Temperature acclimation in Tribolium and Musca at locomotory, metabolic, and enzyme levels

Insect Physiol., 1971, Vol. 17, pp. 2205 to 2219, Pergamon Press. Printed in Great Britain

TEMPERATURE ACCLIMATION IN TRIBOLIUM AND MUSCA AT LOCOMOTORY, METABOLIC, AND ENZYME LEVELS* R. L. ANDERSON? Department

and J. A. MUTCHMOR

of Zoology and Entomology, (Received

Iowa State University, Ames, Iowa 50010

17 April

1971)

Abstract-The results of this study indicate that metabolic acclimation precedes locomotory acclimation. The effect of acclimation on the two species varies with respect to enzyme (Mga+-activated ATPase) activity. Tribolium confusum shows acclimative differences in ATPase activity throughout its normal temperature range. Enzyme activity of warm-acclimated beetles transferred to a low temperature showed little acclimation after more than 900 hr at the new temperature. This suggests that a rapid acclimation of ATPase activity is not essential to this insect and, perhaps, that other mechanisms are more important. The ATPase activity of Musca domestica shows no substantial acclimative differences in the temperature range of 15 to 30°C but, at low temperatures near the chill-coma temperature, acclimative differences are evident. These differences increase the insect’s sensitivity to temperature and are similar to interspecific differences found with the same enzyme in other insects. The results of this study also suggest that the rate of acclimation to temperature is dependent on the thermal environment of the insect being studied. Insects that live in a relatively stable thermal environment, like T. confusum, seem to acclimate slowly. M. domestica, which lives in a highly variable thermal environment, acclimates more rapidly. INTRODUCTION

THERE is substantial evidence, based on differences in chill-coma temperatures, respiration rates, and enzyme activity that insects show both interspecific (adaptive) and intraspecific (acclimative) differences in response to temperature. Adaptive differences have been correlated to the normal habitat temperatures of the insect, and acclimative differences have been shown to be influenced by the temperature history of the insect. Much of this work has been reviewed by BURSELL(1964) and MUTCHMOR(1967). Although these differences are well documented, there have been no integrative studies on the rate of acclimation in insects. Workers who do report rate* Journal Paper No. J-6798 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 1647. This work was supported by the United States Department of Agriculture, Agricultural Research Service, Market Quality Research Division, Grant No. 12-14-100-8017 (51). t Present address: Department of Biochemistry, Health Sciences Center, University of Louisville, Louisville, Kentucky 40202. 2205

2206

R. L.

ANDERSON

AND

J. A. MUTCHMOR

of-acclimation data usually are concerned only with one aspect of the temperature effects. Our study is the first known attempt to correlate the rate of temperature acclimation at the locomotory, metabolic, and enzyme activity levels in insects. Rate of acclimation at the locomotory level has been studied by BELEHRADEK (1935), who indicated that differences in chill-coma temperatures occurred after 3 days at a new temperature. In the blowfly, Calliphora erythrocephala, the tsetse fly, Glossina palpalis, and the bedbug, Cimex lectularis, chill-coma differences could be seen after 20 hr at a new temperature (MELLANBY,1939). Mellanby also reported that, for the Oriental cockroach, Blatta orientalis, 2 to 3 days were necessary for acclimation after transfer from 30 to 15°C. COLHOUN(1960) transferred the German cockroach, Blattella germanica, from high to low temperatures and from low to high temperatures. He found that the rates of acclimation depended on the size and range of the temperature change. For example, if the insect was transferred from 25 to 15°C acclimation was complete within 2 to 3 hr. A transfer from 35 to 25°C or from 35 to 15°C required more than 24 hr for completion. A similar relationship was found in transfers of insects from low to higher temperatures. The transfer of insects from 15 to 25°C was complete in 16 hr, from 25 to 35°C in 20 hr, and from 15 to 35°C in 40 hr. EDWARDS(1958) maintained the confused flour beetle, Tribolium confusum, at 18, 30, and 38°C and measured the oxygen consumption of the beetles at a series He examined the temperature-rate (T-R) curves of beetles from of temperatures. the three maintenance temperatures with respect to differences in the beetles’ sex, weight, and water content. As a part of his study, insects from 30°C were transferred to 18°C and to 38°C. He then recorded the oxygen consumption at daily intervals and reported that no acclimation occurred. After 24 hr, the rate for beetles transferred to 18°C was the same as that of beetles maintained and tested at 18°C. The transfer of beetles from 30 to 38°C resulted in an increase in oxygen consumption that lasted for 3 days before returning to the baseline rate established for insects maintained and tested at 38°C. HUNTER (1968) studied the effect of temperature on oxygen consumption in several species of Drosophila from different thermal habitats. She found that stenothermal flies showed no adaptive response to temperature, but that eurythermal flies did show an adaptive response to temperature. There have been few studies on the rate of acclimation of enzymes in insects. MUTCHMORand RICHARDS(1961) maintained the American cockroach Periplaneta americana, at 30 and 10 or 15°C for periods of 3 to 10 days, and Tendwio molitor at 35 and 15°C for a similar time before they tested for Mg2+-activated adenosine triphosphatase (ATPase) activity. APPLEBAUMet al. (1964) transferred T. molitor from 23 to 13°C and then studied the activity of the midgut proteases and amylases. They found a lag of about 4 days after transfer, during which the protease activity was lower than in insects maintained and tested at 23°C. The activity then increased and was at its maximum in 8 days. They could find no compensation in midgut amylase activity.

