Pergamon J. therm. Biol. Vol. 20, No. l/2, PP. 4348, 1995

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HEAT SHOCK RESPONSE AND THERMAL ACCLIMATION EFFECTS IN THE GILLS OF ANODONTA CYGNEA:

CILIARY ACTIVITY, STRESS PROTEINS AND MEMBRANE FLUIDITY

K. Y. H. LAGERSPETZ, I. ANNELI KORHONEN and A. J. TIISKA

Laboratory of Animal Physiology, Department of Biology, University of Turku, FIN-20500 Turku, Finland

Abstract-l. After the transference of fresh water mussels (Anodonta cygnea) from 4 to 20-24°C the heat resistance of ciliary activity in gills is increased significantly in 1 day and then further in l-4 days to a new steady level.

2. If gills isolated from animals at 4°C are transferred to 24°C the heat resistance of ciliary activity increases in 18-24 h to a level steady for 3 days. This level is equal to that found in gills of whole mussels after 1 day at 20-24°C.

3. The response of isolated gills is not due to isolation, but probably to heat shock. 4. The transfer of isolated gills to the higher temperature induces in 24 h the synthesis of 90 kDa stress

protein, but does not affect the cell membrane fluidity. 5. The heat shock response occurs also in isolated gills, but the thermal resistance acclimation of ciliary

activity only occurs in the gills of whole mussels.

Key Word Index: Heat shock, acclimation, heat resistance, ciliary activity, stress proteins, membrane fluidity, mussels, Anodonta cygnea

INTRODUCPION

The paired gills of bivalve mussels are suitable for the study of a cellular function, ciliary activity. The gills of many mussels can be isolated and kept in isolation for hours or days without the impairment of ciliary activity. The immediate effects of different chemical compounds and temperature on this cellular function can therefore be easily studied. Also the chemical and thermal history of the mussels from which the gills are taken may be reflected in the ciliary activity or in its thermal resistance.

When bivalve molluscs (freshwater: Anodonta cygnea, A. anatina; marine: Mytilus edulis) are trans- ferred from low temperatures (4-14°C) to 21-24°C the heat resistance of the coordinated activity of frontal gill cilia is increased in 4-10 days to a new steady level (Lagerspetz and Dubitscher, 1966, Senius, 1975, 1977). This change can be reversed by moving the animals back to cold, after which the original level of heat resistance is attained in a few days (Senius, 1975, 1977). This is a case of heat resistance acclimation, observable at the cellular level.

The activity of gill cilia continues in isolated gills of Anodonta cygnea for 2-3 days after the isolation.

If the paired median gills are isolated from an animal kept previously at 4°C and then one of the gills is stored at 4°C and the other at 24°C the heat resist- ance of the ciliary activity in the latter gill increases in 18 h to a new steady level (Lagerspetz et al., 1970). The question arises whether this shows that the thermal resistance acclimation of ciliary activity in the gills of Anodonta is a direct cellular phenomenon, or is it the effect of a heat shock on the tissue level?

A short-time exposure to elevated temperature causes in many ectothermic animals and in cultured cells a rapid but transient synthesis of stress proteins (heat shock proteins, hsp) (Lindquist, 1986, Lindquist and Craig, 1988). This has been observed also in marine bivalve molluscs (Sanders, 1993). The syn- thesis of stress proteins is in many animals and cells correlated with increased heat resistance (acquired thermotolerance, heat hardening). This connection has not been studied in bivalve mussels.

Studies on the effects of thermal acclimation and of membrane fluidizing substances show that low fluidity of cell membranes correlates with high heat resistance of ciliary activity in gills of Anodonta (Lagerspetz, 1985). We suggested that the thermal resistance acclimation of ciliary activity depends on the partial homeoviscous adaptation of membranes

43

44 K. Y. H. LAGERSPETZ et al.

of gill cells. The effects of heat shock on membrane fluidity have not been studied in molluscs or in other Metazoa, except in cultured mammalian cells (Lepock et al., 1981).

Using whole mussels and isolated gills we have now studied four variables in which heat shock response and thermal acclimation may differ: (1) the time- course of change in the heat resistance of ciliary activity; (2) the magnitude of the change; (3) the occurrence of stress protein synthesis, and (4) the change in cell membrane fluidity.

MATERIALS AND METHODS

Maintenance of animals and isolated gills

The mussels Anodonta cygnea (shell length lo-16 cm) were collected at several occasions from a lake in SW Finland. They were stored in aerated charcoal-filtered tap water at 8-10°C without feeding, and subsequently transferred to similar conditions at 4 or at 20--24°C for thermal acclimation to these two temperatures for times given below.

Median gills (which do not contain developing glochidia) were isolated and used immediately or kept, one at 4°C and the other at 20-24°C in Petri dishes in charcoal-filtered tap water for times indicated below.

