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Populational analysis of thermal responses—I. Changes in the heat resistance of muscle tissue and contractile muscle models of Salamandra salamandra larvae
J. therm. Biol. Vol. I1, No. 3, pp. 167-173, 1986
0306-4565/86 $3.00 + 0.00 Pergamon Journals Ltd
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POPULATIONAL ANALYSIS OF THERMAL RESPONSES--I. CHANGES IN THE HEAT RESISTANCE OF MUSCLE TISSUE A N D CONTRACTILE MUSCLE MODELS OF S A L A M A N D R A S A L A M A N D R A LARVAE B. P. USHAKOWtV~and I. M. PASHKOVA Laboratory of Comparative Cytology, Institute of Cytology, Academy of Sciences of the U.S.S.R., Leningrad 194064, U.S.S.R. (Received 25 October 1985; accepted in revised form 10 February 1986) Abstract--l. Study has been made of the heat resistance of m. rectus superficialis and their contractile
models of larvae of 12 families of Salamandra salamandra kept at three different temperatures: 14, 21 (optimal) and 27°C. The heat resistance of the organism has been studied only in larvae kept at 21°C. 2. An inverse linear relation has been found between the level of the average heat resistance of muscles for offspring, of the same family, under optimal conditions and its increase caused by a change in environmental temperature. A similar relationship was observed in contractile muscle models. This implies that a population responds to a change in environmental temperature as a functional system. 3. The analysis of individual differences in the pattern of response of muscles, and their contractile models, to changes in environmental temperature allows the systemal and individual components to be isolated. The extent of co-ordination in responses of different individuals of the population can be evaluated from the proportion of the systemal component. The value of this component varies from 0.60 to 0.90. 4. Selective advantage during thermal selection belongs to individuals with lower heat resistance of muscles. Key Word Index--Salamandra salamandra; larvae; sib analysis; heat resistance; muscle tissue; contractile muscle models; selective value of the resistance of muscles and contractile muscle models during thermal selection; functional structure of the population.
INTRODUCTION
It remains a paradox that being an experimental science, physiology when elaborating the problems of evolution, is still confined to the study of formation of functions of the organism in phylogenesis. Little attempt is made to elucidate the mechanisms of evolutionary process. The study of individual responses of genetically heterogenous individuals from natural populations, to changes in ecological factors, makes it possible for physiologists to analyse the mechanisms of evolutionary processes and particularly the process of speciation. Synthetic theory of evolution is based on the study of selection of genetic markers whose phenotypical expression is practically independent of environmental changes. Such an approach significantly facilitates the study of the result of selection and makes it unnecessary to analyse the process of selection itself. However, with the use of such genetic markers for the analysis of microevolution, an important point is overlooked: changes in selected characteristics occurring during selection can affect its result. Therefore, it is necessary to consider labile physiological characteristics, whose phenotypical expression is affected by environment and whose adaptive value is doubtless. It is evident that without the participation of physiologists the analysis of the kind can hardly be effective. More advanced in this field of investigation are the physiologists engaged in the studies of resistanceadaptations (see: classification of adaptations accord167
ing to: Precht, 1958). They study the effect of ecological factors on survival of individuals and the criterion of resistance used is the dose of injurious effect killing 50% of individuals. Experiments of this kind are aimed at the study of the limiting effect of ecological factors and the resulting selection. Traditionally the results of such experiments are erroneously interpreted as if they were made on a single individual, "typical" of the population studied, and not on a group of genetically heterogenous individuals. During the study of populational responses the attention is shifted from the resistance level, average for a group of specimens, and focused on the specificity of individual responses, i.e. the variability of this characteristic and its reaction norm in a group of individuals of the same population. This method of investigation is termed here as populational analysis. This study is concerned with the populational analysis of thermal responses of individuals in relation to the problem of microevolutian. The first communication considers the effect of temperature on the heat resistance of muscle tissue and contractile muscle models of salamanders and the selective value of this characteristic. MATERIALS AND METHODS
A study of individual variability of physiological characteristics is not quite a common task for a
168
B.P. USHAKOVand I. M. PASHKOVA
physiologist, therefore it seems worthwhile to consider in more detail the quantitative evaluation of this characteristic and the methods for the analysis of its components. The criterion of the heat resistance used in our study was time to the onset of heat shock or loss of some of cell functions during continual exposure to injurious temperature. However, this criterion has two disadvantages: first, its statistical distribution differs from normal, which complicates the statistical processing of the data obtained: and second, standard deviation of this criterion depends on the temperature used in the experiment, Both disadvantages are avoided if logarithmic values of the heat resistance are used instead of their absolute values. In this case the distribution becomes normal and independent of the temperature at which the heat resistance is measured (Ushakov et al., 1968). Therefore, logarithms of inactivation times are used as quantitative characteristics of the heat resistance of the organism and its cells; and their standard deviations show the individual variability of the heat resistance. When studying individual variability a question arises to what extent the variability of the heat resistance levels are actually due to differences between the individuals, and to what extent it is due to the error of methods. On the leech Hirudo medicinal)s, which is a convenient object for obtaining a large number of muscle preparations, it has been shown that the share of individual variability in the total phenotypical variability constitutes 87%, and only 13% is due to the error of methods. The share of the latter is higher during the determination of the heat resistance levels of glycerinated frog muscles, but even then it is two times lower than individual variability (31 and 69% respectively). These, and similar data, (Ushakov et al.. 1968) imply that the methods used in our experiments are valid for the study of the individual variability of the heat resistance. When elaborating the problems of evolution it is not sufficient to study individual variability alone, it is also necessary to determine the share of genetic component in the total phenotypic variability. It can be done on the offsprings of a number of families* reared separately under identical and constant conditions. In this case the environmental component of variability is minimal and the share of genetic variability (between the families) in the total phenotypic variability of a physiological characteristic in a population can be more clearly seen (Lush, 1949). In this case the genetic share of variability determines the upper limit of heritability of a characteristic. It also involves the variability caused by dominance. It should be pointed out that the extent of the variation caused by environmental changes is the significant point. The study concerns the same physiological characteristic in offsprings of the same families (i.e. the individuals with practically identical gene pool) under conditions of variable environment. Thus, changes in the share of genetic variability of a characteristic are due to an increase in the environ-
*In this paper the term "family" is used to denote a group of individuals arising from a single female.