TEMPERATURE ACCLIMATION

IN TRIBOLIUM

AND MUSCA

2207

The objective of our research was to explore the rate of acclimation, at the locomotory, metabolic, and enzymic levels of T. confusum, a cold-intolerant species, and of M. domestica, a cold-tolerant species. Change in chill-coma temperature was used as a measure of acclimation. The usual method for the determination of chill-coma is to watch the insect respond to a mechanical stimulus, such as a gentle prodding, at a series of low temperatures. After acclimation, the cold-acclimated insect will exhibit movement at a lower temperature than an insect not cold-acclimated. It seemed very likely that the acclimative process involved metabolic adjustments, so a measurement of oxygen consumption was made. In the enzyme study, the Mg2+-activated ATPases of insect muscle were chosen because these enzymes are considered to be one of the chief immediate sources of energy needed for contraction and the rates of acclimation of these enzymes might reasonably be related to the rate of acclimation at the locomotory level. MATERIALS

AND METHODS

Rearing and acclimation T. confusum was reared and maintained on a diet of 50% white flour and 50% corn meal supplemented with 5% brewers’ yeast. The beetles were cultured in battery jars or in 1 pint mason jars at 30°C or 18°C for 6 months before testing. Adult M. domestica 1 day after emergence were obtained from the Iowa State University Insectary where they had been reared at 26°C. The flies were maintained at 30 or 15°C on a diet of 10% sucrose for 4 days before the females were removed for experimentation. Acclimation was evidenced by a difference in chillcoma temperature after the 4 day period. In all rearings, the photoperiod was 12 hr light and 12 hr dark per day. The relative humidity was not controlled, but varied between 40 and 70 per cent. Locomotion

Chill-coma temperatures of 9 to 10°C for beetles acclimated to 18°C and of 3 to 4°C for flies acclimated to 15°C were determined by the method of ANDERSON and MUTCHMOR(1968). Samples of beetles acclimated to 30 and 18°C were each transferred to the opposite environment, and samples of flies acclimated to 30 and 15°C likewise were transferred to opposite environments. Subsamples of 3 beetles or 4 flies then were removed from the transferred samples and tested at 11 or 4*5”C, respectively, for 1 hr periods for presence or absence of insect activity. The hour was divided into four 900 set segments, and the number of insects active in each time segment was recorded. Hour tests of subsamples were made at regular intervals for the first 50 hr and then at irregular intervals up to 383 hr after transfer of the samples to opposite environments. Oxygen consumption Baseline respiratory rates for the two species at their respective acclimation temperatures had to be established before the rate of acclimation could be determined.

