A temperature-induced mitochondrial cycle in the earthworm Eisenia foetida (Savigny)

J. therm. Biol. Vol. 16, No. 4, pp. 217-221, 1991

0306-4565/91 $3.00 + 0.00 Copyright © 1991 Pergamon Press pie

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A TEMPERATURE-INDUCED MITOCHONDRIAL CYCLE IN THE EARTHWORM EISENIA FOETIDA (SAVIGNY) ATTILIO ARILLOand FEDERICOMELODIA Istituto di Zoologia dell'Universitd di Genova, Via Balbi 5, 1-16126 Genova, Italy

(Received 18 August 1990; accepted in revisedform 16 March 1991) Abstract--l. The respiration rate and the acceptor control index values of earthworm mitochondria are

usually very low. 2. An improvement in mitochondrial coupled respiration occurs when earthworms are subjected to an in vivo appropriate thermal cycle. 3. Variations in mitochondrial aerobic functions caused by the naturally-occurring thermal cycles may play an important role in earthworm biology and reproduction.

Key Word Index: mitochondria; acclimation; respiration; earthworm; Eiseniafoetida

INTRODUCTION

Eisenia foetida is an earthworm preferentially living in compost and manure heaps, where oxygen, because of fermentative processes, is scarce. Mitochondrial properties of such a microaerobic species are markedly atypical, especially when organelles are tested in a normally oxygenated medium (220#M oxygen): acceptor control index (ACI) values are very low; respiration is largely insensitive to common inhibitors; the ADP-stimulated respiration, if any, remained linear and does not return to state 4 at the expected point of complete conversion of ADP to ATP (AriUo and Melodia, 1987). During spring, however, the laboratory-reared earthworms show a sudden and transient improvement in their aerobic mitochondrial metabolism, so that their mitochondrial properties become more comparable to those found in higher aerobic animals. This mitochondrial improvement also occurs in nature, although aerobic performance may be shifted to different spring-months, depending on climatic conditions prevalent at sites where earthworms are collected. Factors underlying such a transient optimization in mitochondrial function remain unknown. Certainly, photoperiod is not responsible for this phenomenon, which occurs even when earthworms are kept in a dark container. Under laboratory conditions, soil and food qualities are apparently the same during each season. The only environmental parameter undergoing some seasonal variation is ambient temperature: e.g. in our laboratory the temperature reaches 17-18°C during winter, 21-22°C in spring and 25-27~'C during summer. Therefore, temperature appears to be a factor capable of controlling earthworm mitochondrial functions. We have now explored this suggestion by studying mitochondrial respiration of earthworms subjected to different thermal cycles. MATERIALS AND METHODS

Earthworms were collected from a horse-compost heap, and transported in their native soil to our ra 16/*~:

217

laboratory. Temperature of their native soil, at the time of collection, was 26°C. Some specimens w e r e immediately (last days of October) sacrificed in order to determine the mitochondrial respiration of o u r earthworm population before starting the experiments. The remaining earthworms were subdivided into six groups, which were separately placed in darkened and thermostated incubators. Each group was then subject to different thermal cycles, taking into account the temperature preferendum of the species under test (Edwards and Lofty, 1977). It is universally accepted that Eisenia foetida prefers soils from 16 to 23°C. However, a considerable variation in estimates of upper lethal temperatures is provided by different workers: on the whole, however, we can state that exposure to 26-27°C represents a state cause of severe stress for this species. Concerning low temperatures, it is known that earthworms freely tolerate temperatures of about 10°C. On this basis, our experimental groups were as follows: Group I. Animals were kept for 2 months (November, December) at 10°C; subsequently they were maintained for 4 months (January, February, March, April) at 23°C; and finally 26°C (May, June, July). Group 2. Animals were exposed for 1 month (November) at 10°C; 5 months (December, January, February, March, April) at 23°C; 3 months at 26°C (May, June, July). Group 3. Earthworms were kept at 23°C from October to April and then at 26°C (May, June, July). Group 4. Earthworms were kept for 1 month at 16°C; 4 months at 22°C; 3 months at 26°C. Group 5. Animals were maintained at 18°C (from October to March); at 21°C for April and then at 26°C (May, June, July). This thermal cycle simulates fluctuations in temperature usually occurring in our laboratory, where earthworms are normally reared. Group 6. Earthworms were kept at 10°C for 3 months (November, December, January). Soil temperatures w e r e m e a s u r e d by a mercury-bulb thermometer at a 5 cm depth. Mitochondriai respiration was determined on the last day of each month.