mental component of the Iotal variability (Ushakox et al., 1977a). The determination of heat resistance of an or.ganism usually results m the death or injury of experimental animals, therefore it is impossible to follow in one individual the dynamics of the changcs in this character. For this reason, in out studies we used agamic clones of animals, or siblings, progeny tH the same parents (the method of sib analysis). In this study the method of sib analysis is used for the examination of changes in the heat resistance of abdominal muscles of salamander larvac lind their contractile models caused by their maintenance at high and low temperature. Moreover. the selective value of the characteristic of the heal resistance of muscles and their models is considered. Pregnant females o[" S a l a m a m b a ~aiamandra I. were captured in October in the outskirts of the town of Uzhgorod (the Carpathians). Each of thc 12 females was opened, the larvae removed and divided into three parts, one of which was grown at 14 C. and the two others at 21 and 27 C They were kept in glass vials (2 1), 6 9 individuals in each, lit 10 h of daylight, fed daily with mosquito larvae and sludge worm. The salamander larvae used in the experiments were of the same size (40 45 mm from nose oritices to the tip of the tail). This size is characteristic of larvae in the middle of premctamorphosis (Polushina. 1966; Pyastolova and lvanova, 1974). The survival of larvae in the laboratory was 100%, which means that there was no selection during development, and hence, the results obtained were only due to changes in the functional state of animals The heat resistance of lhe organism ~as dolormined in water at 34 + 0.1 C. The criterion cH the heat resistance used was the time to the ~mset of heat shock, which was judged by the loss of coordination. Records were made of the moment when the larwte turned on their backs (and remained so for more than 1 2min). In contrast to adult animals, m larvae, when such a loss of coordination is observed it results in death. Since in salamanders the number of offsprings per family is rather small, the organismal resistance was determined only in one series of experiments (at 21' C). The time to the onset of heat shock was recorded in 6--9 individuals and the average value calculated, which served as a characteristic of the heat resistance of the organism~ average for a family. Preparations of muscle tissue were made of M. rectus superficialis which was cut in two along the medial line. One half was used for the determination of the heat resistance of muscle tissue, the other for that of actomyosin complex of contractile muscle models. Immediately after preparation the muscles were put into Ringer's solution at 36 + 0.1 C . The criterion of the heat resistance of muscles used was the time to loss of excitability in response to electrical stimulus from a portable stimulator (Arzumanov and Kusakina, 1960). To record the moment of loss of excitability the muscles were periodically taken out of heated Ringer's solution for 5- 7 s and subjected to electrical stimulation (frequency 50 Hz, duration 1 s or less). The total number of such testings usually was 10 12. However, it was considered that such frequent testing might affect the rate of development of heat injury
Populational analysis of thermal responses--I and thereby our assessment of the heat resistance of muscles. Such an effect is more pronounced during the first half of the heat exposure. To avoid this, prior to experiment, a preliminary determination of the time to loss of excitability was made on 2-3 muscles by means of repeated testings. Such preliminary data allowed the testing of the muscle excitability to be confined to the latter half of heat exposure and so to reduce the total number of testings to 3~5. Contractile muscle models were prepared by putting stretched intact muscles into solution containing 50% glycerol, 0.12M KCI, 0.01 M Trismaleic buffer (pH 7.0). Glycerinization was carried out at - 5 to 7°C for 3 to 5 days. Glycerol was washed out in a solution of 0.12 M KCI, 0.005 M MgC12 and 0.01 M of the same buffer. The criterion of the heat resistance of contractile models used was the time from their immersion into washing solution at 36 _ 0.1 °C to the loss of contractility on addition of 0.005 M ATP, pH 7.0. Contractile muscle models are only capable of one-time contraction in response to ATP. Therefore, prior to the determination of the heat resistance the muscles washed out of glycerol are cut into small pieces and only then put into heated testing solution. From time to time these pieces of tissue are taken out with a pipette and put on a glass on which ATP-containing solution is added. The contractile muscle models, not yet injured by heat, contract immediately on addition of ATP by more than 1/3 of their initial length. The moment of loss of such contraction is recorded during the determination of the heat resistance of contractile muscle models. The contraction was observed under the microscope ( x 16). The genetic share in the total phenotypic variability in the heat resistance was calculated using one-factor analysis of variance (Snedecor, 1957). The differences between the average values were considered statistically significant at P ~<0.05. The analysis of the variability of the heat resistance of the organism, muscles and contractile muscle models at the populational level was our major concern. The experimental material obtained allows the share of variability within a family in the total phenotypical variability of these characteristics in a population to be determined. This share appeared to be quite similar: 39, 32 and 37% for the organism, muscles and contractile muscle models respectively. The variations in the heat resistance within a family are due to three factors: first, genetic differences between offspring; second, differences caused by nonuniformity of environment (the effect of this factor is minimal); and third, the error of methods. These data allow an estimation of the error of the methods used,
169
which according to our data does not exceed 15-20% of the total variation of these characteristics in a population. This conclusion is supported by relatively high coefficients of correlation between the heat resistance level of the organism, on one hand, and those of muscles and their contractile models, on the other, observed under optimal environmental conditions. RESULTS
As is known from literature, salamander larvae develop at 21°C in nature (Andreev, 1956; Polushina, 1966; Gegani and Mendelssohn, 1980), therefore this temperature was considered to be optimal for their development. Changes in the heat resistance of muscle tissue In larvae kept at high temperature (27°C) the average heat resistance of muscles was 55% higher than in those kept at optimal temperature (21°C) (from 141 min at 21°C to 218min at 27°C). This increase in tissue heat resistance practically did not have any effect either on the total phenotypical variability of this characteristic or its genetic component (Table 1). In larvae kept at low temperature (14°C) a 58% decrease in the heat resistance of muscles was recorded (from 141 min at 21°C to 59min at 140C). This decrease in resistance was accompanied by a statistically significant 20% decrease in standard deviation of the characteristic studied, i.e. a narrowing in its phenotypical variability (from 0.184 to 0.148). Simultaneously, a double decrease in the genetic share of variability was recorded. This implies that the effect of cold results in phenotypical masking of genetic differences in the heat resistance of muscle tissue of different individuals in a population. As has been shown earlier, an increase in the heat resistance of muscle tissue of each single individual during heat acclimation depends on its initial (preacclimational) level. It was of interest to compare the responses to heat of muscle resistance level, average for a family, of "warm" and "cold" individuals and their siblings developed at optimal temperature (Fig. 1). As can be seen in the figure, the increase in muscle resistance to heat in "warm" larvae is the highest in the families with initially less resistant muscle tissue. Families with initially more resistant muscles practically do not show any changes in the resistance after heat effect. Thus, an increase in the heat resistance of muscles observed after heat effect characteristic of the population as a whole, occurs at the expense of individuals with initially less resistant muscle tissue.
