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Populational analysis of thermal responses—II. Changes in the rate of development of Salamandra salamandra larvae
J. therm. Biol. Vol. I1, No. 3, pp. 175-180, 1986 Printed in Great Britain
0306-4565/86 $3.00+ 0.00 PergamonJournals Ltd
POPULATIONAL ANALYSIS OF THERMAL RESPONSES--II. CHANGES IN THE RATE OF DEVELOPMENT OF S A L A M A N D R A S A L A M A N D R A LARVAE B. P. USHAKOW,I~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 revisedform 10 February 1986) Abstract--l. Study was made of the rate of development of Salamandra salamandra larvae in relation to the heat resistance of the organism, muscles and contractile muscle models. The offspring of 12 families were examined at 14, 21 and 27°C. 2. A linear relation is observed between the rate of development of salamander larvae at optimal temperature and its change caused by the effect of high and low temperature. 3. An increase in the rate of development caused by heat (27°C) was the highest in the families in which it was low initially; and vice versa, its decrease caused by cold (14°C) was more significant the higher it was initially. 4. A positive correlation is observed between the rate of development of larvae and the best resistance of their muscles (or contractile muscle models). Such a correlation can be explained by the dependence of these characteristics on the thyroid function. 5. Under optimal thermal conditions the population is in a state of "ecological rest" which is characterized by maximum range of individual variability and its genetic share, and maximum values of correlation coefficients between different physiological characteristics of an organism. Key Word Index--Salamandra salamandra; larvae; sib analysis; rate of development; heat resistance of the organism and muscles; linear relation; correlation; population as a functional system; systemal component of the populational response; ecological rest of a population, phenotypical masking of genetic differences.
INTRODUCTION
The previous paper dealt with the populational analysis of changes in the heat resistance of muscles of Salamandra salamandra larvae and their contractile muscle models caused by their maintenance at high and low temperatures. Larvae of the same body length (40-45 mm) were compared (the middle of premetamorphosis). The results obtained show the relationship between the temperature of maintenance of larvae and the development rate of progeny. The progeny of 12 females were exposed to 14, 21 (optimal) and 27°C. This paper deals with the populational analysis of the data obtained.
the larvae developed at 14 and 21°C (Table 1) the distribution curves of their development rates expressed in logarithms were normal. When the larvae developed at 27°C the distribution curve was also close to normal, whereas with the use of arithmetic values it was far from normal. Such normalization is not unexpected, since the characteristics of the rate of biological processes show log-normal distribution (Koch, 1966). Therefore, in this study the logarithmic values of development rates were used.
MATERIALS AND METHODS
The methods are described in detail in the previous paper. The criterion of the development rate used was an inverse value of time until the body length of larvae reached 40-45 mm. The time was measured in days and for convenience of calculations multiplied by 1000. As known, Fisher's statistical analysis is only applicable in the case when the distribution curve of a characteristic studied is normal. In this study, when *In this paper the term "family" is used to denote a group of individuals arising from a single female. 175
RESULTS
The relation between the development rate at optimal temperature and its change caused by the effect of heat or cold When maintained at 21°C (optimal temperature) the body length of larvae reached 40--45 mm in 35 days, whereas at 27°C it took 21 days. Thus, the development rate increased on average by 37.7%. However, offspring of different families* increased their development rates to a different extent. In larvae with lower development rate this increase was more than 35-40%, whereas in those with higher development rate it almost remained unchanged. Hence, an inverse relation is observed between the rate of development at optimal temperature and its increase caused by heat effect (Fig. 1). Regression, calculated by the method of least squares is described by an
176
B. P. USHAKOV a n d I. M. PASHKOVA Table 1. The evaluation of normality of the curves of statistical distribution of the developmenl rates ol ",ahlmal}del lar~ac (Z" method) 1" i',h,~-~,~.~
{ f ' -: t t O I )
Temperature (C)
Time o f development (days)
N u m b e r of larvae
Arithmetic value
Logarithmic value
Arilhmetic ~alue
l ~,guxitlmm v;ihlc
14 21 27
43 35 21
72 67 67
37.03 83.33 76.02
15.54 875 20.5 ]
225 "- ~
2(I.~ 20 :' I x ~,
2 :',
~F Fisher's criterion.
equation
The effect o! environmental temperature ~,n the cffectiveness qf sib selection hv the rate qf development
AV27 = 1.36-0.08. V:I where Vz~ is development rate at 21 C, average for offsprings of each single family (log); A 1/27 is the difference between these characteristics at 27'C and at 21'C. Maintenance at low temperature (14"C) results in a decrease in development rate by 16.7% on average. This decrease occurs at the expense of families with higher development rate at 21"C. Families with lower development rates show very slight changes in this characteristic, if any (Fig. 1). The relationship between the rate of development at 21"C and its decrease caused by the effect of cold is described by AVI4 = 0.99 - 0.72. l~t where AVe4 is the difference between the average development rates (log) of the offsprings of a family at 14 and 21°C. Thus, the determination of the development rate at optimal temperature allows the prognosis of this characteristic at high and low environmental temperature. It is of interest that heat- and cold-induced changes in the development rate occur at the expense of different families of the population studied.
Earlier studies on acclimation perlbrmed m our laboratory considered the question of the effect of temperature on the possible result of thermal selection (Ushakov, 1977). As shown in the paper by Skholl and Pasynkova (1982), the results of selection by the heat resistance of muscles of Microtus suharyalis are in good agreement with those theoretically expected. It seems worthwhile to estimate the effect of exposure to heat and cold on the effectiveness of artificial selection of larvae by their development rate. Changes in the effectiveness of selection (6E) can be induced by shifts in one of the three parameters: first, standard deviation (a) of a characteristic indicating its phenotypical variability; second, heritability coefficient (h 2) showing the share of genetic variability in the total phenotypical variability of a characteristic; and third, rank correlation coefficient (p) between the development rate of larvae at optimal temperature and its increase caused by the effect of heat and cold, which shows changes in the selective rank of families by the characterislic studied. A change in the effectiveness of sib selection can be calculated from the equation 6E
0.30 "6 0
O2O
oo
0.10
.-= $
° °
o.oo
go => -o.Io -0.20 1.30
1.40
1.50
q,60
Rate of development at 21*C ( log d ~ s
.1000)
Fig. 1. The relation of increase in the rate of development of salamander larvae caused by heat (O) and cold (O) to the rate of development at optimal temperature (21~'C). Abscissa: rate of development at 21C (log l/days. 1000). Ordinate: the difference between the logarithms of the rates of development of larvae of the families studied in experiment (27 and 14°C) and in control (2VC).