Heat resistance of ciliary activity

The heat resistance time of ciliary activity (HRT) was determined as previously described (Senius and Lagerspetz, 1978). The median gills of mussels were cut in strips of 2-4mm in width. lo-15 strips from each gill were incubated in 40 ml of charcoal-filtered tapwater at 39.o”C. HRT was defined as the mean time (in min) from the beginning of the incubation to the cessation of coordinated transport of graphite particles by frontal gill cilia. Student’s t-test was used for the assessment of the statistical significance of the differences between mean values.

Amino acid incorporation

Gills were isolated from the animals at the two temperatures and used immediately or kept at 4 or 24°C for different times. A piece weighing about 200mg was cut from the gill and placed in an incubation dish containing 200 PCi [3SS]methionine (from Amersham) per ml water.

After incubation of 2 h at 4°C the tissues were washed and put in cryotubes to liquid nitrogen. Frozen tissues were homogenized manually in a glass homogenizer in a buffer consisting of 5 mM Tris-HCl, pH 7.1, 0.1 mM phenylmethylsulfonyl fluoride (from Sigma), and 1% 2-mercaptoethanol (from Fluka). Homogenates were centrifuged at

12,OOOg for 5 min. Supematants were diluted with loading buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS (from Bio-Rad), 10% glycerol, 0.2% 2-mercap- toethanol and 0.00125% bromophenol blue) and heated at 95°C for 5 min. The chemicals were obtained from Merck, unless another origin is given above.

Gel electrophoresis andjuorography

Proteins were separated using one dimensional discontinuous SDS-polyacrylamide gel electrophor- esis (SDS-PAGE) according to Laemmli (1970) in a Bio-Rad MiniProtean II dual slab cell following the instructions of the manufacturer. After electrophoresis the gels were fixed in acetic acid : water: isopropanol (25 : 65 : 10) and impregnated in Amplify (Amersham). The gels were dried on Whatman filter paper. The fluorography was carried out using Fuji RX X-ray film and intensifying screens. Exposures were done at -70°C for 12 h to 3 days.

Membrane Juidity

Microsomal membranes were prepared from the excised gills as described earlier (Lagerspetz, 1985) and stored at - 70°C. Steady state fluorescence polar- ization technique with 1,6-diphenyl- 1,3,5-hexatriene (DPH) as the fluorescent probe molecule was used for the assessment of membrane fluidity with a Perkin-Elmer LS-50 luminiscence spectrometer as described before (Lahdes et al., 1993).

RESULTS

Equilibration of shell cavity temperature with environ - ment

Two mussels kept at 4°C were transferred to 2O”C, and the temperature of their shell cavity was moni- tored by a 0.5 mm thermocouple. In the cold room (4°C) a 1 mm hole was bored with a finger drill through the center of the shell of one of the mussels at the level of the dorsal edge of the gills and the thermocouple inserted. The temperature inside the shell cavity was that of the surrounding water. The mussel was transferred immersed in water from 4 to 20°C. Frequent monitoring of the shell cavity tem- perature showed that it was equilibrated with the surrounding water in 40 min.

A similar hole was drilled in the second mussel after it had been in 20°C water for 50 min. The temperature of the water inside the shell cavity was the same as that of the surrounding water. Both mussels kept their valves closed during the exper- iment.

Heat shock response in the gills of A. cygneu 45

150-

E E

P P

.v glOO-

iir

z ____________________~~~~~~-~~~~~~~~~~~~~~~~_ .-

al

ii

---_________________~~~~~~~~~~~~~~~~~~~~~~~~~

3 50-

t!

z I"

0

-I

5 10 15 20 41-48

Acclimation time at 20-24’C (days)

Fig. I. Heat resistance time of ciliary activity (HRT) in the gills of mussels at different times after the transference of animals from 4 to 20-2&C. Means f SEM for 4-7 animals. The broken lines give the upper

and lower 95% confidence limits of HRT of the 39 control animals kept at 4°C.

The shell length of the two A. cygneu individuals

tested was 13.1 and 15.7 mm, and the mass of the animals (including the shell weight, the soft organ weight and the water content of their shell cavities), was 175 and 270 g, respectively. It seems to take less than 1 h for the temperature of the shell cavity in A. cygnea of the size used in this study to equilibrate with a 16°C rise in environmental temperature.

DeveIopment of heat resistance in whole animals

Figure 1 shows the development of heat resistance of ciliary activity in the gills of animals, which have been kept at 4°C and then transferred to 20-24°C. The increase in HRT is statistically significant (P ~0.01) in 1 day with the mean value of 87.0 f 5.2 min (N = 5). A steep increase in the HRT follows during the second day at the higher tem- perature, and HRT attains a new steady level in at least 5 days. This level is unchanged at least for 6-7 weeks.