Table 1. Populational characteristics of the heat resistance level of muscles and their contractile models of salamander larvae at different temperatures of maintenance
Object of investigation Muscles Contractile muscle models
Temperature of maintenance CC)
Number of observations (n)
14 21 27 14 21 27
72 67 66 67 67 64
Average time of loss of functional activity log10 min
min
P
1.77 _ 0.01 2.15 + 0.01 2.34+0.01 1.13+-0.01 1.35 + 0.02 1.50 _+0.01
59 141 218 13 22 32
<0.05 <0.05 <0.05 < 0.05
Standard deviation (SD) 0.148 + 0 . 0 1 0.184 __.0 . 0 1 0.178+_0.01 0.103+-0.01 0.157 + 0.02 0.126 +- 0.01
P <0.05 <0.05 <0.05 < 0.05
Heritability coetficient and its SEM (h: + SEM) 0.37 + 0.11 0.68 + 0.04 0.56_+0.08 0.58+-0.05 0.63 _+0.05 0.26 +- 0.09
<0.05 >0.05 >0.05 < 0.05
170
B.P. USHAKOVand I. M. PASHKOVA
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-0.5 -0.6 2.0
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2.2
2.3
2.4
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Fig. 1. The relation of increase in the heat resistance of muscles of salamander larvae caused by heat (Q) and cold (O) to its level at optimal temperature (21°C). Abscissa: heat resistance, average for a family, at 21°C (log rain). Ordinate: the difference between the logarithms of the resistance values in experiment and in control (21°C). In "cold" larvae the relation is just the opposite: the decrease in muscle resistance is more pronounced in siblings with initially more resistant muscle tissue. In those with initially less resistant muscles this decrease was less significant. Thus, in "cold" larvae a decrease in the average populational level of muscle 'heat resistance occurs at the expense of individuals with initially more resistant muscles. As can be seen from Fig. 1, changes in the heat resistance of muscles of salamander larvae caused by thermal changes follow a linear equation
heat resistance of contractile muscle models was accompanied by a narrowing in the individual variability of this characteristic and a decrease in its genetic share. Thus, an increase in the heat resistance of contractile models after heat effect resulted in a phenotypic masking of genotypic differences in this characteristic, as was the case in "cold" larvae. The effect of cold resulted in a 40% decrease in the heat resistance of contractile muscle models. In this case too, a significant decrease in individual variability of this characteristic accompanied by a reduction in its genetic component was observed (Table I). As can be seen in Fig. 2, an increase in the heat resistance of contractile muscle models caused by heat occurs mainly at the expense of families with low resistance level of muscle models at optimal temperature. A decrease in this characteristic caused by cold occurs mainly at the expense of families with high resistance level of contractile muscle models at optimal temperature. In both cases some of the families did not show any statistically significant changes in the heat resistance of contractile muscle models. The range of change in the heat resistance of muscle models caused by both heat and cold depends on the level of their resistance under optimal conditions. As in the experiments with muscles, a linear relation is observed between an increase in resistance of muscle models and its control level. In the equation showing the effect of heat: a = -0.51 + 0.02: b = 0.83 + 0.02; and in that for the effect of cold: a = - 1.00 + 0.06; b = 1.11 _+ 0.09. The equations for calculating a change in the heat resistance read as follows: At27 = 0.83 - 0.51 -t:~
A/j4 = 1.11 - tzl.
A t = a " t2~ + b
where t21 is time to loss of muscle contraction in response to electrical stimulus at 21°C, average for a family (log min); A t ~ i f f e r e n c e between these times at low or high temperature (14 or 27°C) and at optimal one (21°C) (log min); a and b----constants. In the equation showing the effect of heat a=-0.99_0.06; b=2.23_+0.16, and in that showing the effect of cold a = - 1.04 + 0.07 and b = 1.85-I-0.16. Since constants a in both cases are statistically indistinguishable from - 1 , the above equation can be simplified At27 = 2.23 - t21 for heat effect Ate4 = 1.85 - t2~ for cold effect. These equations allow an approximate determination of changes in the heat resistance of muscles caused by changes in environmental temperature. For that it is only necessary to know the level of the heat resistance of muscles, average for a family, under optimal conditions. C h a n g e s in the heat resistance o f contractile m u s c l e models
Changes in the heat resistance of contractile muscle models during temperature changes are very similar to those just described for muscles. After heat effect the average time to loss of contraction increased by 41% (Table 1). An increase in the average level of the
Summing up the data obtained both on muscles and contractile muscle models it should be pointed out that an increase in heat resistance caused by heat and its decrease caused by cold occur at the expense of different individuals of the population. The relation of individual increases in the resistance of muscles and their contractile models to their control levels follows a linear equation. A
0,4
~-
0.2
=
]
•
~ -~~
"-...o °
o.o
--01
.~_ - 0 2 ~-0.3 --0.4 1.1
1,3
H e a t resistance
1.5
1.7
at 2 1 * C (loq rain)
Fig. 2. The relation of increase in the heat resistance of contractile muscle models of salamander larvae caused by heat (Q) and cold (©) to its level at optimal temperature (21°C). Designations as in Fig. I.
Populational analysis of thermal responses--I
171
DISCUSSION It has already been demonstrated on the muscle tissue of Asellus aquaticus that a linear relation is observed between the initial level of cellular heat resistance and its increase due to heat acclimation (Ushakov and Pashkova, 1984). The present study supports this conclusion extending it to the effect of cold. Differences in the responses of muscle resistance level to thermal changes are due, on one hand, to the linear relation, common to all specimens of the population, and, on the other, to the specificity of the response of each single individual. Hence, total variance (a2op) characterizing variability of responses in the population can be resolved into two components, first, variance due to this linear relationship (a~.3, 2 and second, variance characterizing differences in individual responses of single specimens (Cri2nd):
o o=
..q
(7 2pop ~ (7 2syst .~ O. 2rid.
Total variance of the responses (trOop) can be calculated using differences between the logarithmic values of time to loss of muscle contraction before and after thermal effect for all the families, or individuals, of the experimental population. The individual component of variability (O'~nd) c a n be quantitatively characterized by variance of differences between the logarithms of actual time to loss of muscle contraction and its theoretically expected value calculated from the linear equation. Having determined these two variances from the above equation, it is possible to calculate the variance which corresponds to systemal component of variability (%~0. 2 When the individual component is low, the points representing experimental data are closer to the straight line showing the theoretically expected values of an increase in resistance calculated from the hyperbolic equation (Figs 1 and 2). If there were no individual component, these points would have fallen precisely on the straight line. If there were no systemal component, these points would have formed a chaotic accumulation, which would mean a complete lack of co-ordination in the responses of single specimens in the population. If both parts of the above equation are divided by total variance (a:pop), the equation reads as follows O.~nd 2 0"syst ~-- t~O.2nd 2 __ : ~- ~ + 60"syst - - 1. O'pop O'po p
This equation allows quantitative evaluation of the shares of systemal (ra~y,t) 2 and individual (&r~,d) components in the total variance of responses of different families of the population. Of more interest here is the share of systemal component, since it allows the evaluation of the extent of co-ordination in the responses of different families (individuals) of the population to environmental changes. It is equal to the squared correlation coefficient between the heat resistance in control and its increase caused by thermal changes (Urbach, 1963)
O'~nd
cSO'~y~t= 1 -- _-ST-=
r2.