218

ATTILIO ARILLOand FEI)ERI('OMELOI)IA

During the experimental time, soils of earthworm terraria were regularly supplemented with horse manure, minced vegetables and water. Mitochondrial function In order to remove gross intestinal content, earthworms were kept for 24 h in washed, sterile and fine sand before starting mitochondrial extraction. Mitochondria were then isolated from earthworms (whole body) and assayed according to procedures described in Arillo and Melodia (1987). Acceptor control index (ACI) and oxygen consumption were polarographically determined by a Clark-type electrode in the following medium: NaCI (100 mM), TES (25 mM), fatty acid-free albumin (Sigma, 16yM), phosphate buffer (3.5mM), MgC12 (5raM), rotenone (1 izM). All the solutions were adjusted at pH 7.15. Succinate (3raM) was used as a substrate. The state 3 of oxidative phosphorylation was determined by the addition of 200nmol of ADP to the incubation mixture (1.8 ml). Polarographic measurements were carried out at 2ZC. Protein concentration in mitochondrial preparations was determined according to Lowry et al. (1951). Cytochrome-c oxidase Cytochrome-c oxidase activity was spectrophotometrically assayed according to Wharton and Tzagoloff (1967), by following the oxidation of ferrocytochrome-c through the decrease in the absorbance of its A-band at 550 nm. Oxygen concentration in the assay mixture Respiratory properties of earthworm mitochrondria were determined in assay mixtures containing 90 or 220/1 M oxygen, the latter concentration being the normal oxygen level present in air-exposed water solutions. The 90/~ M concentration was obtained by gently flushing N z into the assay mixture until the desired oxygen level was reached.

..~ "~-~

RESt LTS Oxygen consumption and (Tables I 2 and Fig. l )

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2.8

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2.8

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G4

2.4

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(B)

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2.4

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1.2

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:"J .,~.~ja3,,,,,~ /I ."f / " ~ - ~ ' ~ - ...~*, ~ / '~ / ~ ~ " ~ ~ td ' '""~' ..~

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control index

Initial respiration. At the beginning of the experiments (last days of October), earthworm mitochondria displayed a relatively low oxygen consumption and a marked ADP insensitivity (ACI = 1.05 ± 0.02). Respiration did not return to state 4 when the added ADP was exhausted. AC! values were slightly improved by using assay mixtures containing low oxygen concentrations (90/~ M). Mitochondria displaying such properties will be called henceforth hypofunctional mitochondria (HF mitochondria). Group I. A 10°C exposure for 1 month (November) causes an increase in both the state 3 and state 4 respiration. With lasting cold exposure, a further increase in state 3 respiration could be observed, together with a decrease in state 4 respiration (December). As a consequence, the ACI values displayed a sudden increase. After a 1-month exposure to 23 C (January), an additional increase in state 3 respiration takes place, so that ACI reached its maximum value in January (2.2 _+ 0.05 and 1.7 _+0.03 at low or at normal oxygen tension, respectively). Mitochondria displaying a relatively good ACI also possessed other more typical properties, such as a near-to-normal inhibitor sensitivity; furthermore, state 4 respiration was usually restored when ADP is exhausted. In this paper, such organelles will be called norreally-functioning mitochondria (NF mitochondria). Respiratory performance was only transient, because in February a decrease occurred both in mitochondrial oxygen consumption and in ACI values. This decrease progressively continued during the subsequent months so that in April mitochondria again fell in a hypofunctional state. Exposure to 2 6 C caused no further change in mitochondrial functions.