2208

R. L. ANDERSON ANDJ. A. MUTCHMOR

Respiration was measured with Gilmont differential syringe manometers in a water-bath controlled to rt O*l”C. Ten unsexed beetles or 1 female housefly of known age were placed in Warburg flasks and allowed to equilibrate for a minimum of 1 hr before readings were begun, Readings were made at 1 hr intervals for a maximum of 6 hr/sample. The insects then were weighed to the nearest 0.1 mg on a torsion balance, and oxygen consumption was expressed as ,IJ 0, consumed/mg of insect per hr. Preliminary tests indicated that, if flies were allowed free movement in the flasks, the respiration rates would vary considerably. To reduce movement, flies were confined in small Nylon sacs in the respirometers. Also, because of their relatively short life span, flies less than 8 days old were used in all tests. Enzyme study Homogenate preparation. Samples of 20 to SO unsexed T. confusum adults were lightly anaesthetized with CO, and weighed on a torsion balance to the nearest 0.1 mg. The insects were homogenized at 0°C in a Potter-Elvehjem homogenizer with a small known amount of cold deionized water. Final homogenate concentrations for T. confusum were 50 mg of tissue/ml. Because of the larger size of the housefly, it was possible to use thoraces rather than whole insects. For each test, samples of 6 to 8 thoraces were homogenized in a manner similar to that used with T. confusum. Final homogenate concentrations were 30 mg of tissue/ml. Enzyme activity determinations. ATPase activity determinations followed the procedures and reaction mixture used by MUTCHMORand RICHARDS(1961). Reaction mixtures were O-2 ml of 0.02 M MgCla.6 H,O; 1.56 ml of O-038 M sodium barbital buffer, pH 7.8; and 0.02 ml of 0.02 M ATP as the disodium salt (Nutritional Biochemical Co.). The amount of phosphorous in replicate tests and reaction blank were determined using a Coleman Universal spectrophotometer (Model 14) at 600 w. Values were corrected for blank, replicates averaged, and results expressed as pg P/mg insect per min. The initial rate of change was determined and was proportional to fresh weight. Similar results have been previously obtained by KENNEY and RICHARDS(1955) and MUTCHMOR and RICHARDS(1961). RESULTS

Locomotion When warm-acclimated insects were transferred to the lower-temperature environments (18°C for beetles, 15°C for flies) and subsamples thereafter were subjected to 1 hr periods at the test temperatures (11 “C for beetles, 4*5”C for flies), all insects in the subsamples initially were inactive during all four 900 set segments As the insects became cold-acclimated, activity of the hour at test temperatures. first occurred in the fourth segment of the hour at test temperatures. As acclimation continued, activity eventually began to occur in the first segment of the hour in which subsamples were subjected to the test temperatures. The insects were

TEMPERATURE ACCLIMATION

IN TRIBOLIUM AND MUSCA

2209

considered cold-acclimated when all insects in the subsamples at test temperatures were active in the first segment of the hour tests. When cold-acclimated insects were transferred to the higher-temperature environment (30°C) and subsamples thereafter were subjected to the test temperatures, all insects in the subsamples initially were active during all four segments of the 1 hr tests. As the insects became acclimated to the warmer environment, lack of activity in the subsamples subjected to the test temperatures first occurred in the first 900 set segment of the hour and, as acclimation continued, eventually in the fourth segment of the hour at test temperatures. The insects were considered acclimated to the warm environment and to have lost their cold tolerance when all insects in the subsamples subjected to the test temperatures were inactive in the fourth segment of the hour tests. Locomotory ability was recorded in all the time intervals. The number of insects active in the appropriate time intervals is reported in Table 1. TABLE I-LOCOMOTORY ACCLIMATION IN T. confusum

AND

M. domestica

Transfer Time after transfer * (hr) 0

5 10 15 20 25 30 40 53 97 173 383

Warm to cold? T. confusum

M. domestica

Cold to warm: T. confusum

M. domestica

2 1 3

0 0 0 -

-

0 0 0 0 1 1 1 2

1 1 1 3 1 0 1 1

* Zero to 40 hr are 1 hr, others exact. t First time period (see Materials and Methods). $ Fourth time period (see Materials and Methods).

In the transfer of T. confusum from 30 to lS”C, acclimation was evident after about 50 hr but was not completed in 383 hr when the experiment was terminated. In the transfer of 18°C beetles to 3O”C, acclimation was seen in less than 20 hr in the fourth time interval. Acclimation in the fourth interval was not complete in 383 hr. Again, the experiment was terminated at this point. In the transfer of 30°C M. domestica to 15°C acclimation to 15°C was evident at about 20 hr and was complete in 40 hr. In the transfer of 15°C flies to 3O”C,

2210

R.L.

ANDERSONANDJ.A.MUTCHMOR

acclimation was evident as early as 5 hr in the fourth interval and was complete within 25 hr. Oxygen consumption Average rates of oxygen consumption at 30 and 18°C for T. confusum and at 30 and 15°C for M. domestica are in Table 2. These averages are indicated in Figs, 1 and 2 by horizontal lines labelled 30 at 30 and 18 at 18 for T. confusum, and 30 at 30 and 15 at 15 for M. domestica. These constitute the oxygen consumption baselines. TABLE 2-AVERAGE

fj~l O,/mg per hr) VALUES FOR T. confusum AND M. domestica Acclimation temperature 0-J)

No. of hourly readings

Average values + 1 S.D.