O"pop
In this study the share of systemal component is rather high, ranging from 0.60 to 0.90 (Table 2). In our previous study on heat acclimation of Asellus
-s
e~oo
O
~o ~E ~o=
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~ o.~=
"d
172
B. P. USHAKOVand I. M. PASHKOVA
aquaticus it is equal to 0.87 (Ushakov and Pashkova, 1972). Hence, it can be concluded that all the individuals of the population follow the same functional relationship. In other words, to a change in environmental temperature the population responds as a functional system. Thermal acclimation and selection
The survival of individuals during selection caused by heating is determined by the heat resistance of the organism and not by that of muscle tissue or contractile muscle models. Thus, in salamander larvae the heat resistance of the organism appeared to be almost 2°C lower than that of muscles and their contractile models. That is why changes in the result of thermal selection of muscles and their contractile models can be of interest only in relation to the problem of artificial selection of these characteristics (Amosova, 1967; Skholl, 1981). In order to determine the selective value of the heat resistance of muscles and their contractile models, a correlation between these characteristics and the organismal resistance should be considered. Sib analysis allows this problem to be solved experimentally, Unfortunately, in salamanders the number of offspring per family is rather small. Therefore, the organismal resistance was determined only in one series of experiments (at 21'C). In Fig. 3 an inverse relation can be seen between the organismal heat resistance, average for a family, and that of muscles and their contractile models. More heat resistant individuals are characterized by lower heat resistance of their muscles and muscle models. The correlation coefficients for muscles and muscle models are - 0 . 6 8 and --0.71 respectively (P <0.05). Hence, under optimal conditions the individuals with higher selective rank show lower heat resistance of muscles and their contractile models. As has already been pointed out, it is at the expense of these individuals that the total increase in the heat resistance in the population occurs. Thus, selective advantage during thermal selection belongs to individuals with lower heat resistance of muscle tissue, which are capable of
E
i=
2.4
2.3 E
22 O
~ 2.'1 g T
1 14
1 'L6
_
I
1
1.8
1.9
HeQf resistance of orgQnism (log min)
Fig. 3. The relation between the heat resistance levels of the organism and muscles of salamander larvae in the families studied. Abscissa: average time of the onset of heat shock of the organism (log min). Ordinate: average time of loss of excitability of muscles in response to electrical stimulus (log min).
raising it more significantly in response to changes in environmental temperature. Such a relation between the selective rank of individuals during thermal selection and the heat resistance of their muscle tissue has been first observed on tadpoles (Ushakov et al., 1972) and adult Rana temporaria (Dzhamusova and Chernokozheva, 1976) and more recently, on Bt4fo riridis (Pashkova, 1984). It can therefore be assumed that this relation is characteristic of all amphibians. However, in Asellus aquaticus the picture was quite different: individuals with higher heat resistance of muscles possessed higher selective rank (Pashkova, 1977). Thus, it can be concluded that the differences in the pattern of changes in the heat resistance levels of muscles and contractile muscle models of different individuals caused by heat are not random. The increase in cellular heat resistance at elevated temperature and its decrease at low temperature occur at the expense of different individuals of the population. At high temperature it is due to individuals with more heat sensitive cells, whereas at low temperature it is due to those with less heat sensitive cells and protein complexes.
REFERENCES
Amosova 1. S. (1967) Selection of the blowfly Calliphora erythrocephala by the heat resistance of muscle tissue. In Variability in the Heat Resistance qf Animal Cells in Ontoand Phylogenesis (Edited by Ushakov B. P.), pp. 66 70. Academy of Sciences, Moscow (summary in English). Andreev I. F. (1956) Spotted salamander. Scient. Pap. Kishinev State Univ. 23, 144 146. Arzumanov V. N. and Kusakina A. A. (1960) A portable stimulator for field investigations. T~itolog(va 2, 501 503. Dzhamusova T. A. and Chernokozheva I. S. (1976) A relation of the heat resistance of different muscles with the survival of the organism of the frog during thermal selection. Tsitologiya 18, 114(~ 1143. Gegani G. and Mendelssohn H. (1980) Seasonal activity ot adult and juvenile Salamandra salamandra at the southern limit of their distribution. Br. J. Herpet. 6, 79 81. Lush J. L. (1949) Cited from P. F. Rockitsky, 1974. Pashkova I. M. (1977) The heat resistance of muscles and the survival of Asellus aquaticus during selection caused by injurious temperature. 7~'itologo'a 19, 689 691. Polushina N. A. (1966) Reproduction in spotted salamander (Salumandra salamandra) and its dependence on tern perature. Zool. Zh. Ukr 65, 144 147. Precht H. (1958) Concepts of the temperature adaptation of unchanging reaction systems of cold-blooded animals. In Physiological Adaptation (Edited by Prosser C. L.), pp. 52 78. Am. Physiol. Soc., Washington, D.C. Pyastolova O. A. and Ivanova N. A. 11974) Experimental study of the rate of growth and development of larvae of Salamandra salamandra. Ekolog(va 2, 52 56. Snedecor G. W. (1957) Statistical Methods. Iowa State College Press, Ames, Iowa. Skholl E. D. (1981) The effect of heat acclimation on the artificial selection for muscle tissue heat resistance in the field mouse Microtus subarvalis. Tsitolog(va 23, 404 409. Urbach V. Yu. (1963) Mathematical Statisticsfi)r Biolo~,dst.~ and Physicians. Academy of Sciences, Moscow Ushakov B., Amosova I., Pashkova 1. and Chernokozhewi I. (1968) Quantitative evaluation of individual variability in the heat resistance of cells and their contractile models. J. exp. Zool. 167, 381 390
Populational analysis of thermal responses--I Ushakov B, P., Amosova I. S., Chernokozheva I. S., Dregolskaya I. N., Pashkova I. M. and Skholl E. D. (1977a) The relation of changes in the organismal heat resistance to its initial level during heat acclimation. J. therm. Biol. 2, 9 17. Ushakov B. P., Amosova I. S., Chernokozheva I. S., Dregolskaya I. N., Pashkova I. M. and Skholl E. D. (1977b) Heat acclimation and the population response to selection caused by heating. J. therm. Biol. 2, 17-22. Ushakov B. P. and Pashkova I. M. (1972) The dynamics of individual changes in the heat resistance of muscle tissue
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during heat acclimation of hog slaters, Asellus aquaticus L. Zh. obshch. Biol. 33, 387-396. Ushakov B. P., Pashkova I. M. and Chernokozheva I. S. (1972) Changes in the heat resistance of the organism and muscle tissue of frog tadpoles during heat acclimation as a stabilizing adaptation. Dokl. Akad. Nauk SSSR 203, 935-939. Ushakov B. P. and Pashkova I. M. (1984) The relation of changes in the individual levels of the heat resistance of muscle tissue to their initial values during heat acclimation of Asellus aquaticus. J. therrn. Biol. 9, 303-309.