¢~'P h~xp . . . . . . w~-'l
where the parameters with the subscript c (control) signify their value at optimal (control) temperature, and with a subscript exp (experimental) their value at high and low temperatures (Ushakov, 1977). The data of Table 2 show that at the effect of heat the effectiveness of sib selection by the rate of development decreases 10 times (0.10), which is mainly due to a change in the selective rank of families. Spearman's coefficient is reduced to 0.19. The heritability coefficient decreases almost 2 times (0.59). Phenotypical variability of development rates also significantly decreases, however this decreases does not show so strongly the effectiveness of sib selection. At low temperature the decrease in the effectiveness of sib selection is not so strong, but in this case too, it is more than 3 times lower (0.29) than at optimal temperature. Such a decrease is also due to a reduction of the genetic share in the total phenotypical variability (Table 2). A decrease in phenotypical variability is statistically significant as well, but its influence on the effectiveness of sib selection is also weak. Thus, during sib selection o1" salamander larvae by the rate of their development, the maximum effect is
177
Populational analysis of thermal responses---II
observed with the cultivation of animals at constant optimal temperature. When the larvae are cultivated at temperatures different from the optimal one, the effectiveness of sib selection decreases. A correlation between the development rate and the
V V
~~ ~.o
heat resistance of muscle tissue of salamander larvae The heat resistance of muscles, as well as the development rate, was determined at all the three temperatures. At all the three temperatures a statistically significant positive correlation was found between the development rate and the heat resistance of muscle tissue (Fig. 2). The correlation coefficient was the highest at 21°C (0.70), whereas at 27 ° it was lower (0.59), and at 14°C--statistically not significant
(0.45). Similarly, in contractile muscle models, maximum value of this coefficient (0.83) was recorded at 21°C,
vv
~
I.
-
2.30
-
2.20
-
(o) O
+1 +1
m.,~-
~ N
2.40
.~'~ ~N
o
2.10
•
o O
i~r.,
I
r~e~
N
I 1.30
¢:
I
I
1.40
1.50
1 1.60 (b)
2.50 e~
o r',l~
~'~
2.40 E 2.30
VV
o
2.L~O =..
+1 +1
I 1.40
'I-
I I. 5 0
I 1.60
1 1.70
~-r.~
1.90
e o
~
ee
(c)
oo
2
1.80
O
+t +1
o
•
1.70
•
1.60
i 1.30 Rote
I
I
I
1.40
I. 5 0
I. 6 0
of
( log ~
dovl)lopmont .1000 )
Fig. 2. The relation of the heat resistance of muscles to the rate of development of larvae at different temperatures. Abscissa: rate of development of larvae (log l/days. 1000). Ordinate: time to loss of electrical excitability of muscle tissue (log min) at 21 ° (a); 27 ° (b) and 14°C (c).
178
B.P. USHAKOVand I. M. PASHKOVA
whereas at 14 and 27~C it was statistically not significant (0.38 and 0.15 respectively). Thus, a correlation between the development rate and the heat resistance of muscles (or their contractile muscle models) is more pronounced at a temperature, optimal for the development of salamander larvae. Since the number of offspring per family is rather small, the organismal heat resistance was determined only in the larvae kept at optimal temperature. When studying a correlation between the development rate of larvae and the heat resistance of the organism a statistically significant negative correlation coefficient was obtained (-0.58). This is what could be expected since as has been found earlier, at this stage of ontogenesis the heat resistance of the organism and that of the muscle tissue of larvae are in inverse relation (see Part I). DISCUSSION
a decrease in correlation coefficient between different physiological characteristics of an organism within a population during changes in environmental temperature. In view of the above, the state of ecological rest of t, population can be characterized, first, by maximum individual variability of physiological characteristics: second, maximum heritability coefficient: and third, maximum correlation coefficients between different characteristics of an organism. A change in environmental conditions results in a narrowing in individual variability and a decrease in the heritability cofficient of physiological characteristics, thus leading to a decrease in the genetic effectiveness of selection. Moreover, it results in a change in the selective rank of individuals, which hinders selection by physiological characteristics dependent on environmental conditions.