T. confusum

18 30

68 77

O-742 f O-176 2.199 + 0.476

M. domestica

15 30

86 46

O-841 + O-338 3.060 + 0.882

Species

Each point in Fig. 1 is the average of from 4 to 10 hourly readings. After the transfer of the beetles from 30 to 18°C oxygen consumption initially increased. This was not the expected response. The increase reached a peak after about 18 hr, and then the consumption began to decrease. Consumption reached the baseline (18 at 18) rate about 60 hr after transfer. The transfer of insects from 18 to 30°C also produced an unexpected result. In this instance, there was a decrease in oxygen consumption for the first 20 hr followed by an increase to the baseline rate in about 80 to 90 hr. Fig. 2 illustrates the oxygen consumption of 15 and 30°C-acclimated M. domestica following their transfer to 30 and 15”C, respectively. Each point is an average of from 4 to 18 hourly readings. After the transfer of insects from 30 to 15X!, there was an immediate drop in oxygen consumption from the 30°C baseline to the 15°C baseline. No obvious increase in oxygen consumption similar to that found in T. confvsum occurred. After the transfer of insects from 15 to 3O”C, there was a slow increase in oxygen consumption from the 15°C baseline toward the 30°C baseline. The 30°C baseline was crossed between 12 and 15 hr. The oxygen consumption continued to increase in an overshoot and finally reached a maximum at about 25 to 30 hr. A slow decrease in oxygen consumption then took place, until the baseline rate was reached between 70 and 80 hr. Enzyme study Baseline rates of the Mg 2+-activated ATPase are given in Table 3.

TEMPERATURE ACCLIMATION

28-

IN TRIBOLIUM

AND MUSCA

Tconfusum

l

0=301018 .=I* to30

. .

2A* 23-

o

c

l

t .*

2;

;

o &

w

. .

.

k

r

.

301130 *

.

1.6- 3

FIG. 1. Rate of acclimation as indicated by measurement of the rate of oxygen consumption by T. confusum. Ordinate: Oxygen consumption in pl/mg insect per hr. Abscissa: Time in hours at the indicated transfer temperature. Thirty at 30 and 18 at 18 indicate baseline rates.

M.domestica

.

40-

0:3oto15 l=15to30

=2

. .

l. ..

.

l

32-

%

:

l *

.

l *-

l*

Fd

.

ZA-

. .

.

.

l *

30at 30 .

. .

.

FIG. 2. Rate of acclimation as indicated by measurement of the rate of oxygen consumption by M. domestica. Ordinate: Oxygen consumption in pl/mg insect per hr. Abscissa: Time in hours at the indicated transfer temperature. Thirty at 30 and 15 at 15 indicate baseline rates.

2211

2212

R.L.

ANDERSON

ANDJ.

A.MUTCHMOR

Statistical examination of the rates for M. domestica indicated that the enzyme rates of lYC!-acclimated flies tested at 15°C were not different from those of 3O”Cacclimated flies tested at 15°C. A similar result was found for 30”C-acclimated flies. This indicated that acclimation did not occur between 1.5 and 30°C. Because of the absence of any acclimative difference in enzyme activity, no rate of acclimation study with respect to enzyme activity was done with this species, TABLE 3-ABASE

RATES &g P/mg insect per min) OF WARM- AND COLD-ACCLIMATED T. confusum AND M. domestica Acclimation temperature W)

Test temperature CC)

No. of tests

T. confusum

18 18 30 30

18 30 30 18

12 12 13 12

0.274 + 0.062 l-058 + 0.234* 0.697 + 0.098 * O-195 rt O-082

M. domestica

15 15 30 30

15 30 30 15

18 21 15 13

1.326 5.280 s-730 1.566

Species

Average values f 1 S.D.

III0.578 f 0.324 + o-955 + 0.232

* P
A similar examination of the enzyme rates for T. confusum showed a significant difference (PC O-01) between the lS”C-acclimated beetles tested at 30°C and the 30”Gbeetles tested at 30°C. Because of this difference, beetles acclimated at 30°C were transferred to lS”C, and homogenates were tested at 30°C. The results of this experiment are shown in Fig. 3. As the insects became acclimated to 18”C, the enzyme rate was expected to approach the baseline rate previously established for 18”C-acclimated insects tested at 30°C (upper baseline, Fig. 3). But, there was an initial decrease in enzyme activity to levels below the 30°C baseline. This reduced rate existed for about 150 hr. A slow increase in activity was then evident. After the insects had been at the new temperature for about 900 hr, the enzyme rate had reached the 30°C baseline. A determination was made at 1500 hr (62.5 days; not shown in Fig. 3), but the rate was still near the 30°C baseline. The ATPase activity in these two insects indicates a basic difference in response to temperature after acclimation. To obtain more information about the enzymic response to temperature, rate determinations were carried out at a series of temperatures, extending from near the chill-coma temperatures to about 38°C with homogenates of cold- and warm-acclimated insects. The results of these tests are shown in Fig. 4. Fig. 4 indicates that the general relationships shown in the statistical analysis extend to higher and lower temperatures in both species. Near the chill-coma temperature, a difference, however, could be seen in the pattern of enzyme activity. To define these differences, temperature coefficients were