3.0 -- (A) --

aeceptor

~

~

i N

/ D

/ J

, F

I ~'~ M A

I M

, J

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Months Fig. l. Mitochondrial acceptor control index displayed by earthworms subjected to different thermal cyles. G 1-5 = groups I 5 of Tables l and 2; (A, B) values obtained by using normoxic or hypoxic assay-mixtures, respectively.

Mitochondrial cycle in the e a r t h w o r m

219

Table 1. Oxygen consumption detected in earthworm mitoehondda tested under normoxie assay-mixtures (200/~M oxygen) October

, f State 3 ~.State 4 10°C

• /"State 3 Novemoer t State 4

23°C 14.3_+1.0 13.9-+ 0.7

18.9 _+ 1.3 (a) 17.2 + 1.1 (a)

I . I'State 3 cemoer ~State 4

23°C

January

.fState 3 ~ State 4

29.5 -+ 0.9 (b*) 17.0 -+ 0.8

19.5 _+0.5 14.6 _+0.4 (b)

10oc1 16.1 _+0.9 (c) 14.7 _+0.8

I

123oc 22.6 -+ 0.7 (c*) 13.5 -+ 0.7

I

Group 6 , f State 3 February ~.State 4

27.3 + 1.0 (c) 20.3 -+ 0.9 (c)

23°C 15.0 -+ 1.1 (d) 11.5 -+ 0.6 (d)

./'State 3 I State 4

14.1 + 0.8 12.8 -+ 0.4 (de)

fState 3 ~ State 4

26.2 -+ 1.5 20.1_+1.1

13.9_+0.7 13.1 _+0.6

, f State 3 ~.State 4

13.4 _+0.6 (f) 12.7 -+ 0.5 (d)

15.6_+ 0.7 15.4 -+ 0.6 (c)

16.5 -+ 0.7 (de) 15.4 -+ 0.6 (de)

16.4_+0.9 14.6_+0.8

18°C

14.2_+ 0.3 (e) 14.0_+ 0.2

12.1 _+0.5 (f) ll.3 -+ 0.6 (f)

18°C 21.6_+1.3 19.2 -+ l.I

22°C 25.4-+ 1.0 (d*) 12.4 -+ 0.6 (d)

18°C 24.2-+ 1.0(d) 20.1 _+ I.l

22°C 17.1 _+0.8 (e) 14.5 _+0.6 (e)

21°C 34.1 _+ l.I (e*) 17.9_+0.8

26°C 15.2 -+ 0.5 14.3_+0.4

26~'C

26"C 14.2 _+0.6 (df) 13.5 -+ 0.5 (df)

18.2 -+ 0.7 (b) 14.4+0.5

26°C 12.9-+ 0.4 12.2-+0.3

19.5 _+0.6 (b) 17.5 _+0.4 (b)

22°C

23°C

26'~C 15.0_+0.7 13.7_+0.5

17.6 _+0.6 15.3 _+0.3

23°C

23°C

26~'C

July

16.5 _ 0.6 (a) 15.7 -+ 0.5 (a)

22°C

23°C

23°C

26°C

May

15.0 -+ 0.4 14.2 _+0.2

23°C

23°C

April

16.9 _+0.8 (a) 15.7 -+ 0.5 (a)

18°C

] 23°C

10°C[ De

16°C

26'~C 16.3+0.6 15.0_+ 0.5

26oc 14.8 -+ 0.7 (f) 13.8+0.7

26°C 14.1 _+0.7 (f) 13.0-+ 0.5 (f)

I

I

I

I

I

Group 1

Group 2

Group 3

Group 4

Group 5

Values (natoms/min/mg protein) represent the mean + SE of data obtained from 4 different mitochondrial preparations. Earthworms were subdivided into 6 groups, which were exposed to different thermal cycles, as indicated near the vertical lines. For experimental details see Materials and Methods. Statistically significant differences by Student's t test (P <0.05) against respective data detected in (a) October; (b) November; (c) December; (d) January; (e) February; (f) April. *Maximum respiration rate found within the experimental group considered.