The heat resistance 0["cells and the/unction ~[ thyroid
The state of "ecological rest'" of a population The results of experiments on cultivated Daphnia magna and their comparison with the data from literature led us to conclude that individual variability and the genetic share in the total phenotypical variability of organismal and cellular heat resistance are integral characteristics of the functional state of individuals in a population (Ushakov and Pashkova, 1983). Both these characteristics have maximum values under optimal conditions. Maximum range of individual variability and its genetic share in the heat resistance of muscle tissue and contractile muscle models was observed in salamander larvae at optimal temperature (21°C) (see Part I). The same result was obtained by the studies of the rate of development of salamanders (Table 2). This enables us to assume that such a relation is typical of physiological characteristics dependent on environmental conditions. We qualify such a state of the organism as the state of "ecological rest". Under optimal conditions owing to the widening of the range of individual variability and its genetic share, a correlation between different physiological characteristics of an organism is more pronounced. Thus, correlation coefficients between the rate of development of larvae and the heat resistance of muscles (or their contractile models) had maximum values at an optimal temperature. Similarly, the correlation coefficients between the heat resistance of muscles and their contractile models were statistically significant only at an optimal temperature (Table 3). A change in environmental temperature results in a responsive narrowing in the individual variability and an increase in the environmental component of variability in physiological characteristics, thus masking genetic correlations between them. Hence, phenotypical masking of genetic differences is the reason for
Table 3. Coefficients of correlation between average heat resistance values of muscles and contractile muscle models in offsprings of several families of salamanders at different temperatures Temperature ( C )
p
P
27 21 14
0.15 0.83 0.38
>0.05 <0.05 >0.05
The rate of development is controlled by the thyroid function. As has already been shown in this study, a positive correlation is observed between the rate of development and the heat resistance of muscles average for a family. A question arises, could not this correlation be due to the intensity of thyroid function, which differs from family to family. If it is so, the activation of the thyroid should result not only in acceleration of development but also in an increase in the heat resistance of muscles. In fact, feeding mature Rana temporaria with thyroxine as well as injecting them with thyrotrophic hormone or thyroxine result in an increase in the heat resistance of their muscle tissue (Pashkova, 1967). It appears that different muscles of an organism increase their heat resistance to a different extent: less resistant muscles (m. ileofibularis) show a more significant increase in their heat resistance level, whereas more resistant ones (m. sartorius, m. gastrocnemius) do not change it at all, and the most resistant (m. lingua) can even show a decrease in the resistance level (Dzhamusova, 1971). On Rana temporaria tadpoles it has been shown that acceleration in development caused by putting them in thyroxine solution is accompanied by an increase in the heat resistance of tail muscles. At a temperature, optimal for this species (12°C) thyroxine induced a 51 96% increase in the resistance of muscles, whereas at elevated temperature--only a 15% increase (Chernokozheva. 1970). On mature frogs Rana temporaria it has been demonstrated that a rise in environmental temperature results in activation of the thyroid and a simultaneous increase in the heat resistance of muscles (Pashkova, 1965). Similarly, in our experiments on salamanders a rise in environmental temperature leads to an increase in the development rate of larvae and a rise in the heat resistance of their muscle tissue. All the data considered above allow the conclusion that both the rate of development and the heat resistance of muscles are controlled by the thyroid function. A shift in environmental temperature results in a change in the thyroid function, which, in its turn, leads to changes in both the rate of development and the heat resistance of muscles tissue. However, it should be remembered that the effect of temperature on the heat resistance of cells was also observed in the
P o p u l a t i o n a l analysis o f t h e r m a l r e s p o n s e s - - I I
179
Table 4. Systemal component in the response of the population to changes in environmental factors
Environmental factors 1 Temperature (cultivation at temperatures, different from the optimal ones)
Species 2 Salamandra salamandra (larvae) Salamandra salamandra (larvae) Salamandra salamandra (larvae) Rana temporaria (tadpoles) Rana temporaria (tadpoles) Spirostomum ambiguum Oxytricha minor Opalina ranarum
Temperature (heat acclimation)
Hydra oligactis Strongyloeentrotus droebachiensis (blastula) Asellus aquaticus Drosophila melanogaster Rana temporaria (tadpoles) Misgurnus fossilis (larvae) Asellus aquaticus Mytilus galloprovincialis
Salinity
Paramecium aurelia Paramecium caudatum
Quinine
Paramecium caudatum
Physiological characteristic studied 3 Heat resistance of muscles Heat resistance of contractile muscle models Rate of development Heat resistance of muscles Heat resistance of the organism Heat resistance of the organism Heat resistance of the organism Heat resistance of the the orgamsm Heat resistance of the the orgamsm Heat resistance of the the orgamsm Heat resistance of the the orgamsm Heat resistance of the the orgamsm Heat resistance of the the orgamsm Heat resistance of the the orgamsm Heat resistance of muscles Heat resistance of the cilitated epithelium Salt resistance of the organism Salt resistance of the organism Quinine resistance of the organism
cases when neuro-humoral effect on cells was not possible. Thus, the heat resistance changes caused by heat acclimation were observed in isolated ciliated epithelium cells of molluscs (Friedrich, 1967; Lagerspetz and Dubischer, 1966; Skholl, 1971). Hence, in addition to the hormonal regulatory mechanism of the heat resistance of cells, at least in ciliated epithelium, there exists some other intracellular mechanism, independent of the organism. The response o f the population as a functional system The data obtained in this study on the rate of development of salamander larvae support the conclusion made in Part I that to a change in environmental temperature a population responds as a functional system. Individual responses of single families of the population are to a great extent co-ordinated. To evaluate the extent of this co-ordination, we used a systemal component of variability in responses of single families of the population to changes in environmental temperature (60"syst). 2 It shows the share of phenotypical variability associated with general relationship between the responses of single families (or individuals) of the population to environmental changes. The less stochastic the response of the population, the higher the value of systemal component of variability. In this study on the rate of development the systemal component in the response of the population is equal to 0.46 and 0.37 for the effect of heat and cold respectively, which is lower than in the study
Systemal component of the response of the population 4
Authors 5
0.60-0.63 0.654).94
Ushakov and Pashkova (this study) Ushakov and Pashkova (this study)
0.37-0.46 0.70-0.91 0.59
