, FIN-20014 Turku, Finland Received 25 September 2002; accepted 7 March 2003
Abstract 1. In the fresh water isopod Asellus aquaticus the critical thermal maximum (CTMax) after 2 weeks was similar in animals transferred directly from 9 C to 22 C and in animals acclimated gradually (1 C/day). 2. A sudden change of ambient temperature caused immediate changes in the locomotor behaviour of Asellus that did not persist, and especially an increase of the frequency and angle of turning (klinokinesis). 3. Asellus, acclimated to 12 C, preferred temperatures of 11–15 C in a thermal gradient, and showed klinokinetic heat avoidance reactions at effective heat shock temperature (28 C). r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Asellus aquaticus; Crustacea; Heat shock; Thermal acclimation; Avoidance; Preference; Motor activity; Klinokinesis
1. Introduction Thermal acclimation of poikilotherms can be observed in laboratory experiments but in nature acclimation is merely a part of seasonal acclimatization, and so occurs in response to slowly changing environmental temperature. Thus, thermal acclimation may occur in nature as a response to a prolonged change in the weather or in the microclimatic conditions. The changes concerned will often be relatively slow, but the organism’s responses may be made complex because they would be capable of behavioural thermoregulation, i.e. to move to a favourable temperature, and so to avoid or offset the progressive changes in ambient temperature. However, rapid changes in environmental temperature may occur in nature, e.g., when an animal crosses a thermocline in a lake, or moves from shade to sunshine, or when exposed to air on the ebb of the tide.
*Tel.: +358-2-2328566; fax: +358-2-3336590. E-mail address: kari.lagerspetz@utu.fi (K.Y.H. Lagerspetz).
In most existing experimental studies on thermal acclimation of ectotherms, animals have been transferred directly from one temperature to the other or allowed only a few hours for the temperature change to occur. The temperature difference used has been typically 5–20 C in magnitude. The effects of a heat shock on heat tolerance were generally recognized first in the 1980s (Lindquist, 1986). A shift of only 5 C (from 20 C to 25 C for 1 h) was sufficient to increase heat shock protein (HSP) production in intertidal limpets (Sanders et al., 1991). Heat shock effects occur when animals are exposed to temperatures close to the critical thermal maximum (CTMax) of the species (Tomanek and Somero, 1999). In some habitats a rapid change of environmental temperature may cause a heat shock to the animals so that occasional heat shock events would not be expected to alter the longer term effects of acclimatization, but its effect on heat tolerance would be short-term (hours). The animals are left long enough (from 5 days to several weeks) at the new temperature before the assessment of acclimation status, so that the immediate effects of a possible heat shock must be over (Sanders, 1993).
0306-4565/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4565(03)00027-5
ARTICLE IN PRESS 422
K.Y.H. Lagerspetz / Journal of Thermal Biology 28 (2003) 421–427
However, the question remains whether an initial heat shock has some effects necessary for the onset of thermal acclimation processes under laboratory conditions. As the conditions for thermal acclimation and heat shock and the additivity of their effects on thermal tolerance are well known in the freshwater isopod Asellus aquaticus (Lagerspetz and Bowler, 1993; Korhonen and Lagerspetz, 1996), the effects of a slow gradual change of environmental temperature were compared with the effects of an abrupt temperature change on CTMax and on the survival of Asellus. In addition, the initial effects of a temperature change on motor activity in Asellus were studied by a sudden transfer of the animals to a new temperature. For this, a method for the measurement of locomotor activity was developed. The thermal preference of animals was also studied using a thermal gradient.