TEMPERATURE

ACCLIMATION

IN

TRIBOLIUM

AND MUSCA

2213

T confusum

18a130

-LOO-

i’

.60-

.40-

, ,t 30a,30

1

BO-

t

t

t

t ’

t

t

t

t

tt’

I’ t+ t

+ 1

.20-

I

2’0

40

I

60

I

I

I

300

lb0

1

600

900

HOURS

FIG. 3. Rate of ATPase acclimation in T. confusum. Ordinate: Enzyme rate in pg P/mg insect per min. Abscissa: Acclimation time, in hours, at 18°C; log-scale. Eighteen at 30 and 30 at 30 indicate baseline rates. Vertical lines at each point indicate one standard deviation.

Motsmostica 3-

*=15'

-2-

-3-

32

33

34

35

36

l/TAO4

32

33

34

35

36

FIG. 4. Comparison of the effect of temperature on the rate of ATPase activity in cold- and warm-acclimated M. dontestica and T. confusum. Ordinate: Natural logarithm of rate @g P/mg insect per min) of ATPase activity. Abscissa : Reciprocal of absolute temperature (X 103. The small numbers above the abscissa are reference temperatures, in “C.

2214

R. L.

ANDERSON AND J. A. MUTCHMOR

calculated for the temperature range extending from the chill-coma temperature to a temperature 5” higher. The results of these calculations are summarized in Table 4. TABLE &TEMPERATURE

COEFFICIENTSOF WARM-

AND COLD-ACCLIMATED

T. confusum AND M. domestica

temperature

Temperature range

(“C)

(“C)

Temperature coefficient (calculated)

T. confusum

30 18

11-16 11-16

31,000 37,000

M. domestica

30 15

S-10 S-10

40,500 45,500

Acclimation Species

A comparison of the p-values over similar physiological temperature ranges indicates that the cold-acclimated insects of both species have a higher value than the warm-acclimated insects. The higher p-values correspond to a steeper slope and represent a greater sensitivity to temperature change. This is the same relationship found in interspecific comparisons in other insects (MUTCHMOR and RICHARDS, 1961). The results of our study are summarized in Table 5. TABLES-SUMMARY

OF THE RATE OF TEMPERATUREACCLIMATION (in hr) OF T. confusum AND M. domestica WITH RESPECTTO LOCOMOTION, OXYGEN CONSUMPTION, AND ATF’AsE ACTIVITY Species

Transfer

Locomotion

Respiration 60 90

T. confusum

30 to 18 18 to 30

383 383

M. domestica

30 to

15

40

10

15 to 30

25

80

ATPase 1500

No

test

DISCUSSION

It already has been indicated that insects show adaptive and acclimative differences in chill-coma temperature, respiration, and enzyme rates. These differences can be correlated with the normal habitat temperatures and with the temperature history of the insect. To understand the interspecific differences found in this study, it is important to consider differences between the normal habitats of the two test species. T.confv.wm is a cold-intolerant, long-lived, general feeder on farinaceous foods. It is one of the worst insect pests of prepared cereal foods. It is usually found in granaries, mills, warehouses, and in grain shipments. The insect essentially does