Ushakov and Pashkova (this study) Glushankova and Chernokozheva, Ushakov et aL, 1972
0.81
Irlina, 1960
0.49
lrlina, 1960
0.77
Sukhanova, 1962
0.21-0.85 0.36-0.66
Dregolskaya, 1977; Ushakov et al., 1977a Ushakov et al., 1977a
0.34-0.92
Ushakov et al., 1977a
0.03-0.46
Ushakov et al., 1977a
0.23-0.88
Ushakov et al., 1977a
0.59-0.79
Pashkova et al., 1981
0.65-0.90 0.42
Ushakov and Pashkova, 1984 Skholl, 1971
0.81
Gause, 1939
0.56
Smaragdova and Gause, 1939
0.50
Smaragdova and Gause, 1939
on the heat resistance of muscles and their contractile models made on the same families of salamanders (0.60-0.94). The data are shown in Table 4. The values of this component were as high in the study on the heat resistance of muscles of Rana temporaria tadpoles which developed at elevated temperature (Glushankova and Chernokozheva, 1965; Ushakov et al., 1971, 1972). High values of systemal component were also obtained by the studies on the organismal heat resistance of tadpoles of the same families (Ushakov et al., 1972) and on three species of protozoans (Irlina, 1960; Sukhanova, 1962). Thus, when the animals develop under thermal conditions, diflerent from the optimal ones, the population responds to thermal changes as a functional system. Under such conditions the individual (stochastic) component of the populational response is very small. The data of several earlier studies allow systemal component of variability during short-term temperature acclimation to the determined (Table 4). At earlier stages of thermal acclimation it is very small or even absent, but in the course of acclimation it significantly increases (Table 4). The data of the studies by Gause (1939) and Smaragdova and Gause (1939) allow determination of the systemal component in the populational response of infusorians to changes in the chemical composition of environment. In this case too, the values of this component are rather high (Table 4). These data allow the conclusion that the population
B. P. USHAKOVand I. M. PASHKOVA
180
responses as a functional system, not only to changes in temperature, but also in chemical composition of the environment. Thus, the same pattern of populational response is observed during the study of such different physiological characteristics as heat resistance, salt resistance and the rate of development. It should be pointed out that all these characteristics are directly or indirectly related to the survival of individuals under variable environmental conditions. Systemal component of variability shows the extent to which the response of each single individual follows the general relationship characteristic of the population. High values of systemal component serve as evidence that the responses of single individuals are to a great extent determined by their genetic programme. Apparently, these responses are of epigenotypic origin. All the individuals of the population show a distinct relation between an increase in resistance to injury caused by environmental factors and its initial level. This relation is the reason for phenotypic masking of genotypic differences between the individuals during changes in ecological factors (Ushakov et al., 1977a, 1977b; Ushakov, 1977, 1982). Phenotypic masking of genotypic differences results in a decrease in the genetic effectiveness of selection, which facilitates maintenance of constant genetic structure of the population. However, it should be remembered that such a response of the population does not involve interaction of individuals, therefore the question of whether or not a population is an integral system (as is, for instance, organism or biocoenosis) remains so far open. REFERENCES
Chernokozheva I. S. (1970) Effect of the rearing of tadpoles in thyroxine solutions on the thermostability of their muscle tissue. Tsitologiya 12, 119-123. Dregolskaya I. N. (1977) Heritabi]ity coefficient of the heat resistance of the organism of Hydra oligactis Pall. under optimal cultivation temperature and after heat acclimation. Zh. obshch. Biol. 38, 440~45. Dzhamusova T. A. (1971) Changes in the heat resistance level of different frog muscles caused by thyroxine injections. Ekologiya 5, 53-58. Fisher R. A. (1954) Statistical Methods for Research Workers. Oliver & Boyd, London. Friedrich L. (1967) Experimentelle Untersuchungen zum Problem zellul/irer nichtgenetischer Resistenzanderungen bei der Miesmuschel Mytilus edulis L. Kieler Meeresforsch. 23, 105 126. Gause G. F. (1939) The adaptation of Paramecium aurelia to the increased salinity of the medium. Zool Zh. Ukr. 18, 631~41. Glushankova M. A. and Chernokozheva I. S. (1965) The thermostability of muscle fibres and some tissue proteins in the tadpoles of Rana temporaria L. in connection with cultivation temperature. In Heat Resistance of Cells of Animals (Edited by Ushakov B. P.), pp. 153 160. Academy of Sciences, Moscow. lrlina I. S. (1960) Changes in thermostability of some free-living Protozoa under the influence of preliminary temperature regime. Tsitologiya 2, 227 234.
Koch A. L. (1966) The logarithm in biology. 1. Mechanisms generating the log-normal distribution exactly. J. theoret. Biol. 12, 276-290. Lagerspetz K. and Dubischer J. (1966) Temperature acclimation of the ciliary activity in the gills of Anodonta. Comp. Biochem. Physiol. 17, 665-671. Lerner J. M. (1954) Genetic Homeostasis. Oliver & Boyd, London. Pashkova I. M. (1965) Relationships between muscle thermostability and activity of thyroid gland of Rana temporaria L. at different seasons of the year. In Heat Resistance of Cells o f Animals (Edited by Ushakov B. P.), pp. 82-89. Academy of Sciences, Moscow (summary in English). Pashkova I. M. (1967) Analysis of seasonal changes in thermostability of frog muscles, in The Cell and Environmental Temperature (Edited by Troshin A. S.), pp. 225.-231. Pergamon Press, Oxford. Pashkova I. M., Amosova I. S., Skholl E. D., Chernokozheva I. S. (1981) Changes in the response of a population of Misgurnus fossilis L. to thermal selection after a short-term heat acclimation. Zh. obshch. Biol. 42, 556-563. Skholl E. D. (1971) A correlation between the initial heat resistance level of isolated ciliated epithelium of Mytilus galloprovincialis and its change caused by heat effect. Ekologiya 6, 69 73. Skholl E. D. and Pasynkova R. A. (1982) The assumed and realized in F~ result of selection for heat resistance of myofibrillar ATPase in the field mouse. Genetika 18, 956-959. Smaragdova N. P. and Gause G. F. (1939) A comparative investigation of adaptation of Paramecium caudatum to the increased salinity of the medium and to quinine solutions. Zool. Zh. Ukr 18, 642-655. Sukhanova K. M. (1962) Temperature adaptations in Opalina ranarum Ehrenberg (Opalinidae) during its life cycle. Tsitologiya 4, 644-651. Ushakov B. P. (1977) The effect of heat acclimation on the intensity and the genetic effectiveness of selection caused by heating. J. therm. Biol. 2, 177-182. Ushakov B. P. (1982) Temperature adaptations of animals and their significance in evolution. Usp. sovren. Biol. 93, 302--319. Ushakov B. P., Glushankova M. A., Salmenkova E. A. and Chernokozheva I. S. (1971) The relation between the initial level of heat resistance of cells and proteins and the direction of phenotypical shift caused by temperature adaptation of frog tadpoles. Ekologiya 3, 9--18. 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., Amosova I. S., Chernokozheva I. S., Dregolskaya 1. 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 1. S., Chernokozheva 1. 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. (1983) The analysis o1 the increase in heat resistance of Daphnia magna in the process of cultivation. Genetika 19, 1251-1256. 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. therm. Biol. 9, 303 309.