0306-4565/86 $3.00 + 0.00 Pergamon Journals Ltd
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POPULATIONAL ANALYSIS OF THERMAL RESPONSES--I. CHANGES IN THE HEAT RESISTANCE OF MUSCLE TISSUE A N D CONTRACTILE MUSCLE MODELS OF S A L A M A N D R A S A L A M A N D R A LARVAE B. P. USHAKOWtV~and I. M. PASHKOVA Laboratory of Comparative Cytology, Institute of Cytology, Academy of Sciences of the U.S.S.R., Leningrad 194064, U.S.S.R. (Received 25 October 1985; accepted in revised form 10 February 1986) Abstract--l. Study has been made of the heat resistance of m. rectus superficialis and their contractile
models of larvae of 12 families of Salamandra salamandra kept at three different temperatures: 14, 21 (optimal) and 27°C. The heat resistance of the organism has been studied only in larvae kept at 21°C. 2. An inverse linear relation has been found between the level of the average heat resistance of muscles for offspring, of the same family, under optimal conditions and its increase caused by a change in environmental temperature. A similar relationship was observed in contractile muscle models. This implies that a population responds to a change in environmental temperature as a functional system. 3. The analysis of individual differences in the pattern of response of muscles, and their contractile models, to changes in environmental temperature allows the systemal and individual components to be isolated. The extent of co-ordination in responses of different individuals of the population can be evaluated from the proportion of the systemal component. The value of this component varies from 0.60 to 0.90. 4. Selective advantage during thermal selection belongs to individuals with lower heat resistance of muscles. Key Word Index--Salamandra salamandra; larvae; sib analysis; heat resistance; muscle tissue; contractile muscle models; selective value of the resistance of muscles and contractile muscle models during thermal selection; functional structure of the population.
INTRODUCTION
It remains a paradox that being an experimental science, physiology when elaborating the problems of evolution, is still confined to the study of formation of functions of the organism in phylogenesis. Little attempt is made to elucidate the mechanisms of evolutionary process. The study of individual responses of genetically heterogenous individuals from natural populations, to changes in ecological factors, makes it possible for physiologists to analyse the mechanisms of evolutionary processes and particularly the process of speciation. Synthetic theory of evolution is based on the study of selection of genetic markers whose phenotypical expression is practically independent of environmental changes. Such an approach significantly facilitates the study of the result of selection and makes it unnecessary to analyse the process of selection itself. However, with the use of such genetic markers for the analysis of microevolution, an important point is overlooked: changes in selected characteristics occurring during selection can affect its result. Therefore, it is necessary to consider labile physiological characteristics, whose phenotypical expression is affected by environment and whose adaptive value is doubtless. It is evident that without the participation of physiologists the analysis of the kind can hardly be effective. More advanced in this field of investigation are the physiologists engaged in the studies of resistanceadaptations (see: classification of adaptations accord167
ing to: Precht, 1958). They study the effect of ecological factors on survival of individuals and the criterion of resistance used is the dose of injurious effect killing 50% of individuals. Experiments of this kind are aimed at the study of the limiting effect of ecological factors and the resulting selection. Traditionally the results of such experiments are erroneously interpreted as if they were made on a single individual, "typical" of the population studied, and not on a group of genetically heterogenous individuals. During the study of populational responses the attention is shifted from the resistance level, average for a group of specimens, and focused on the specificity of individual responses, i.e. the variability of this characteristic and its reaction norm in a group of individuals of the same population. This method of investigation is termed here as populational analysis. This study is concerned with the populational analysis of thermal responses of individuals in relation to the problem of microevolutian. The first communication considers the effect of temperature on the heat resistance of muscle tissue and contractile muscle models of salamanders and the selective value of this characteristic. MATERIALS AND METHODS
A study of individual variability of physiological characteristics is not quite a common task for a
168
B.P. USHAKOVand I. M. PASHKOVA
physiologist, therefore it seems worthwhile to consider in more detail the quantitative evaluation of this characteristic and the methods for the analysis of its components. The criterion of the heat resistance used in our study was time to the onset of heat shock or loss of some of cell functions during continual exposure to injurious temperature. However, this criterion has two disadvantages: first, its statistical distribution differs from normal, which complicates the statistical processing of the data obtained: and second, standard deviation of this criterion depends on the temperature used in the experiment, Both disadvantages are avoided if logarithmic values of the heat resistance are used instead of their absolute values. In this case the distribution becomes normal and independent of the temperature at which the heat resistance is measured (Ushakov et al., 1968). Therefore, logarithms of inactivation times are used as quantitative characteristics of the heat resistance of the organism and its cells; and their standard deviations show the individual variability of the heat resistance. When studying individual variability a question arises to what extent the variability of the heat resistance levels are actually due to differences between the individuals, and to what extent it is due to the error of methods. On the leech Hirudo medicinal)s, which is a convenient object for obtaining a large number of muscle preparations, it has been shown that the share of individual variability in the total phenotypical variability constitutes 87%, and only 13% is due to the error of methods. The share of the latter is higher during the determination of the heat resistance levels of glycerinated frog muscles, but even then it is two times lower than individual variability (31 and 69% respectively). These, and similar data, (Ushakov et al.. 1968) imply that the methods used in our experiments are valid for the study of the individual variability of the heat resistance. When elaborating the problems of evolution it is not sufficient to study individual variability alone, it is also necessary to determine the share of genetic component in the total phenotypic variability. It can be done on the offsprings of a number of families* reared separately under identical and constant conditions. In this case the environmental component of variability is minimal and the share of genetic variability (between the families) in the total phenotypic variability of a physiological characteristic in a population can be more clearly seen (Lush, 1949). In this case the genetic share of variability determines the upper limit of heritability of a characteristic. It also involves the variability caused by dominance. It should be pointed out that the extent of the variation caused by environmental changes is the significant point. The study concerns the same physiological characteristic in offsprings of the same families (i.e. the individuals with practically identical gene pool) under conditions of variable environment. Thus, changes in the share of genetic variability of a characteristic are due to an increase in the environ-
*In this paper the term "family" is used to denote a group of individuals arising from a single female.