2. Materials and methods 2.1. Animals and acclimation Adults of A. aquaticus (6–12 mm long) were collected from a shallow pond near Turku. They were kept in laboratory in charcoal-filtered tap water at 9–10 C or 12–13 C with decaying alder leaves for food. For thermal acclimation, the animals were kept either in groups of 5–40 in containers with 2–5 l of water, or for CTMax experiments individually in clear plastic vials (diameter 4.5 cm) with 40 cm3 of water at the acclimation temperature (Lagerspetz and Bowler, 1993). In these vials a thin layer of fine sand and pieces of decaying alder leaves were present for attachment and food. A total of 5 cm3 of water was changed daily. 2.2. CTMax determination Individuals from the group of animals were subjected to an increase of environmental temperature from 9 C to 22 C (1 C/day), which was slow enough as not to induce a heat shock responds. Individuals from another group were transferred abruptly from 9 C to vials at 22 C for the same period of time (13–14 days). The control animals remained at 9 C in individual containers. CTMax was determined by subjecting each animal in a plastic vial with 40 cm3 of water, with a little sand but no food to a temperature rise of 12 C/h in a Lauda RC 20-RCS thermocontrolled bath, until they could not right themselves within 60 s after being turned on their backs using a small paint brush (Lagerspetz and Bowler, 1993). Temperature close to the animal was measured by a 0.5 mm thermocouple and a calibrated Ellab thermogalvanometer with an accuracy of 0.1 C. This was done at intervals of 5 min and more often close to the CTMax.
2.3. Measurement of motor activity For the measurement of motor activity one animal at time was transferred to a clear plastic Petri dish of 14 cm in diameter with water at the desired temperature, which was monitored with a thermocouple. The water depth of 6–9 mm allowed for some vertical swimming, but this did not occur in our experiments. The animals walked often along the perimeter of the dish keeping one of the antennae in contact with the wall. The illumination of the dishes was measured by a quantum counter (Li-Cor, model Li-189) and adjusted to be in the same range in all experiments. Pilot experiments showed that a change from 0.12 to 3.5 mmol of quanta s1 m2 did not affect the behaviour of the animals. A paper marked with eight equal sectors and a circle half of the diameter of the dish was placed beneath the dish. This allowed the tracing of the movements of animals on similar charts (Fig. 1). Using a stopwatch, each minute was marked on the chart, as were all points where animals stopped moving. These charts were later measured with a map-measurer. The speed of the animals could be analysed for each minute, and the number of times they stopped as well as the number and angle of turnings they made could be estimated. These data were obtained from 6 min sample recordings of motor activity made at different intervals. Another method for retrieving data from the track charts for 6 min periods was to measure linear movement and turning angles separately for each sector of the chart and to add them up. In this study, two measures of locomotor activity were used: (1) linear velocity (LV, cm/min) which was calculated from the length of the track, and (2) the rate of change of direction (angular velocity (AV) of the movement, /min). In calculating this, the changes of direction caused by animals following the perimeter of the dish were not taken into account. After the animal had been accustomed to the experimental conditions for half an hour, it could be transferred to another similar dish with water at the same temperature without a change in its LV and with a relatively small change in AV (Table 2). In experiments on the effects of temperature change, one animal at time was transferred in 1 min to a similar dish with water of a different temperature. Its subsequent motor activity was measured immediately and later for periods of 6 min. After the experiment, the animal was transferred to 18 C, where all animals recovered. 2.4. Thermal preference and avoidance A gradient apparatus similar to that of Hodgson (1951) was used, but with two sources of flowing water
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of different temperatures. A thermal gradient from 9 C to 33 C was established in the glass tube. The water phase in the tube was 33 cm long and 1.5 cm deep. Temperatures in the different parts of the tube were measured by thermocouples inserted through holes on the upper surface of the tube. The position of the animals was recorded at 5 min intervals. The direction of the gradient was changed between the experiments. The total water flow through the gradient tube was 50 cm3/min, at which rate the flow did not disturb the animals. Six experiments of 1 h with ten 12 C-acclimated animals in each gave 720 position records. Four experiments with each of four animals were done in order to decide, if the heat avoidance responses of Asellus depended on klinokinetic or klinotactic orientation mechanism (see Section 4). A 13 C-acclimated animal was held for 30 min in the conditions used in measuring motor activity, before a plastic bag containing 10 cm3 water of 30 C was introduced and punctured, allowing the warmer water to reach the animal. The subsequent movements of the animal were recorded.