0306-4565/86 $3.00+ 0.00 PergamonJournals Ltd
POPULATIONAL ANALYSIS OF THERMAL RESPONSES--II. CHANGES IN THE RATE OF DEVELOPMENT OF S A L A M A N D R A S A L A M A N D R A LARVAE B. P. USHAKOW,I~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 revisedform 10 February 1986) Abstract--l. Study was made of the rate of development of Salamandra salamandra larvae in relation to the heat resistance of the organism, muscles and contractile muscle models. The offspring of 12 families were examined at 14, 21 and 27°C. 2. A linear relation is observed between the rate of development of salamander larvae at optimal temperature and its change caused by the effect of high and low temperature. 3. An increase in the rate of development caused by heat (27°C) was the highest in the families in which it was low initially; and vice versa, its decrease caused by cold (14°C) was more significant the higher it was initially. 4. A positive correlation is observed between the rate of development of larvae and the best resistance of their muscles (or contractile muscle models). Such a correlation can be explained by the dependence of these characteristics on the thyroid function. 5. Under optimal thermal conditions the population is in a state of "ecological rest" which is characterized by maximum range of individual variability and its genetic share, and maximum values of correlation coefficients between different physiological characteristics of an organism. Key Word Index--Salamandra salamandra; larvae; sib analysis; rate of development; heat resistance of the organism and muscles; linear relation; correlation; population as a functional system; systemal component of the populational response; ecological rest of a population, phenotypical masking of genetic differences.
INTRODUCTION
The previous paper dealt with the populational analysis of changes in the heat resistance of muscles of Salamandra salamandra larvae and their contractile muscle models caused by their maintenance at high and low temperatures. Larvae of the same body length (40-45 mm) were compared (the middle of premetamorphosis). The results obtained show the relationship between the temperature of maintenance of larvae and the development rate of progeny. The progeny of 12 females were exposed to 14, 21 (optimal) and 27°C. This paper deals with the populational analysis of the data obtained.
the larvae developed at 14 and 21°C (Table 1) the distribution curves of their development rates expressed in logarithms were normal. When the larvae developed at 27°C the distribution curve was also close to normal, whereas with the use of arithmetic values it was far from normal. Such normalization is not unexpected, since the characteristics of the rate of biological processes show log-normal distribution (Koch, 1966). Therefore, in this study the logarithmic values of development rates were used.
MATERIALS AND METHODS
The methods are described in detail in the previous paper. The criterion of the development rate used was an inverse value of time until the body length of larvae reached 40-45 mm. The time was measured in days and for convenience of calculations multiplied by 1000. As known, Fisher's statistical analysis is only applicable in the case when the distribution curve of a characteristic studied is normal. In this study, when *In this paper the term "family" is used to denote a group of individuals arising from a single female. 175
RESULTS
The relation between the development rate at optimal temperature and its change caused by the effect of heat or cold When maintained at 21°C (optimal temperature) the body length of larvae reached 40--45 mm in 35 days, whereas at 27°C it took 21 days. Thus, the development rate increased on average by 37.7%. However, offspring of different families* increased their development rates to a different extent. In larvae with lower development rate this increase was more than 35-40%, whereas in those with higher development rate it almost remained unchanged. Hence, an inverse relation is observed between the rate of development at optimal temperature and its increase caused by heat effect (Fig. 1). Regression, calculated by the method of least squares is described by an
176
B. P. USHAKOV a n d I. M. PASHKOVA Table 1. The evaluation of normality of the curves of statistical distribution of the developmenl rates ol ",ahlmal}del lar~ac (Z" method) 1" i',h,~-~,~.~
{ f ' -: t t O I )
Temperature (C)
Time o f development (days)
N u m b e r of larvae
Arithmetic value
Logarithmic value
Arilhmetic ~alue
l ~,guxitlmm v;ihlc
14 21 27
43 35 21
72 67 67
37.03 83.33 76.02
15.54 875 20.5 ]
225 "- ~
2(I.~ 20 :' I x ~,
2 :',
~F Fisher's criterion.
equation
The effect o! environmental temperature ~,n the cffectiveness qf sib selection hv the rate qf development
AV27 = 1.36-0.08. V:I where Vz~ is development rate at 21 C, average for offsprings of each single family (log); A 1/27 is the difference between these characteristics at 27'C and at 21'C. Maintenance at low temperature (14"C) results in a decrease in development rate by 16.7% on average. This decrease occurs at the expense of families with higher development rate at 21"C. Families with lower development rates show very slight changes in this characteristic, if any (Fig. 1). The relationship between the rate of development at 21"C and its decrease caused by the effect of cold is described by AVI4 = 0.99 - 0.72. l~t where AVe4 is the difference between the average development rates (log) of the offsprings of a family at 14 and 21°C. Thus, the determination of the development rate at optimal temperature allows the prognosis of this characteristic at high and low environmental temperature. It is of interest that heat- and cold-induced changes in the development rate occur at the expense of different families of the population studied.
Earlier studies on acclimation perlbrmed m our laboratory considered the question of the effect of temperature on the possible result of thermal selection (Ushakov, 1977). As shown in the paper by Skholl and Pasynkova (1982), the results of selection by the heat resistance of muscles of Microtus suharyalis are in good agreement with those theoretically expected. It seems worthwhile to estimate the effect of exposure to heat and cold on the effectiveness of artificial selection of larvae by their development rate. Changes in the effectiveness of selection (6E) can be induced by shifts in one of the three parameters: first, standard deviation (a) of a characteristic indicating its phenotypical variability; second, heritability coefficient (h 2) showing the share of genetic variability in the total phenotypical variability of a characteristic; and third, rank correlation coefficient (p) between the development rate of larvae at optimal temperature and its increase caused by the effect of heat and cold, which shows changes in the selective rank of families by the characterislic studied. A change in the effectiveness of sib selection can be calculated from the equation 6E
0.30 "6 0
O2O
oo
0.10
.-= $
° °
o.oo
go => -o.Io -0.20 1.30
1.40
1.50
q,60
Rate of development at 21*C ( log d ~ s
.1000)
Fig. 1. The relation of increase in the rate of development of salamander larvae caused by heat (O) and cold (O) to the rate of development at optimal temperature (21~'C). Abscissa: rate of development at 21C (log l/days. 1000). Ordinate: the difference between the logarithms of the rates of development of larvae of the families studied in experiment (27 and 14°C) and in control (2VC).