mental component of the Iotal variability (Ushakox et al., 1977a). The determination of heat resistance of an or.ganism usually results m the death or injury of experimental animals, therefore it is impossible to follow in one individual the dynamics of the changcs in this character. For this reason, in out studies we used agamic clones of animals, or siblings, progeny tH the same parents (the method of sib analysis). In this study the method of sib analysis is used for the examination of changes in the heat resistance of abdominal muscles of salamander larvac lind their contractile models caused by their maintenance at high and low temperature. Moreover. the selective value of the characteristic of the heal resistance of muscles and their models is considered. Pregnant females o[" S a l a m a m b a ~aiamandra I. were captured in October in the outskirts of the town of Uzhgorod (the Carpathians). Each of thc 12 females was opened, the larvae removed and divided into three parts, one of which was grown at 14 C. and the two others at 21 and 27 C They were kept in glass vials (2 1), 6 9 individuals in each, lit 10 h of daylight, fed daily with mosquito larvae and sludge worm. The salamander larvae used in the experiments were of the same size (40 45 mm from nose oritices to the tip of the tail). This size is characteristic of larvae in the middle of premctamorphosis (Polushina. 1966; Pyastolova and lvanova, 1974). The survival of larvae in the laboratory was 100%, which means that there was no selection during development, and hence, the results obtained were only due to changes in the functional state of animals The heat resistance of lhe organism ~as dolormined in water at 34 + 0.1 C. The criterion cH the heat resistance used was the time to the ~mset of heat shock, which was judged by the loss of coordination. Records were made of the moment when the larwte turned on their backs (and remained so for more than 1 2min). In contrast to adult animals, m larvae, when such a loss of coordination is observed it results in death. Since in salamanders the number of offsprings per family is rather small, the organismal resistance was determined only in one series of experiments (at 21' C). The time to the onset of heat shock was recorded in 6--9 individuals and the average value calculated, which served as a characteristic of the heat resistance of the organism~ average for a family. Preparations of muscle tissue were made of M. rectus superficialis which was cut in two along the medial line. One half was used for the determination of the heat resistance of muscle tissue, the other for that of actomyosin complex of contractile muscle models. Immediately after preparation the muscles were put into Ringer's solution at 36 + 0.1 C . The criterion of the heat resistance of muscles used was the time to loss of excitability in response to electrical stimulus from a portable stimulator (Arzumanov and Kusakina, 1960). To record the moment of loss of excitability the muscles were periodically taken out of heated Ringer's solution for 5- 7 s and subjected to electrical stimulation (frequency 50 Hz, duration 1 s or less). The total number of such testings usually was 10 12. However, it was considered that such frequent testing might affect the rate of development of heat injury
Populational analysis of thermal responses--I and thereby our assessment of the heat resistance of muscles. Such an effect is more pronounced during the first half of the heat exposure. To avoid this, prior to experiment, a preliminary determination of the time to loss of excitability was made on 2-3 muscles by means of repeated testings. Such preliminary data allowed the testing of the muscle excitability to be confined to the latter half of heat exposure and so to reduce the total number of testings to 3~5. Contractile muscle models were prepared by putting stretched intact muscles into solution containing 50% glycerol, 0.12M KCI, 0.01 M Trismaleic buffer (pH 7.0). Glycerinization was carried out at - 5 to 7°C for 3 to 5 days. Glycerol was washed out in a solution of 0.12 M KCI, 0.005 M MgC12 and 0.01 M of the same buffer. The criterion of the heat resistance of contractile models used was the time from their immersion into washing solution at 36 _ 0.1 °C to the loss of contractility on addition of 0.005 M ATP, pH 7.0. Contractile muscle models are only capable of one-time contraction in response to ATP. Therefore, prior to the determination of the heat resistance the muscles washed out of glycerol are cut into small pieces and only then put into heated testing solution. From time to time these pieces of tissue are taken out with a pipette and put on a glass on which ATP-containing solution is added. The contractile muscle models, not yet injured by heat, contract immediately on addition of ATP by more than 1/3 of their initial length. The moment of loss of such contraction is recorded during the determination of the heat resistance of contractile muscle models. The contraction was observed under the microscope ( x 16). The genetic share in the total phenotypic variability in the heat resistance was calculated using one-factor analysis of variance (Snedecor, 1957). The differences between the average values were considered statistically significant at P ~<0.05. The analysis of the variability of the heat resistance of the organism, muscles and contractile muscle models at the populational level was our major concern. The experimental material obtained allows the share of variability within a family in the total phenotypical variability of these characteristics in a population to be determined. This share appeared to be quite similar: 39, 32 and 37% for the organism, muscles and contractile muscle models respectively. The variations in the heat resistance within a family are due to three factors: first, genetic differences between offspring; second, differences caused by nonuniformity of environment (the effect of this factor is minimal); and third, the error of methods. These data allow an estimation of the error of the methods used,
169
which according to our data does not exceed 15-20% of the total variation of these characteristics in a population. This conclusion is supported by relatively high coefficients of correlation between the heat resistance level of the organism, on one hand, and those of muscles and their contractile models, on the other, observed under optimal environmental conditions. RESULTS
As is known from literature, salamander larvae develop at 21°C in nature (Andreev, 1956; Polushina, 1966; Gegani and Mendelssohn, 1980), therefore this temperature was considered to be optimal for their development. Changes in the heat resistance of muscle tissue In larvae kept at high temperature (27°C) the average heat resistance of muscles was 55% higher than in those kept at optimal temperature (21°C) (from 141 min at 21°C to 218min at 27°C). This increase in tissue heat resistance practically did not have any effect either on the total phenotypical variability of this characteristic or its genetic component (Table 1). In larvae kept at low temperature (14°C) a 58% decrease in the heat resistance of muscles was recorded (from 141 min at 21°C to 59min at 140C). This decrease in resistance was accompanied by a statistically significant 20% decrease in standard deviation of the characteristic studied, i.e. a narrowing in its phenotypical variability (from 0.184 to 0.148). Simultaneously, a double decrease in the genetic share of variability was recorded. This implies that the effect of cold results in phenotypical masking of genetic differences in the heat resistance of muscle tissue of different individuals in a population. As has been shown earlier, an increase in the heat resistance of muscle tissue of each single individual during heat acclimation depends on its initial (preacclimational) level. It was of interest to compare the responses to heat of muscle resistance level, average for a family, of "warm" and "cold" individuals and their siblings developed at optimal temperature (Fig. 1). As can be seen in the figure, the increase in muscle resistance to heat in "warm" larvae is the highest in the families with initially less resistant muscle tissue. Families with initially more resistant muscles practically do not show any changes in the resistance after heat effect. Thus, an increase in the heat resistance of muscles observed after heat effect characteristic of the population as a whole, occurs at the expense of individuals with initially less resistant muscle tissue.