TEMPERATURE ACCLIMATION

IN TRIBOLIUM

AND MUSCA

2215

not fly; its only means of self-dispersal within the grain or stored products is by walking. In this insect’s normal habitat, the temperature variation is minimal, at least on a short-term basis. COTTON(1963) measured the temperature at the centre of a 2740 bushel bin of wheat during a Kansas winter and found that it took 2 months for the temperature to drop from 25 to 18°C. The results of our study indicate that about 17 days were required for locomotory acclimation to occur. The insect thus would have had ample time to acclimate in conjunction with such Storage facilities in cold areas would cool more slow decreases in temperature. rapidly than those in Kansas. WATTERS(1966) reported that, in unheated warehouses in the prairie provinces of Canada, temperatures fluctuated between zero and l.S’C, depending on the severity of the outdoor temperatures. ERNST and MUTCHMOR(1969) acclimated T. confwum at 30 and 15°C and tested their disThe 30”C-acclimated insect showed persal ability as a function of temperature. no dispersal below lO”C, a temperature slightly lower than the &ii-coma temperature as determined in our study. The 15”C-acclimated insect showed some dispersal activity to 5°C. Because bulk-stored grain cools slowly and the insect is able to acclimate as the temperature changes, locomotion should be possible through two-thirds of the low temperature range normally found in the Canadian grain facilities. Presumably, dispersability after acclimation would be even less limited in storage facilities in much of the United States. Aquatic poikilotherms inhabit an environment similar to that of T. confusum with respect to thermal stability. SPOOR (1955) determined the loss and gain of heat tolerance in the fresh-water crayfish, Astaczrs. He transferred crayfish from a maintenance temperature (23-24°C) to 4°C and measured the loss of heat tolerance. After 24 days at 4°C there was no change in the heat tolerance. Gradually, the heat tolerance decreased until, at 16 days, the crayfish responded as a 4°C animal. Spoor also transferred crayfish from the maintenance temperature to 12°C and found that 14 days were necessary for the crayfish to attain the same heat tolerance as crayfish maintained at 12°C. In another study, MCLEE~E (1956), using the American lobster, Homarus americanus, found that lobsters transferred from 14.5 to 23°C required approximately 22 days to re-acclimate. M. domestica is a relatively short-lived, cold-tolerant insect with cosmopolitan distribution. If acclimation to temperature is to be of adaptive value to this insect, it must occur in a relatively short time. In our study, locomotory acclimation to cold took approximately 40 hr. A lag of about 20 hr occurs after the change in temperature from 30 to 15°C before locomotory acclimation changes can be detected. The lag may be due to metabolic acclimations that must occur before locomotory acclimation can be expressed. The lag may also be of importance in reducing the effect of very short-term environmental temperature changes. There have been no other studies on the rate of locomotory acclimation with M. domestica, but several studies with physiologically similar (with respect to cold tolerance) species have shown similar rates of acclimation. MUTCHMOR and RICHARDS(1961), THIESSENand MUTCHMOR(1967), and ANDERSONand MUTCHMOR (1968) acclimated the American cockroach, P. americana, for a minimum of 3 days

2216

R. L. ANDERSON AND J. A. MUTCHMOR

and found 3 to 5” difference in chill-coma temperature. THIESSENand MUTCHMOR (1967) also acclimated the housefly and could show chill-coma differences after 3 days. As already noted, in our present study, 2i days were required for locomotory acclimation. Oxygen consumption

There is little information pertaining to the rate of acclimation of oxygen consumption in insects. EDWARDS(1958) determined the oxygen consumption of T. confusum after a transfer from 30 to 18°C and from 30 to 38°C. He found that, after the 30 to 18°C transfer, the insects reached the rate characteristic of lS”Cacclimated insects tested at 18°C in 24 hr. He concluded that no respiratory compensation occurred and that only a ‘passive’ lowering of respiration had taken place. From the information in his paper, however, it is evident that the oxygenconsumption rates were determined at 1 day intervals. In our study, the oxygen consumption was determined at 1 hr intervals during the first 13 hr after transfer, and the results obtained differ from those of Edwards. The first 24 hr after a transfer from 30 to 18°C is not a period of ‘passive’ lowering of respiration. Instead, there is a substantial increase in respiration. This increase may be a metabolic compensation for the decrease in temperature, or it may be that a behavioural compensation has influenced the respiration of the insect. Although critical evidence is lacking, the latter seems to us more likely. For example, activity is a major cause of increased oxygen consumption (PROSSER,1961). An increase in activity after a transfer from a warm to a cooler environment would be a distinct advantage to a cold-intolerant insect, such as T. confusum. As the environmental temperature decreased, the increase in the insect’s activity would give it at least some chance to move away from the cooler area. EDWARDS(1958) reported, for the transfer from 30 to 38°C an overshoot that lasted for 2 to 3 days, followed by a decrease in respiration. In our study, the transfer from 18 to 30°C resulted in a decrease in respiration that lasted for approximately 24 hr. A slow increase then occurred that required 80 to 90 hr to reach the 30°C baseline. It is not possible to compare in detail the results of Edwards’ transfer of SO”C-acclimated insects to 38°C with those of our study because of the differences in temperature ranges used. Edwards reports that the 38”C-insects exhibited poor motor co-ordination, indicating a possible deleterious effect of the high temperature. The transfer of insects from 18 to 30°C does not stress the insect, but actually represents a return of the insect to a temperature nearer to its optimum. At present, it is not possible to ascribe any adaptive significance to the decrease in respiration found in the transfer of insects from 18 to 30°C. The data of Figs. 1 and 2 reveal that it is difficult to show acclimation differences with respect to oxygen consumption. To show such differences, it is necessary to determine the rate of oxygen consumption of two differently acclimated insects at But our results suggest that the insect starts to acclimate a series of temperatures. to the test temperature during the test. Naturally, this tends to obscure the results and makes interpretations difficult at best.