Groups 2-4. Mitochondrial functions showed a cycle similar to that described for group 1, but in this case the maximum improvement in the energy-linked reactions occurred 1 month earlier (group 2) or 1 month later (group 4). Group 3. Mitochondria remained in a hypofunctional state throughout the experimental period. Group 5. In this group a continuous and progressive improvement in mitochondriai function occurred, and maximum aerobic optimization was reached in April. However, the exposure to a thermal stress-producing temperature (26°C) caused a sudden worsening (within a week, data not shown) in the mitochondriai aerobic functions. Group 6. After a slight and transient improvement (December), mitochondria fell rapidly in a hypofunctional state (January).

Cytochrome-c oxidase Cytochrome-c oxidase activity remained constant in all experimental groups, in spite of both the seasonal period and the functional state of mitochondria (data not shown).

DISCUSSION

Mitochondrial properties Results show that earthworm mitochondria possess a poor aerobic ability, their oxygen consumption and ACI values being usually very low for a large part of the worm annual cycle. The reasons underlying these atypical mitochondrial properties are at present unknown. It is possible that a low mitochondrial permeability to exogenous substrates or the occurrence of an extensive lipid peroxidation may influence worm mitochondrial functions. Moreover, it is to be emphasized that earthworm mitochondria can perform some alternative energyyielding anaerobic reactions that may overlap normal substrate respiration. In this connection, we recall that such anaerobic pathways are widespread in primitive bilateral metazoa (Cheah and Bryant, 1966; Mendis and Evans, 1984; Schottler, 1977; Takamiya et al., 1984; Von Brand, 1979). In these animals, the electron flow generated by N A D H is directed (via b-type cytochromes) to fumarate, which in turn is converted to succinate. The latter compound is

220

ATTILIO ARILLO a n d FEDERICO MELODIA Table 2. Oxygen consumption detected in earthworm mitochondria tested under hypoxic assay-mixtures (90 rum (,xygen)

October

f State 3 ~State 4 10'~C

N

, (State 3 ovemner "~State 4

23 C 9.6 +_ 0.3 8.8+0.5

12.5 _+ 0.6 (a) 10.0 -+ 0.4 (a)

I

lo'cl 12.8 + 0.5 8.4 _+ 0.3 (bl

. f State 3 December / State 4

[ 23~C 23.7 + 0.3 (b*) 8.2 +- 0.2 (b)

I lo'cI January

fState 3 ~.State 4

9.9 +_ 0.7 (c) 8.2 _+ 0.8

[

14.5 +- 0.8 (c*) 6.8 +- 0.8 (c)

Group 6

,/'State 3

February ~ S t a t e 4

18,1 _+ 0.9 (c) 11.2 + 0.5 (c)

23°C 7.7 + 0.4(d) 4.9 -- 0.2 (d) 23C

April

f State 3 ~.State 4

6.9 -+ 0.3 (de) 5.3 _+ 0.2 (d)

J'State 3 ~ State 4

6.9 _+ 0.7 5.1 +_0.6

12.0 + 0.5 (d) 8.1 + 0 . 4 ( d )

J'State 3 ~ State 4

7.1 +- 0.3 (d) 5.6 +- 0.2 (d)

7.0 + 0.3 (c) 6.5 + 0.2(c)

7.2__.0.3 6.3 _+ 0.2 23~C 6.9+0.2 6.5__.0,3

10.6 + 0.7 8.5-+0.5

2ff~C 7.2 +- 0.4 6.8-+0,3

26C 11.3 +_ 0.7 (d) 8.1 -+ 0.3 (d)

26 C 7.2+_0.3 6.5+-0.2

18 ( 9.1) :~_0.3 (a) 7 ~ :- 0.1(a)

23 C 12.8+_0.5 7.1 + 0.3 (b)

18 C 12.5 + 0.2 (b] 10.4 +- 0.5 (b)

22 C 12.1 + 0 , 8 5.7 _+0.4 (c)

23°C

26'~C

26' C

July

23C

23"C

26C

May

9.4+0.3 8.6+-0.2

2YC 17.8+0.5 11.8 + 0.5

12.7 t 0.4 (a) 11.1 ± 0 . 4 ( a )