¢~'P h~xp . . . . . . w~-'l
where the parameters with the subscript c (control) signify their value at optimal (control) temperature, and with a subscript exp (experimental) their value at high and low temperatures (Ushakov, 1977). The data of Table 2 show that at the effect of heat the effectiveness of sib selection by the rate of development decreases 10 times (0.10), which is mainly due to a change in the selective rank of families. Spearman's coefficient is reduced to 0.19. The heritability coefficient decreases almost 2 times (0.59). Phenotypical variability of development rates also significantly decreases, however this decreases does not show so strongly the effectiveness of sib selection. At low temperature the decrease in the effectiveness of sib selection is not so strong, but in this case too, it is more than 3 times lower (0.29) than at optimal temperature. Such a decrease is also due to a reduction of the genetic share in the total phenotypical variability (Table 2). A decrease in phenotypical variability is statistically significant as well, but its influence on the effectiveness of sib selection is also weak. Thus, during sib selection o1" salamander larvae by the rate of their development, the maximum effect is
177
Populational analysis of thermal responses---II
observed with the cultivation of animals at constant optimal temperature. When the larvae are cultivated at temperatures different from the optimal one, the effectiveness of sib selection decreases. A correlation between the development rate and the
V V
~~ ~.o
heat resistance of muscle tissue of salamander larvae The heat resistance of muscles, as well as the development rate, was determined at all the three temperatures. At all the three temperatures a statistically significant positive correlation was found between the development rate and the heat resistance of muscle tissue (Fig. 2). The correlation coefficient was the highest at 21°C (0.70), whereas at 27 ° it was lower (0.59), and at 14°C--statistically not significant
(0.45). Similarly, in contractile muscle models, maximum value of this coefficient (0.83) was recorded at 21°C,
vv
~
I.
-
2.30
-
2.20
-
(o) O
+1 +1
m.,~-
~ N
2.40
.~'~ ~N
o
2.10
•
o O
i~r.,
I
r~e~
N
I 1.30
¢:
I
I
1.40
1.50
1 1.60 (b)
2.50 e~
o r',l~
~'~
2.40 E 2.30
VV
o
2.L~O =..
+1 +1
I 1.40
'I-
I I. 5 0
I 1.60
1 1.70
~-r.~
1.90
e o
~
ee
(c)
oo
2
1.80
O
+t +1
o
•
1.70
•
1.60
i 1.30 Rote
I
I
I
1.40
I. 5 0
I. 6 0
of
( log ~
dovl)lopmont .1000 )
Fig. 2. The relation of the heat resistance of muscles to the rate of development of larvae at different temperatures. Abscissa: rate of development of larvae (log l/days. 1000). Ordinate: time to loss of electrical excitability of muscle tissue (log min) at 21 ° (a); 27 ° (b) and 14°C (c).
178
B.P. USHAKOVand I. M. PASHKOVA
whereas at 14 and 27~C it was statistically not significant (0.38 and 0.15 respectively). Thus, a correlation between the development rate and the heat resistance of muscles (or their contractile muscle models) is more pronounced at a temperature, optimal for the development of salamander larvae. Since the number of offspring per family is rather small, the organismal heat resistance was determined only in the larvae kept at optimal temperature. When studying a correlation between the development rate of larvae and the heat resistance of the organism a statistically significant negative correlation coefficient was obtained (-0.58). This is what could be expected since as has been found earlier, at this stage of ontogenesis the heat resistance of the organism and that of the muscle tissue of larvae are in inverse relation (see Part I). DISCUSSION
a decrease in correlation coefficient between different physiological characteristics of an organism within a population during changes in environmental temperature. In view of the above, the state of ecological rest of t, population can be characterized, first, by maximum individual variability of physiological characteristics: second, maximum heritability coefficient: and third, maximum correlation coefficients between different characteristics of an organism. A change in environmental conditions results in a narrowing in individual variability and a decrease in the heritability cofficient of physiological characteristics, thus leading to a decrease in the genetic effectiveness of selection. Moreover, it results in a change in the selective rank of individuals, which hinders selection by physiological characteristics dependent on environmental conditions.