TEMPERATURE

ACCLIMATION

IN TRIBOLIUM

AND MUSCA

2217

Enzyme study PROSSER (1961) reported that the usual manifestation of cold acclimation or adaptation in poikilotherms is translation and rotation of T-R curves, resulting in a decreased slope. The interpretation, in terms of adaptive or acclimation advantage, is that the reduced slope would allow rate-processes in the cold-acclimated poikilotherm to be less temperature dependent. An alternative response is translation and rotation of the T-R curve in the opposite direction, resulting in an increase in the slope and, therefore, in increased temperature sensitivity, SCHOLANDER et d. (1953) measured the oxygen consumption of arctic and tropical poikilotherms and reported increases in slope in molluscs, spiders, and insects. They did not consider these changes to be adaptive; they considered only reduced temperature dependence to have adaptive value. MUTCHMORand RICHARDS(1961) measured Mg2+ -activated ATPase activity and reported greater slopes in coldtolerant insects than in cold-intolerant insects. Graphs of the ATPase baseiine rates (not shown here) or an examination of the rates indicates that, with T. confusum, there is a translation and rotation of the cold-acclimated enzyme activity to the left. This shift indicates that the process has become more sensitive to temperature changes. Through this temperature range, a small increase in temperature would allow a substantial increase in ATPase rate. M. domestica baseline rates show a different response from that of T. confusum. The warm- and cold-acclimated flies show only slight differences in enzyme activity. Therefore, with this insect, a small increase in temperature between 15 and 30°C would not substantially affect the enzyme rates. To obtain more information about the ATPase activity throughout the normal range of habitat temperatures of the test insects, a series of enzyme rate determinations was made at temperatures extending from the chill-coma temperatures of each insect to 38°C. As indicated previously, these results are shown in Fig. 4. To compare the ATPase rates through similar physiological temperatures, p-values were calculated in the range from the chill-coma temperature to the chill-coma temperature plus 5”. In this low temperature range, the relationships for T. confusum found in the 18 to 30°C range persist, with the cold-acclimated beetles having higher p-values and an increased temperature sensitivity. But with iVl. domestica, relationships found in the 15 to 30°C temperature range have changed in the lower range. At the low temperatures, the cold-acclimated fly has a substantially higher p-value, thus indicating a greater temperature sensitivity and dependence. The results we found confirm the interspecific differences found by MUTCHMOR and RICHARDS(1961). The more cold-tolerant insect, M. domestica, has a greater temperature sensitivity than does T. confusum, in the low temperature range. Also, it can be assumed that, through the temperature range of 15 to 30°C J!. domestica has ample enzyme activity to serve the needs of the fly irrespective of temperature, and it is only at the lower temperatures that differences can be seen and are important. T. confusum, which is exposed to only slow temperature changes, shows a temperature dependence throughout its normal temperature range.

2218

R. L.

ANDERSON

ANDJ. A. MUTCHMOR

The differences in p-values found at the low temperatures also are reflected in gross observations of both insects in chill-coma as they are slowly warmed. M. domestica recovers from chill-coma more quickly than does T. confuwm, and its movements become very vigorous far more quickly than do those of T. confusum. The biochemical events that occur during metabolic acclimation and result in locomotory acclimation have not been determined in insects. Work with other poikilotherms (HOCHACHKA,1967; RAO, 1967) h as outlined changes in metabolic pathways and described isozymic changes with acclimation. MILLS and COCHRAN (1967) found four different ATPases in the thoracic muscles of P. americana, each showing a different temperature tolerance. They held homogenates at 0°C for 2, 4, and 6 hr and found that ATPase IV lost 24, 67, and 100 per cent of its activity after exposure to the low temperature for the three time intervals. ATPase II showed no loss in activity during the same low temperature exposure. The other two ATPases were intermediate in loss of activity. The events that occur upon transfer of these two species from one temperature to another may be summarized: Initially, there is a metabolic adjustment followed by a change in the locomotory ability at the new temperature. Behavioural responses to the temperature change may be superimposed on the metabolic adjustment, as shown by T. confusum. Baseline Mga+ -activated ATPase activity rates

indicated that, if the temperature change persists for a relatively long time, changes in the enzyme itself occurs. This time period is greater than 1500 hr. Therefore, an acclimative process that proceeds from the metabolic and locomotory levels and ends with changes at the enzymic level would be consistent with the results of this study. Also, if one may say so from a rather limited number of species, the greatest rates of acclimation are found in species that carry out their life process in highly variable environments. Those functioning in thermally stable habitats acclimate more slowly.