3. Results 3.1. CTMax after acclimation with or without heat shock Animals subjected to a slow rise (1 C/day, from 9 C to 22 C) in their environmental temperature had a CTMax of 34.070.2 C (n ¼ 12), which was similar to CTMax in animals directly transferred from 9 C to 22 C, after 2 weeks of individual acclimation to the latter temperature (34.570.2 C; n ¼ 12). The mean values of both groups were similar to those reported earlier for abruptly transferred Asellus, but differed significantly (t-tests, Po0:001) from the controls kept in individual vials for the same time at 9 C (CTMax 30.470.4 C; n ¼ 16; Lagerspetz and Bowler, 1993). 3.2. Motor activity
Fig. 1. Three examples of records of 6 min tracks of Asellus in the Petri dish arena (diameter 14 cm). Arrowheads mark stops, transverse bars minutes 1–6. (A) 13 C, 30–36 min after the start of the experiment (Exp. A10:2, LV 30 cm/min, AV 20 /min). (B) The same animal 0–6 min after transfer to 25 C (Exp. A10:3, LV 27 cm/min, AV 173 /min). (C) Another animal at 5 C, 6–12 min after transfer from 13 C (Exp. A18:4, LV 1 cm/min, AV 30 /min).
3.2.1. Control experiments The transfer of an animal from the acclimation aquarium to the Petri dish used for the measurement of motor activity caused at first a high explorative activity of the animals. Therefore, each experiment was preceded by 36 min to allow animals to adapt to the new measurement conditions. This adaptation to the new measurement conditions was seen as a decrease of the initially frequent and large turnings by the animals (Table 1), which occurred in about 6 min in the experimental dish (data not shown). The motor activity of individual animals measured at 0–6 min and at 30–36 min in the experiment conditions (Table 1) did not show statistically significant
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Table 1 Adaptation of individual Asellus aquaticus to the conditions for measurement of motor activity at 13 C (n ¼ 21)
Table 3 Effect of a sudden change of temperature on the linear velocity (LV, cm/min) in Asellus (n ¼ 4; for 24 C n ¼ 5)
Time (min)
LV
AV
LV at 13 C
24719
3673b
2875
2479
0–6 30–36
35.072.6 30.574.7
191.4726.9a 46.9712.2a
New temp.
5 C
18 C
24 C
28 C
0–6 min 6–12 min 30–36 min
874 775 14713
4871b 4678 46710
31710 4578 2377
2778 3574 23711n
Note: LV—linear velocity (cm/min), AV—angular velocity ( /min). Mean7S.E.M. Statistically significant differences (t-test) between data in each column are marked by superscripts: a Po0:001; bPo0:01; cPo0:02; dPo0:05:
Table 2 Immediate effects on motor activity of the transfer of Asellus aquaticus from one recording arena to another at the same temperature (13 C)
Before Transfer After
Note: Other explanations as in Table 1. n At 60–66 min.
Table 4 Effect of a sudden change of temperature on the angular velocity (AV, /min) in Asellus (n ¼ 4; for 24 C n ¼ 5) AV at 13 C
19711c,c
38714b
29713d
109747c
LV
AV
New temp.
5 C
18 C
24 C
28 C
3672.8
34719.9d
3874.4
74727.5d
0–6 min 6–12 min 30–36 min
58720c 105734c 38728
206740b 74774 39728
274785d 162771 146762
355774c 1877135 86731n
Note: Data from the last 6 min before and the first 6 min after the transfer (n ¼ 5). Other explanations as in Table 1.
Note: Other explanations as in Table 1. n At 60–66 min.
correlations: for LV measurements r ¼ 0:39; for AV measurements r ¼ 0:13; respectively. Therefore nonpaired t-tests were used throughout this study. As an abrupt change of temperature in this study involved also the transfer of the animal to another similar experiment arena, the changes of motor activity must be compared to the changes observed when an animal is simply transferred from one dish to another at the same water temperature. In these control experiments (Table 2) only a slight transient increase in AV was observed. The change of LV was not significant.
keep an upright position was disturbed by muscle spasms for about 4 (1–6) min. During this period the animals moved by lateral crawling with frequent apparently random turnings. The AV of motor activity was increased. After a transfer of animals from 13 C to 28 C, AV increased for at least 12 min (Table 4). Some motor disturbances occurred at least for half an hour, even for the whole recording period. The duration of the time at 28 C was extended to 60–66 min, as such heat shocks had been previously found to be effective in Asellus (Korhonen and Lagerspetz, 1996).