Table 1. Populational characteristics of the heat resistance level of muscles and their contractile models of salamander larvae at different temperatures of maintenance
Object of investigation Muscles Contractile muscle models
Temperature of maintenance CC)
Number of observations (n)
14 21 27 14 21 27
72 67 66 67 67 64
Average time of loss of functional activity log10 min
min
P
1.77 _ 0.01 2.15 + 0.01 2.34+0.01 1.13+-0.01 1.35 + 0.02 1.50 _+0.01
59 141 218 13 22 32
<0.05 <0.05 <0.05 < 0.05
Standard deviation (SD) 0.148 + 0 . 0 1 0.184 __.0 . 0 1 0.178+_0.01 0.103+-0.01 0.157 + 0.02 0.126 +- 0.01
P <0.05 <0.05 <0.05 < 0.05
Heritability coetficient and its SEM (h: + SEM) 0.37 + 0.11 0.68 + 0.04 0.56_+0.08 0.58+-0.05 0.63 _+0.05 0.26 +- 0.09
<0.05 >0.05 >0.05 < 0.05
170
B.P. USHAKOVand I. M. PASHKOVA
¢:
0.4
E
0.3
o
02
~-
O.O
le
0.1
-oi -0.2 u= c -0.3 -0.4 c u
-0.5 -0.6 2.0
2.1
Heat resistance
2.2
2.3
2.4
at 21*C(10g mini
Fig. 1. The relation of increase in the heat resistance of muscles of salamander larvae caused by heat (Q) and cold (O) to its level at optimal temperature (21°C). Abscissa: heat resistance, average for a family, at 21°C (log rain). Ordinate: the difference between the logarithms of the resistance values in experiment and in control (21°C). In "cold" larvae the relation is just the opposite: the decrease in muscle resistance is more pronounced in siblings with initially more resistant muscle tissue. In those with initially less resistant muscles this decrease was less significant. Thus, in "cold" larvae a decrease in the average populational level of muscle 'heat resistance occurs at the expense of individuals with initially more resistant muscles. As can be seen from Fig. 1, changes in the heat resistance of muscles of salamander larvae caused by thermal changes follow a linear equation
heat resistance of contractile muscle models was accompanied by a narrowing in the individual variability of this characteristic and a decrease in its genetic share. Thus, an increase in the heat resistance of contractile models after heat effect resulted in a phenotypic masking of genotypic differences in this characteristic, as was the case in "cold" larvae. The effect of cold resulted in a 40% decrease in the heat resistance of contractile muscle models. In this case too, a significant decrease in individual variability of this characteristic accompanied by a reduction in its genetic component was observed (Table I). As can be seen in Fig. 2, an increase in the heat resistance of contractile muscle models caused by heat occurs mainly at the expense of families with low resistance level of muscle models at optimal temperature. A decrease in this characteristic caused by cold occurs mainly at the expense of families with high resistance level of contractile muscle models at optimal temperature. In both cases some of the families did not show any statistically significant changes in the heat resistance of contractile muscle models. The range of change in the heat resistance of muscle models caused by both heat and cold depends on the level of their resistance under optimal conditions. As in the experiments with muscles, a linear relation is observed between an increase in resistance of muscle models and its control level. In the equation showing the effect of heat: a = -0.51 + 0.02: b = 0.83 + 0.02; and in that for the effect of cold: a = - 1.00 + 0.06; b = 1.11 _+ 0.09. The equations for calculating a change in the heat resistance read as follows: At27 = 0.83 - 0.51 -t:~
A/j4 = 1.11 - tzl.
A t = a " t2~ + b
where t21 is time to loss of muscle contraction in response to electrical stimulus at 21°C, average for a family (log min); A t ~ i f f e r e n c e between these times at low or high temperature (14 or 27°C) and at optimal one (21°C) (log min); a and b----constants. In the equation showing the effect of heat a=-0.99_0.06; b=2.23_+0.16, and in that showing the effect of cold a = - 1.04 + 0.07 and b = 1.85-I-0.16. Since constants a in both cases are statistically indistinguishable from - 1 , the above equation can be simplified At27 = 2.23 - t21 for heat effect Ate4 = 1.85 - t2~ for cold effect. These equations allow an approximate determination of changes in the heat resistance of muscles caused by changes in environmental temperature. For that it is only necessary to know the level of the heat resistance of muscles, average for a family, under optimal conditions. C h a n g e s in the heat resistance o f contractile m u s c l e models
Changes in the heat resistance of contractile muscle models during temperature changes are very similar to those just described for muscles. After heat effect the average time to loss of contraction increased by 41% (Table 1). An increase in the average level of the
Summing up the data obtained both on muscles and contractile muscle models it should be pointed out that an increase in heat resistance caused by heat and its decrease caused by cold occur at the expense of different individuals of the population. The relation of individual increases in the resistance of muscles and their contractile models to their control levels follows a linear equation. A
0,4
~-
0.2
=
]
•
~ -~~
"-...o °
o.o
--01
.~_ - 0 2 ~-0.3 --0.4 1.1
1,3
H e a t resistance
1.5
1.7
at 2 1 * C (loq rain)
Fig. 2. The relation of increase in the heat resistance of contractile muscle models of salamander larvae caused by heat (Q) and cold (©) to its level at optimal temperature (21°C). Designations as in Fig. I.
Populational analysis of thermal responses--I
171
DISCUSSION It has already been demonstrated on the muscle tissue of Asellus aquaticus that a linear relation is observed between the initial level of cellular heat resistance and its increase due to heat acclimation (Ushakov and Pashkova, 1984). The present study supports this conclusion extending it to the effect of cold. Differences in the responses of muscle resistance level to thermal changes are due, on one hand, to the linear relation, common to all specimens of the population, and, on the other, to the specificity of the response of each single individual. Hence, total variance (a2op) characterizing variability of responses in the population can be resolved into two components, first, variance due to this linear relationship (a~.3, 2 and second, variance characterizing differences in individual responses of single specimens (Cri2nd):
o o=
..q
(7 2pop ~ (7 2syst .~ O. 2rid.
Total variance of the responses (trOop) can be calculated using differences between the logarithmic values of time to loss of muscle contraction before and after thermal effect for all the families, or individuals, of the experimental population. The individual component of variability (O'~nd) c a n be quantitatively characterized by variance of differences between the logarithms of actual time to loss of muscle contraction and its theoretically expected value calculated from the linear equation. Having determined these two variances from the above equation, it is possible to calculate the variance which corresponds to systemal component of variability (%~0. 2 When the individual component is low, the points representing experimental data are closer to the straight line showing the theoretically expected values of an increase in resistance calculated from the hyperbolic equation (Figs 1 and 2). If there were no individual component, these points would have fallen precisely on the straight line. If there were no systemal component, these points would have formed a chaotic accumulation, which would mean a complete lack of co-ordination in the responses of single specimens in the population. If both parts of the above equation are divided by total variance (a:pop), the equation reads as follows O.~nd 2 0"syst ~-- t~O.2nd 2 __ : ~- ~ + 60"syst - - 1. O'pop O'po p
This equation allows quantitative evaluation of the shares of systemal (ra~y,t) 2 and individual (&r~,d) components in the total variance of responses of different families of the population. Of more interest here is the share of systemal component, since it allows the evaluation of the extent of co-ordination in the responses of different families (individuals) of the population to environmental changes. It is equal to the squared correlation coefficient between the heat resistance in control and its increase caused by thermal changes (Urbach, 1963)
O'~nd
cSO'~y~t= 1 -- _-ST-=
r2.