Table 1. Heat resistance time (HRT) of ciliary activity in gills of animals kept at two different temperatures and of the

gills isolated from the animals kept in cold

HRT (min) N

(A) Animals at 4°C 58.8 + 2.2 16 (B) Isolated gills 1 day at 4°C 62.5 f: 3.8 16 (C) Isolated gills 1 day at 24°C 81.9 f 4.3 16 (D) Animals at 24°C 103.6 + 4.3 14

P(A-B) NS, P (AX) < 0.001, P (A-D) < 0.001, P (C-D) < 0.01.

1 day here means 22-28 h.

Development of heat resistance in isolated gills

Table 1 confirms our earlier results (Lagerspetz et al., 1970, Fig. 1, Senius, 1977, Table l), now with a larger material. The gills isolated from animals in cold increase their heat resistance when kept for 1 day at 24°C. This increase of heat resistance is equal to that gained by the gills of whole animals in 1 day after the transference to 20-24°C (see above). It is signifi- cantly smaller than that gained finally through the thermal acclimation of whole animals (Fig. 1 and Table 1).

The high heat resistance of ciliary activity in gills of mussels kept at 24°C is not maintained after the isolation of gills (Table 2). The decrease of heat resistance occurs similarly both at 24 and 4°C. The isolated gills do not maintain the acclimation effect better at 24 than at 4°C.

Stress proteins

Figure 2 shows the gel electrophoresis patterns

Table 2. Heat resistance time (HRT) of ciliary activity in gills of animals kept at 24°C and of the isolated gills

maintained for 1 and 2 days at different temperatures

HRT (min) N

(A) Animals at 24°C 121.6 f 5.4 7 (B) Isolated gills 1 day at 24°C 100.9 * 6.6 7 (C) Isolated gills 1 day at 4°C 98.3 k4.4 6 (D) Isolated gills 2 day at 24°C 76.5 + 8.1 6 (E) Isolated gills 2 day at 4°C 68.3 k 12.1 6

P (A-B) = 0.03, P (8x3) NS, P (D-E) NS, P (A-D) < 0.001.

46 K. Y. H. LAGERSPETZ et al.

106 * 80 *

32.5 w

27.5 *

4 106 - 80

- 495

- 32.5

. 27.5

Fig. 2. Fluorographs of SDS-PAGE patterns of [‘Slmethionine labelled proteins from mussel gills. (1) Ani- mals from 4”C, isolated gills 24 h at 4°C. (2) Animals from 4”C, isolated gills 24 h at 24°C. (3) Gills of animals from 4°C. (4) Animals from 4”C, isolated gills 4 h at 24°C. (5) Gills of animals from 24°C. The positions of standard proteins (Bio-Rad) with different molecular weights (kDa) are given on the left for lanes 1-3 and the right for lanes 4 and 5. The 90 kDa band in lane 2 is indicated by an arrow.

of labelled gill proteins after different temperature treatments. They are remarkably similar. Only gills isolated from mussels stored at 4°C and kept as isolated for 24 h at 24°C (lane 2) show a protein band of about 90 kDa which does not occur in the other fluorograph lanes. The 90 kDa protein has not been synthesized in the gills isolated from 4°C mussels and kept then as isolated at 4°C (lane 1) or for 4 h at 24°C (lane 4). It does not occur in gills of cold- and warm-acclimated mussels (lanes 3 and 5). The heat shock caused by the transfer of isolated gills from 4 to 24°C for 4 or 24 h apparently did not otherwise affect the protein synthesis.

0.300--

P

0.250 -- t

Temperature (‘C)

Fig. 3. Fluorescence polarization of DPH in microsomal membranes prepared from isolated gills kept for 24 h at 5°C (0) and at 24°C (0). Pairs of median gills from 3 animals

(means + SD).

Membrane fluidity

Figure 3 gives the results of membrane fluidity measurements on microsomal fractions prepared from gills of animals originally kept at 4°C and then after isolation for 24 h at 4 or 24°C. The fluorescence polarization values were not significantly different at any of the measurement temperatures which indicates equal fluidity of the membranes.

DISCUSSION

Bivalve mussels react to a change of environmental conditions by the withdrawal of their siphons and the closure of their shells, which may last for hours. Although the exchange of water between the inside and the outside of the shell is interrupted, the tem- perature of the water inside the shell cavity and therefore also of the gill tissues is equilibrated in less than 1 h even in large individuals of Anodonta cygnea. Thermal stress conditions are reached rapidly in spite of the closed shell.

The increase in HRT in isolated gills from Anodonta kept in cold is not an effect of the isolation stress, but related to the temperature in which the gills are kept after the isolation (Table 1). It does not occur in gills isolated from mussels kept previously at the higher temperature. Their isolated gills show a similar decrease in HRT at the two temperatures (Table 2).