3.2.2. Motor activity after a sudden change of temperature When Asellus were transferred from 13 C to 5 C, their LV decreased and stayed at a low level (Table 3), while AV showed a transient increase for at least 12 min (Table 4). The animals assumed a special stance: legs were stretched and telson and caudal abdomen lifted up, so they stood and walked in a stilted fashion. Earlier, Kivivuori (1980) found the same phenomenon in the crayfish Astacus astacus at low temperatures. When the animals were transferred from 13 C to 18 C (Tables 3 and 4), there was a permanent increase in LV and a transient increase (for about 6 min) in AV. When the temperature difference was larger, from 13 C to 24 C (Tables 3 and 4), the ability of animals to
3.2.3. Thermal preference and avoidance The distribution of 12 C-acclimated Asellus in the thermal gradient used is shown in Fig. 2. The animals preferred temperatures of 11–15 C. There is a secondary maximum of position records at 26–31 C. This may depend on heat avoidance reactions which were clear at 28 C. As the low temperature end of the gradient was as high as 9 C, cold avoidance reactions could not be observed. Heat avoidance responses were studied in separate experiments. The animals did not make 180 turns away from water of 30 C, but several smaller random turnings and short stops.
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survived for 52 days at 13 C in laboratory. As these animals (later used in behaviour experiments) were kept at their original temperature, a selection by temperature could not occur among them. The animals fed on the alder leaves, and when housed in groups even reproduced. Some cannibalism probably occurred, as pointed out by Sprague (1963). An addition of animal food would perhaps be necessary to avoid this in laboratory populations of Asellus. 4.3. Motor activity and thermal behaviour
Fig. 2. Thermal preference of Asellus acclimated to 12 C. The distribution of animals in a thermal gradient extending from 9 C to 33 C. Based on position records (PR) taken at 5 min intervals in six 1 h experiments, each with 10 animals. Redrawn from Lehtonen (1960).
4. Discussion 4.1. Thermal acclimation without a heat shock The first experiment confirms that a heat shock is not a necessary prerequisite for thermal acclimation. This result agrees with the earlier studies on fish cell cultures (Merz and Laudien, 1987) and on cellular functions in isolated mussel gills (Lagerspetz et al., 1995) and on CTMax in A. aquaticus (Korhonen and Lagerspetz, 1996), in which the effects of heat shock and thermal acclimation have been shown to be separate because of their different time courses. 4.2. Survival The survival of Asellus in laboratory, especially in acclimation conditions is of interest, because a selection among the original sample of animals taken from a natural population may occur. Sprague (1963) found an average mortality of 28% for Asellus intermedius during 4 weeks of acclimation in groups at 20 C and 30 C. The mortality was higher at more crowded laboratory populations. In the experiments of Lagerspetz and Bowler (1993) with separately housed individuals of A. aquaticus, a loss of about 50% of animals was found during the acclimation period of 15–23 days. However, these animals were subjected at times to an assessment of their CTMax which involved a heat shock. When in this study the animals were acclimated separately, and subjected to either a sudden or gradual transfer from one temperature to another, a loss of 10–20% was observed. Of a group of 40 animals, 20
The possible changes of motor activity caused by a sudden transfer of animals to another temperature have seldom been studied in any invertebrate (Kerkut and Taylor, 1958; Perttunen and Paloheimo, 1964; Marsden, 1976). Also the initial changes in metabolism after temperature change have received little attention (Grainger, 1956, 1958). This is astonishing because of the growing number of heat shock and thermal acclimation studies. The work done by Marsden (1976) on another isopod, Sphaeroma rugicauda, is especially relevant. This work recorded the percentage of animals walking, swimming, inactive, or rolled (total inactivity) at 1 min intervals during the first 10 min and at 30–40 min after the transfer of 10 animals in each group from 10 C to temperatures ranging between 2.5 C and 35 C. At temperatures below 4 C and above 30 C most isopods were inactive, but between 14.5 C and 30 C all isopods showed either walking or swimming