The heat resistance 0["cells and the/unction ~[ thyroid
The state of "ecological rest'" of a population The results of experiments on cultivated Daphnia magna and their comparison with the data from literature led us to conclude that individual variability and the genetic share in the total phenotypical variability of organismal and cellular heat resistance are integral characteristics of the functional state of individuals in a population (Ushakov and Pashkova, 1983). Both these characteristics have maximum values under optimal conditions. Maximum range of individual variability and its genetic share in the heat resistance of muscle tissue and contractile muscle models was observed in salamander larvae at optimal temperature (21°C) (see Part I). The same result was obtained by the studies of the rate of development of salamanders (Table 2). This enables us to assume that such a relation is typical of physiological characteristics dependent on environmental conditions. We qualify such a state of the organism as the state of "ecological rest". Under optimal conditions owing to the widening of the range of individual variability and its genetic share, a correlation between different physiological characteristics of an organism is more pronounced. Thus, correlation coefficients between the rate of development of larvae and the heat resistance of muscles (or their contractile models) had maximum values at an optimal temperature. Similarly, the correlation coefficients between the heat resistance of muscles and their contractile models were statistically significant only at an optimal temperature (Table 3). A change in environmental temperature results in a responsive narrowing in the individual variability and an increase in the environmental component of variability in physiological characteristics, thus masking genetic correlations between them. Hence, phenotypical masking of genetic differences is the reason for
Table 3. Coefficients of correlation between average heat resistance values of muscles and contractile muscle models in offsprings of several families of salamanders at different temperatures Temperature ( C )
p
P
27 21 14
0.15 0.83 0.38
>0.05 <0.05 >0.05
The rate of development is controlled by the thyroid function. As has already been shown in this study, a positive correlation is observed between the rate of development and the heat resistance of muscles average for a family. A question arises, could not this correlation be due to the intensity of thyroid function, which differs from family to family. If it is so, the activation of the thyroid should result not only in acceleration of development but also in an increase in the heat resistance of muscles. In fact, feeding mature Rana temporaria with thyroxine as well as injecting them with thyrotrophic hormone or thyroxine result in an increase in the heat resistance of their muscle tissue (Pashkova, 1967). It appears that different muscles of an organism increase their heat resistance to a different extent: less resistant muscles (m. ileofibularis) show a more significant increase in their heat resistance level, whereas more resistant ones (m. sartorius, m. gastrocnemius) do not change it at all, and the most resistant (m. lingua) can even show a decrease in the resistance level (Dzhamusova, 1971). On Rana temporaria tadpoles it has been shown that acceleration in development caused by putting them in thyroxine solution is accompanied by an increase in the heat resistance of tail muscles. At a temperature, optimal for this species (12°C) thyroxine induced a 51 96% increase in the resistance of muscles, whereas at elevated temperature--only a 15% increase (Chernokozheva. 1970). On mature frogs Rana temporaria it has been demonstrated that a rise in environmental temperature results in activation of the thyroid and a simultaneous increase in the heat resistance of muscles (Pashkova, 1965). Similarly, in our experiments on salamanders a rise in environmental temperature leads to an increase in the development rate of larvae and a rise in the heat resistance of their muscle tissue. All the data considered above allow the conclusion that both the rate of development and the heat resistance of muscles are controlled by the thyroid function. A shift in environmental temperature results in a change in the thyroid function, which, in its turn, leads to changes in both the rate of development and the heat resistance of muscles tissue. However, it should be remembered that the effect of temperature on the heat resistance of cells was also observed in the
P o p u l a t i o n a l analysis o f t h e r m a l r e s p o n s e s - - I I
179
Table 4. Systemal component in the response of the population to changes in environmental factors
Environmental factors 1 Temperature (cultivation at temperatures, different from the optimal ones)
Species 2 Salamandra salamandra (larvae) Salamandra salamandra (larvae) Salamandra salamandra (larvae) Rana temporaria (tadpoles) Rana temporaria (tadpoles) Spirostomum ambiguum Oxytricha minor Opalina ranarum
Temperature (heat acclimation)
Hydra oligactis Strongyloeentrotus droebachiensis (blastula) Asellus aquaticus Drosophila melanogaster Rana temporaria (tadpoles) Misgurnus fossilis (larvae) Asellus aquaticus Mytilus galloprovincialis
Salinity
Paramecium aurelia Paramecium caudatum
Quinine
Paramecium caudatum
Physiological characteristic studied 3 Heat resistance of muscles Heat resistance of contractile muscle models Rate of development Heat resistance of muscles Heat resistance of the organism Heat resistance of the organism Heat resistance of the organism Heat resistance of the the orgamsm Heat resistance of the the orgamsm Heat resistance of the the orgamsm Heat resistance of the the orgamsm Heat resistance of the the orgamsm Heat resistance of the the orgamsm Heat resistance of the the orgamsm Heat resistance of muscles Heat resistance of the cilitated epithelium Salt resistance of the organism Salt resistance of the organism Quinine resistance of the organism
cases when neuro-humoral effect on cells was not possible. Thus, the heat resistance changes caused by heat acclimation were observed in isolated ciliated epithelium cells of molluscs (Friedrich, 1967; Lagerspetz and Dubischer, 1966; Skholl, 1971). Hence, in addition to the hormonal regulatory mechanism of the heat resistance of cells, at least in ciliated epithelium, there exists some other intracellular mechanism, independent of the organism. The response o f the population as a functional system The data obtained in this study on the rate of development of salamander larvae support the conclusion made in Part I that to a change in environmental temperature a population responds as a functional system. Individual responses of single families of the population are to a great extent co-ordinated. To evaluate the extent of this co-ordination, we used a systemal component of variability in responses of single families of the population to changes in environmental temperature (60"syst). 2 It shows the share of phenotypical variability associated with general relationship between the responses of single families (or individuals) of the population to environmental changes. The less stochastic the response of the population, the higher the value of systemal component of variability. In this study on the rate of development the systemal component in the response of the population is equal to 0.46 and 0.37 for the effect of heat and cold respectively, which is lower than in the study
Systemal component of the response of the population 4
Authors 5
0.60-0.63 0.654).94
Ushakov and Pashkova (this study) Ushakov and Pashkova (this study)
0.37-0.46 0.70-0.91 0.59