O"pop
In this study the share of systemal component is rather high, ranging from 0.60 to 0.90 (Table 2). In our previous study on heat acclimation of Asellus
-s
e~oo
O
~o ~E ~o=
oo
r-i [..
~ o.~=
"d
172
B. P. USHAKOVand I. M. PASHKOVA
aquaticus it is equal to 0.87 (Ushakov and Pashkova, 1972). Hence, it can be concluded that all the individuals of the population follow the same functional relationship. In other words, to a change in environmental temperature the population responds as a functional system. Thermal acclimation and selection
The survival of individuals during selection caused by heating is determined by the heat resistance of the organism and not by that of muscle tissue or contractile muscle models. Thus, in salamander larvae the heat resistance of the organism appeared to be almost 2°C lower than that of muscles and their contractile models. That is why changes in the result of thermal selection of muscles and their contractile models can be of interest only in relation to the problem of artificial selection of these characteristics (Amosova, 1967; Skholl, 1981). In order to determine the selective value of the heat resistance of muscles and their contractile models, a correlation between these characteristics and the organismal resistance should be considered. Sib analysis allows this problem to be solved experimentally, Unfortunately, in salamanders the number of offspring per family is rather small. Therefore, the organismal resistance was determined only in one series of experiments (at 21'C). In Fig. 3 an inverse relation can be seen between the organismal heat resistance, average for a family, and that of muscles and their contractile models. More heat resistant individuals are characterized by lower heat resistance of their muscles and muscle models. The correlation coefficients for muscles and muscle models are - 0 . 6 8 and --0.71 respectively (P <0.05). Hence, under optimal conditions the individuals with higher selective rank show lower heat resistance of muscles and their contractile models. As has already been pointed out, it is at the expense of these individuals that the total increase in the heat resistance in the population occurs. Thus, selective advantage during thermal selection belongs to individuals with lower heat resistance of muscle tissue, which are capable of
E
i=
2.4
2.3 E
22 O
~ 2.'1 g T
1 14
1 'L6
_
I
1
1.8
1.9
HeQf resistance of orgQnism (log min)
Fig. 3. The relation between the heat resistance levels of the organism and muscles of salamander larvae in the families studied. Abscissa: average time of the onset of heat shock of the organism (log min). Ordinate: average time of loss of excitability of muscles in response to electrical stimulus (log min).
raising it more significantly in response to changes in environmental temperature. Such a relation between the selective rank of individuals during thermal selection and the heat resistance of their muscle tissue has been first observed on tadpoles (Ushakov et al., 1972) and adult Rana temporaria (Dzhamusova and Chernokozheva, 1976) and more recently, on Bt4fo riridis (Pashkova, 1984). It can therefore be assumed that this relation is characteristic of all amphibians. However, in Asellus aquaticus the picture was quite different: individuals with higher heat resistance of muscles possessed higher selective rank (Pashkova, 1977). Thus, it can be concluded that the differences in the pattern of changes in the heat resistance levels of muscles and contractile muscle models of different individuals caused by heat are not random. The increase in cellular heat resistance at elevated temperature and its decrease at low temperature occur at the expense of different individuals of the population. At high temperature it is due to individuals with more heat sensitive cells, whereas at low temperature it is due to those with less heat sensitive cells and protein complexes.
REFERENCES
Amosova 1. S. (1967) Selection of the blowfly Calliphora erythrocephala by the heat resistance of muscle tissue. In Variability in the Heat Resistance qf Animal Cells in Ontoand Phylogenesis (Edited by Ushakov B. P.), pp. 66 70. Academy of Sciences, Moscow (summary in English). Andreev I. F. (1956) Spotted salamander. Scient. Pap. Kishinev State Univ. 23, 144 146. Arzumanov V. N. and Kusakina A. A. (1960) A portable stimulator for field investigations. T~itolog(va 2, 501 503. Dzhamusova T. A. and Chernokozheva I. S. (1976) A relation of the heat resistance of different muscles with the survival of the organism of the frog during thermal selection. Tsitologiya 18, 114(~ 1143. Gegani G. and Mendelssohn H. (1980) Seasonal activity ot adult and juvenile Salamandra salamandra at the southern limit of their distribution. Br. J. Herpet. 6, 79 81. Lush J. L. (1949) Cited from P. F. Rockitsky, 1974. Pashkova I. M. (1977) The heat resistance of muscles and the survival of Asellus aquaticus during selection caused by injurious temperature. 7~'itologo'a 19, 689 691. Polushina N. A. (1966) Reproduction in spotted salamander (Salumandra salamandra) and its dependence on tern perature. Zool. Zh. Ukr 65, 144 147. Precht H. (1958) Concepts of the temperature adaptation of unchanging reaction systems of cold-blooded animals. In Physiological Adaptation (Edited by Prosser C. L.), pp. 52 78. Am. Physiol. Soc., Washington, D.C. Pyastolova O. A. and Ivanova N. A. 11974) Experimental study of the rate of growth and development of larvae of Salamandra salamandra. Ekolog(va 2, 52 56. Snedecor G. W. (1957) Statistical Methods. Iowa State College Press, Ames, Iowa. Skholl E. D. (1981) The effect of heat acclimation on the artificial selection for muscle tissue heat resistance in the field mouse Microtus subarvalis. Tsitolog(va 23, 404 409. Urbach V. Yu. (1963) Mathematical Statisticsfi)r Biolo~,dst.~ and Physicians. Academy of Sciences, Moscow Ushakov B., Amosova I., Pashkova 1. and Chernokozhewi I. (1968) Quantitative evaluation of individual variability in the heat resistance of cells and their contractile models. J. exp. Zool. 167, 381 390
Populational analysis of thermal responses--I Ushakov B, P., Amosova I. S., Chernokozheva I. S., Dregolskaya I. N., Pashkova I. M. and Skholl E. D. (1977a) The relation of changes in the organismal heat resistance to its initial level during heat acclimation. J. therm. Biol. 2, 9 17. Ushakov B. P., Amosova I. S., Chernokozheva I. S., Dregolskaya I. N., Pashkova I. M. and Skholl E. D. (1977b) Heat acclimation and the population response to selection caused by heating. J. therm. Biol. 2, 17-22. Ushakov B. P. and Pashkova I. M. (1972) The dynamics of individual changes in the heat resistance of muscle tissue
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during heat acclimation of hog slaters, Asellus aquaticus L. Zh. obshch. Biol. 33, 387-396. Ushakov B. P., Pashkova I. M. and Chernokozheva I. S. (1972) Changes in the heat resistance of the organism and muscle tissue of frog tadpoles during heat acclimation as a stabilizing adaptation. Dokl. Akad. Nauk SSSR 203, 935-939. Ushakov B. P. and Pashkova I. M. (1984) The relation of changes in the individual levels of the heat resistance of muscle tissue to their initial values during heat acclimation of Asellus aquaticus. J. therrn. Biol. 9, 303-309.