activity. The response of isopods following 30 min exposure to a new thermal regime was similar to that found immediately after the transfer. When the temperature was increased at a rate of 1 C/min from 2 C to 34 C, and then decreased at a similar rate, the percentage of isopods displaying the various behaviour patterns was generally similar to the results obtained at the same temperature on direct transfer (Marsden, 1976). In that work only walking and swimming were discerned, and the changes of direction were not recorded. These results on Sphaeroma are comparable to those given for Asellus in Table 3 in the present study. A stay for 30 min at the new temperature did not alter the motor activity (LV) from that recorded immediately after the transfer. Mechanical registration and photocell response counting are not useful for smaller animals, and these methods do not differentiate between different types of motor activity. Videotaping with subsequent observation (or suitably programmed imaging) would be as good as the simple chart tracing method used here. It can distinguish between linear locomotory movement, stops, and the frequency and angle of turning. The two first are in this study summated as LV, the two latter as AV.
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The idea of tracing the movements of animals on charts comes from the original work of Fraenkel and Gunn (1961) and their associates, done in 1930s. The use of the rate of change of direction (or AV without regard of the direction of the angle) as a measure of klinokinesis is due to Ullyott (1936) and Amos (1965). The method used here employs a circular arena, in which the animal is moving. For swimming animals, like Daphnia, long tubes have been used, relying on their positive phototaxis (Kobayashi and Gonoi, 1985), and for Daphnia, also counting of the number of strokes of the antennae using a microscope (McKenzie et al., 1992). These methods do not easily allow the measurements of turning, which crawling animals may do frequently. ‘‘Linear movement’’ in the present case was often movement around the circular periphery of the arena. According to Fraenkel and Gunn (1961), ‘‘The term taxis is to-day used for directed orientation reactions. yWe use the word only for reactions in which the movement is straight towards or away from the source of stimulation. yUndirected locomotory reactions, in which the speed of movement or the frequency of turning depend on the intensity of stimulation, we call kineses’’. These commonly adopted definitions have been followed here. The present results show that a sudden transfer of Asellus to another temperature changes its motor behaviour, usually immediately increasing its turning frequency and angles. At temperatures more distant from the holding temperature indicators of increased muscle tonus (the stance of the animals, muscle spasms) appear. The transfer of Asellus to 28 C for 1 h causes a heat shock (Korhonen and Lagerspetz, 1996), but also disturbs at least temporarily its motor behaviour, and so probably the function of its nervous system. This effect is rapid, as shown also by the avoidance responses at 28 C in the thermal gradient. The observed heat avoidance of Asellus seems to be based on klinokinetic behaviour as in Daphnia (Lagerspetz, 2000), and not on klinotactic, stimulus oriented responses. The interindividual variation in the motor activity of Asellus is large. It seems not depend on the size or sex of the individuals. The feeding and molting cycles which were not controlled in the present study are probably important sources of variation. In conclusion, A. aquaticus uses three defence mechanisms against high temperatures: (1) behavioural avoidance reactions which occur by increase of turnings (klinokinesis), (2) induced or increased heat shock protein production, which is associated with an increase of thermal tolerance, and (3) thermal acclimation which is slower but longer lasting and probably depends on CNS and/or endocrine effects. These behavioural, molecular and systemic mechanisms are activated and functioning independently of each other.
Acknowledgements I thank Dr. Liisa A. Vainio for help in collecting the animals and for many discussions, Risto Heikkinen (M.Sc.) and Tapio Haavisto (M.Sc.) for help in statistical treatment of the data, and Aila Martikkala (M.Sc.) (b. Lehtonen) for allowing to use parts of her original M.Sc. thesis (Lehtonen, 1960) material.
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