Ushakov and Pashkova (this study) Glushankova and Chernokozheva, Ushakov et aL, 1972
0.81
Irlina, 1960
0.49
lrlina, 1960
0.77
Sukhanova, 1962
0.21-0.85 0.36-0.66
Dregolskaya, 1977; Ushakov et al., 1977a Ushakov et al., 1977a
0.34-0.92
Ushakov et al., 1977a
0.03-0.46
Ushakov et al., 1977a
0.23-0.88
Ushakov et al., 1977a
0.59-0.79
Pashkova et al., 1981
0.65-0.90 0.42
Ushakov and Pashkova, 1984 Skholl, 1971
0.81
Gause, 1939
0.56
Smaragdova and Gause, 1939
0.50
Smaragdova and Gause, 1939
on the heat resistance of muscles and their contractile models made on the same families of salamanders (0.60-0.94). The data are shown in Table 4. The values of this component were as high in the study on the heat resistance of muscles of Rana temporaria tadpoles which developed at elevated temperature (Glushankova and Chernokozheva, 1965; Ushakov et al., 1971, 1972). High values of systemal component were also obtained by the studies on the organismal heat resistance of tadpoles of the same families (Ushakov et al., 1972) and on three species of protozoans (Irlina, 1960; Sukhanova, 1962). Thus, when the animals develop under thermal conditions, diflerent from the optimal ones, the population responds to thermal changes as a functional system. Under such conditions the individual (stochastic) component of the populational response is very small. The data of several earlier studies allow systemal component of variability during short-term temperature acclimation to the determined (Table 4). At earlier stages of thermal acclimation it is very small or even absent, but in the course of acclimation it significantly increases (Table 4). The data of the studies by Gause (1939) and Smaragdova and Gause (1939) allow determination of the systemal component in the populational response of infusorians to changes in the chemical composition of environment. In this case too, the values of this component are rather high (Table 4). These data allow the conclusion that the population
B. P. USHAKOVand I. M. PASHKOVA
180
responses as a functional system, not only to changes in temperature, but also in chemical composition of the environment. Thus, the same pattern of populational response is observed during the study of such different physiological characteristics as heat resistance, salt resistance and the rate of development. It should be pointed out that all these characteristics are directly or indirectly related to the survival of individuals under variable environmental conditions. Systemal component of variability shows the extent to which the response of each single individual follows the general relationship characteristic of the population. High values of systemal component serve as evidence that the responses of single individuals are to a great extent determined by their genetic programme. Apparently, these responses are of epigenotypic origin. All the individuals of the population show a distinct relation between an increase in resistance to injury caused by environmental factors and its initial level. This relation is the reason for phenotypic masking of genotypic differences between the individuals during changes in ecological factors (Ushakov et al., 1977a, 1977b; Ushakov, 1977, 1982). Phenotypic masking of genotypic differences results in a decrease in the genetic effectiveness of selection, which facilitates maintenance of constant genetic structure of the population. However, it should be remembered that such a response of the population does not involve interaction of individuals, therefore the question of whether or not a population is an integral system (as is, for instance, organism or biocoenosis) remains so far open. REFERENCES
Chernokozheva I. S. (1970) Effect of the rearing of tadpoles in thyroxine solutions on the thermostability of their muscle tissue. Tsitologiya 12, 119-123. Dregolskaya I. N. (1977) Heritabi]ity coefficient of the heat resistance of the organism of Hydra oligactis Pall. under optimal cultivation temperature and after heat acclimation. Zh. obshch. Biol. 38, 440~45. Dzhamusova T. A. (1971) Changes in the heat resistance level of different frog muscles caused by thyroxine injections. Ekologiya 5, 53-58. Fisher R. A. (1954) Statistical Methods for Research Workers. Oliver & Boyd, London. Friedrich L. (1967) Experimentelle Untersuchungen zum Problem zellul/irer nichtgenetischer Resistenzanderungen bei der Miesmuschel Mytilus edulis L. Kieler Meeresforsch. 23, 105 126. Gause G. F. (1939) The adaptation of Paramecium aurelia to the increased salinity of the medium. Zool Zh. Ukr. 18, 631~41. Glushankova M. A. and Chernokozheva I. S. (1965) The thermostability of muscle fibres and some tissue proteins in the tadpoles of Rana temporaria L. in connection with cultivation temperature. In Heat Resistance of Cells of Animals (Edited by Ushakov B. P.), pp. 153 160. Academy of Sciences, Moscow. lrlina I. S. (1960) Changes in thermostability of some free-living Protozoa under the influence of preliminary temperature regime. Tsitologiya 2, 227 234.
Koch A. L. (1966) The logarithm in biology. 1. Mechanisms generating the log-normal distribution exactly. J. theoret. Biol. 12, 276-290. Lagerspetz K. and Dubischer J. (1966) Temperature acclimation of the ciliary activity in the gills of Anodonta. Comp. Biochem. Physiol. 17, 665-671. Lerner J. M. (1954) Genetic Homeostasis. Oliver & Boyd, London. Pashkova I. M. (1965) Relationships between muscle thermostability and activity of thyroid gland of Rana temporaria L. at different seasons of the year. In Heat Resistance of Cells o f Animals (Edited by Ushakov B. P.), pp. 82-89. Academy of Sciences, Moscow (summary in English). Pashkova I. M. (1967) Analysis of seasonal changes in thermostability of frog muscles, in The Cell and Environmental Temperature (Edited by Troshin A. S.), pp. 225.-231. Pergamon Press, Oxford. Pashkova I. M., Amosova I. S., Skholl E. D., Chernokozheva I. S. (1981) Changes in the response of a population of Misgurnus fossilis L. to thermal selection after a short-term heat acclimation. Zh. obshch. Biol. 42, 556-563. Skholl E. D. (1971) A correlation between the initial heat resistance level of isolated ciliated epithelium of Mytilus galloprovincialis and its change caused by heat effect. Ekologiya 6, 69 73. Skholl E. D. and Pasynkova R. A. (1982) The assumed and realized in F~ result of selection for heat resistance of myofibrillar ATPase in the field mouse. Genetika 18, 956-959. Smaragdova N. P. and Gause G. F. (1939) A comparative investigation of adaptation of Paramecium caudatum to the increased salinity of the medium and to quinine solutions. Zool. Zh. Ukr 18, 642-655. Sukhanova K. M. (1962) Temperature adaptations in Opalina ranarum Ehrenberg (Opalinidae) during its life cycle. Tsitologiya 4, 644-651. Ushakov B. P. (1977) The effect of heat acclimation on the intensity and the genetic effectiveness of selection caused by heating. J. therm. Biol. 2, 177-182. Ushakov B. P. (1982) Temperature adaptations of animals and their significance in evolution. Usp. sovren. Biol. 93, 302--319. Ushakov B. P., Glushankova M. A., Salmenkova E. A. and Chernokozheva I. S. (1971) The relation between the initial level of heat resistance of cells and proteins and the direction of phenotypical shift caused by temperature adaptation of frog tadpoles. Ekologiya 3, 9--18. 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., Amosova I. S., Chernokozheva I. S., Dregolskaya 1. 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 1. S., Chernokozheva 1. 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. (1983) The analysis o1 the increase in heat resistance of Daphnia magna in the process of cultivation. Genetika 19, 1251-1256. 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. therm. Biol. 9, 303 309.