23 'C

23C

[2Y'C

16 C

18C 16.2 +- 0.8 (c) 12.4 + 0,6 (c)

22'~C 19.4 + 0.9 (d*) 7.5 -+ 0.3 (d) 22'C 11.3 _+ 0.6(e) 8.1_+0.3

18'C 14.8+0.6 11.2 + 0.5 (d) 21 C 23,1 -+ 1,2 (e*) 8,5 -+ 0,3 (e)

26 'C 11.2 _+ 0.5 8.7+-0.4

26 C 13,3 _+ 0.6 8.9_+0.5

26 C 10.5 z 0.5 7.9 ~ 0.3

26' C 8.9 -+ 0.4 (f) 6.1 +- 0,3 (f)

I

I

I

I

i

Group I

Group 2

Group 3

Group 4

Group 5

See legend to Table 1.

subsequently metabolized to propionate. ATP is formed both in complex I of the respiratory chain and in some reactions leading to propionate. In Tubifex such an anaerobic mechanism is surprisingly maintained even in the presence of oxygen (SeuB et al., 1983). These metabolic features presumably occur also in Eisenia foetida, because the same pattern in mitochondrial metabolites was found in the two Oligochaeta when they were tested in vivo under anoxic and aerobic conditions (Arillo and De Giuli, 1973).

Temperature effects As previously reported, during spring the mitochondrial properties so far described show a sudden/ transient improvement in their aerobic function, and most of the atypical properties detected in other months surprisingly disappeared. Our results point out that a similar change in mitochondrial functions also occurs when earthworms are subjected to an in vivo appropriate thermal cycle. The process observed here can be summarized as follows. At the beginning of the experiments, mitochondria from earthworms inhabiting 26°C soil are clearly in a hypofunctional state; when earthworms undergo a fall in temperature (~< 18°C), an

increase both in ACI values and in oxygen consumption is developed. This increase becomes maximum when the upper levels of normothermic conditions (ULN = 22-23°C) are again established. Such a relative efficiency in aerobic functions can be maintained only for a definite time, because even by lasting normothermic conditions, mitochondria fall again to a hypofunctional state. It is noteworthy that the best mitochondrial functions (highest oxygen consumption and highest ACI) are displayed at different times from the beginning of experiments, depending on the thermal history undergone by the groups tested. When earthworms are exposed to 10°C, maximum mitochondrial improvement occurs 20-30 days after the return of ULN conditions. It the case of the 16~C-exposed animals, maximum increase in both ACI and oxygen consumption takes place about 3 months after re-establishing favorable ULN conditions. Finally, mitochondria from the 18°C-acclimated earthworms reach their maximal aerobic ability after a 4-month exposure to ULN conditions. Evidence suggests that the fluctuation in environmental temperature is strictly necessary to improve aerobic function of mitochondria. In fact, mitichondria from earthworms maintained under a constant temperature from October to April