REFERENCES ANDERSON R. L. and MUTCHMOR J. A. (1968) Temperature acclimation and its influence on the electrical activity of the nervous system in three species of cockroaches. J. Insect Physiol. 14, 243-251. APPLEBAUM S. W., JANKOVIC J., GROZDANOVIC J., and MARINKOVIC D. (1964) Compensation for temperature in digestive metabolism of Tenebrio molitor larvae. Physiol. Ziiol. 34, 90-96.

BELEHRADEK J. (1935) Temperature and Living Matter. Protoplasma-Monographien. Vol. 8. BURSELL E. (1964) Environmental aspects: Temperature. In The Physiology of Insectu (Ed. by ROCKSTEIN M.), 1. Academic Press, New York. COLHOUN E. H. (1960) Acclimation to cold in insects. Entomologia exp. uppl. 3, 27-37. COTTON R. T. (1963) Pests of Stored Grain and Grain Products. Burgess, Minneapolis. EDWARDS D. E. (1958) Effects of acclimation and sex on respiration and thermal resistance in Tribolium (Coleoptera: Tenebrionidae). Can. J. Zool. 36, 363-382. ERNSTS. H. and MUTCHMOR J. A. (1969) Dispersal of three species of grain beetles as a function of thermal acclimation, storage temperature,and larval size. J. stored Prod. Res. 5, 407-412.

TEMPERATURE ACCLIMATION IN

TRIBOLIUM

AND MUSCA

2219

HOC~ACHKAP. W. (1967) Organisation of metabolism during temperature compensation. In Molecular Mechanisms of Temperature Adaptation (Ed. by LADD PROSSER C.), pp. 177-203. American Association for the Advancement of Science, Washington, D.C. HUNTERA. (1968) Effects of temperature on Drosophila-IV. Adaptation of D. immigrans. Comp. Biochem. Physiol. 24, 327-333. KEXNEY J. W. and RICHARDSA. G. (1955) Differences between leg and flight muscle of the giant water bug, Lethocerus americanus. Ent. News 66, 29-36. MCLEESE D. W. (1956) Effects of temperature, salinity and oxygen on the survival of the American lobster. J. fish. Res. Bd Can. 13, 247-272. MELLANBYK. (1939) Low temperature and insect activity. Proc. R. Sot. (B) 127, 473-487. MILLS R. R. and COCHRAND. G. (1967) Adenosinetriphosphatases from thoracic muscle mitochondria of the American cockroach. Camp. Biochem. Physiol. 20, 919-923. MUTCHMORJ. A. (1967) Temperature adaptation in insects. In Molecular Mechanisms of Temperature Adaptation (Ed. by LADDPROSSJXR C.), pp. 165-176. American Association for the Advancement of Science, Washington, D.C. MUTCHMORJ. A. and RICHARDSA. G. (1961) Low temperature tolerance in insects in relation to the influence of temperature on muscle apyrase activity. J. Insect Physiol. 7, 141-158. PROSSERC. L. (1961) Temperature. In Comparative Animal Physiology (Ed. by PROSSER C. L. and BROWN F. A. JR.), 2nd ed., pp. 238-284. Saunders, Philadelphia. RAO K. P. (1967) Biochemical correlates of temperature acclimation. In Molecular Mechanisms of Temperature Adaptation (Ed. by LADD PROSSERC.), pp. 227-244. American Association for the Advancement of Science, Washington, D.C. SCHOLANDER P. F., FLAGG W., WALTERSV., and IRVINGL. (1953) Climatic adaptation in arctic and tropical poikilotherms. PhysioZ. ZoX 26, 67-92. SPOOR W. A. (1955) Loss and gain of heat tolerance by the crayfish. Biol. Bull., Woods Hole 108, 77-87. THIESSENC. I. and MUTCHMORJ. A. (1967) Some effects of thermal acclimation and muscle apyrase activity and mitochondrial number in Periplaneta americana and Musca domestica. J. Insect PhysioZ. 13, 1837-1842. WATTERSF. L. (1966) The effects of short exposures to subthreshold temperatures on subsequent hatching and development of eggs of Tribolium confusum Duval (Coleoptera, Tenebrionidae). J. stored Prod. Res. 2, 81-90.