Mitochondrial cycle in the earthworm (see group 4, Tables 1 and 2), always remain in a hypofunctional state throughout the seasonal cycle. On the other hand, low temperatures slightly activate mitochondria in a transient manner (group 6), eliciting maximal effects only upon return of normothermic conditions (groups 1 and 2). Results also show that exposure to high, stress inducing temperatures (26°C) causes an immediate worsening in mitochondrial aerobic functions, even when maximum mitochondrial performance had been reached shortly beforehand (see Results-group 5). In our opinion, such a temperature-induced variation in mitochondrial functions can play an important role in earthworm biology. In fact, we observed (both in the lab. and in nature) that mitochondrial improvement is always shortly followed by (i) an increase in earthworm reproductive activity and (ii) a subsequent production of cocoons, which are preferentially placed near the soil surface. It is difficult to determine whether a cause and effect relation may exist between the observed improvement in aerobic metabolism and reproductive activity. Nevertheless, evidence confirms that these biological processes are time-linked. On this basis, one can presume that, after a cold season, the favourable spring temperature should facilitate both earthworm aerobic function and reproductive activity. As a consequence, during these periods earthworms can rely on a more efficient energy-yielding (aerobic) metabolism, making it possible to face the reproduction-induced waste of energy. Furthermore, it is noteworthy that this mechanism (starting with the re-establishment of ULN conditions) occurs shortly before the onset of the warm summer temperatures. Thus, the subsequent production of cocoons naturally occurs at a temperature particularly favourable for earthworm development: in fact, optimal temperatures for the growth of newly-hatched worms are relatively high (25°C or more), and certainly exceed the preferred thermal range of mature earthworms (Edwards and Lofty, 1977). These biological phenomena, on the whole, are so temporally synchronized that they may be well considered as an example of physiological coadaptation. Our opinion that earthworms are capable of operating a shift between HF- and NF-mitochondria by a temperature-dependent mechanism deserves some further discussion. A temperature-induced change in cytochrome-c oxidase activity have been reported in literature (Cherchi et al., 1987; Sidell et al., 1973);

221

hence, it may be presumed that this also occurs in E. foetida, thus influencing worm mitochondrial functions. However, our results show that in earthworms this enzyme is not involved in determining mitochondrial change, because its activity remains constant during all the mitochondrial cycle. Therefore, alternative mechanisms must exist in the species under study. REFERENCES

Arillo A. and De Giuli A. M. (1973) Variazioni del ciclo di Krebs in anaerobiosi. II. Studi su anellidi oligocheti. Boll. Mus. Ist. biol. Univ. Genova 41, 121-130. Arillo A. and Melodia F. (1987) Properties of mitochondria from Eisenia foetida (Savigny): a case for adaptive evolution. In On Earthworm (Edited by Bonvicini Pagliai A. M. and Omodeo P.). Selected Symposia and Monographs U.Z.I. Vol. 2, pp. 15-31. Mucchi, Modena. Cheah K. K. and Bryant C. (1966) Studies on the electron transport system of Moniezia expansa (Cestoda). Comp. Biochem. Physiol. 19, 197-223. Cherchi M. A., Arillo A., Balletto E., Mensi P. and Gaino E. (1987) The seasonal cycle in tortoise mitochondria: evidence for a role in the control of circannual activity. Boll. Zool. 4, 319-324. Edwards C. A. and Lofty J. R. (1977) Biology of Earthworms, p. 333. Chapman and Hall, London. Lowry O. H., Rosebrough N. I., Farr A. L. and Randall R. J. (1951) Protein measurement with folin phenol reagent. J. biol. Chem. 193, 265-275. Mendis A. H. W. and Evans A. A. F. (1984) First evidence for the occurrence of cytochrome-o in a free-living nematode. Comp. Biochem. Physiol. 78B, 729-735. Sehottler U. (1977) The energy-yielding oxidation of NADH by fumarate in anaerobic mitochondria of Tubifex sp. Comp. Biochem. Physiol. 58B, 151-156. SeuB J., Hipp E. and Hoffmann K. H. (1983) Oxygen consumption, glycogen content and the accumulation of metabolites in Tubifex during aerobic-anaerobic shift and under progressing anoxia. Comp. Biochem. Physiol. 75A, 557-562. Sidell B. D., Wilson F. R., Hazel J. and Prosser C. L. (1973) Time course of thermal acclimation in goldfish. J. comp. Physiol. 84, 119-127. Takamiya S., Furushima R. and Oya H. (1984) Electron transport systems of the mitochondrial fractions in the freshwater snail Biomphalaria glabrata. Comp. Biochem. Physiol. 77B, 465-473. Von Brand T. (1979) In Biochemistry and Physiology of Endoparasites, pp. 239-254. Elsevier, Amsterdam. Wharton D. C. and Tzagoloff A. (1967) Cytoehrome oxidase from beef heart mitochondria. In Methods in Enzymology (Edited by Estabrook R. W. and Pullman M. E.), Vol. 10, pp. 245-250. Academic Press, New York.