These results can be compared with the increase of HRT in whole animals (Fig. 1). The HRT is similar after transfer from 4 to 24°C after 1 day in whole animals and in isolated gills. Thereafter, the HRT in whole animals increases further for l-4 days, but stays at the same level in isolated gills (Lagerspetz et al., 1970).

This shows that the response of whole animals to elevated temperature differs both in its extent and in its longer time-course from that found in isolated gills. This seems to differentiate between the early heat shock response and the subsequent thermal acclimation found in whole Anodonta.

In most studies on stress proteins in invertebrates, whole animals have been subjected to heat stress or to ecotoxical compounds. The occurrence of stress proteins has been shown in several molluscs (Sanders, 1993), also in bivalve mussels. The transfer of speci- mens of Myths edulis from lo-15°C to 20°C or higher temperatures for l-28 h induces the synthesis of stress proteins in their gills (Sanders, 1988, Steinert and Pickwell, 1988; Margulis et al., 1989, Veldhuizen- Tsoerkan et al., 1990, 1991).

In the present study, 24 h but not 4 h at 24°C was sufficient to induce the synthesis of 90 kDa protein in

Heat shock response in the gills of A. cygnea 47

gills isolated from cold-acclimated Anodonta cygnea

(Fig. 2). This is apparently the first discovery of a stress protein in freshwater mussels (cf. Sanders, 1993). In this case, the stress protein synthesis was induced in an isolated tissue preparation. However, this took more time than usually found in other invertebrates.

In isolated gills, the occurrence of 90 kDa protein seems to correlate with the increase of heat resistance. We did not find this or other specific proteins in the

thermal acclimation in the gills of Anodontu depends on the nutritive or coordinating systems of the whole animal.

Acknowledgements-The Academy of Finland has sup- ported this work. We wish also to thank Dr K. E. 0. Senius, MS Sirpa Lehti-Koivunen and MS Sinikka Hillgren for their help.

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homeoviscous adaptation or acclimation of cell mem- branes is usually associated with an improved resist- ance of cellular functions to high temperatures (Cossins and Bowler, 1987). On the other hand, the exposure of gills of Anodonta cygneu to the membrane lipid solvent n-hexanol decreased the HRT in relation to its concentration (Lagerspetz, 1985). Homeo- viscous acclimation of gill cell membranes may there- fore be important for the thermal acclimation of HRT.

(decreased fluidity) of the membrane lipid matrix in animals adapted to the higher temperature. Such

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Lagerspetz K. Y. H. and Dubitscher I. (1966) Temperature acclimation of the ciliary activity in the gills of Anodonta.

Lahdes E. O., Kivivuori L. A. and Lehti-Koivunen S. M. (1993) Thermal tolerance and fluidity of neuronal and branchial membranes of an Antarctic amphipod (Orchomene plebs); a comparison with a Baltic isopod (Saduria entomon). Comp. Biochem. Physiol. 105A, 463-470.

Lepock J. R., Massicotte-Nolan P., Rule G. S. and Kruuv J. (1981) Lack of correlation between hyperthermic cell killing, thermotolerance, and membrane lipid fluidity. Radiat. Res. 87, 300-313.

However, it is not so for the increase of HRT found in isolated gills from cold-acclimated Anodontu, kept for 24 h at 24”C, as the microsomal membranes prepared from gills kept at 4°C show equal fluidity

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Lindquist S. (1986) The heat shock response. A. R~u. Biochem. 55. 1151-l 191.

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(1989) 70 kDa heat shock proteins from mollusc and human cells have common structural and functional domains. Comp. Biochem. Physiol. !MB, 621-623.

Sanders B. M. (1988) The role of the stress proteins response in physiological adaptation of marine molluscs. Mar. Environ. Res. 24, 2077210.

SUMMARY

After the transference of Anodontu cygneu mussels or their isolated gills from 4 to 20-24”C, the effects

Sanders B. M. (1993) Stress proteins in aquatic organisms: an environmental perspective. Crit. Rev. Toxic. 23,49-75.

Senius K. E. 0. (1975) The thermal resistance and thermal resistance acclimation of ciliary activity in the Myfilus

of a heat shock response are evident in 1 day. These gills. Camp. Biochem. Physiol. SIA, 957-961.

involve an increase of the heat resistance time of Senius K. E. 0. (1977) Thermal resistance of the ciliary

ciliary activity, and at least in isolated gills, a syn- activity in the gills of the fresh water mussel Anodonta anatina. J. Therm. Biol. 2. 233-238.

thesis of 90 kDa protein, but cause no change in cell s enius K. E. 0. and Lagerspetz K. Y. H. (1978) Effects of membrane fluidity. A further increase of the heat calcium and magnesium on the thermal resistance of

resistance time (thermal acclimation) occurs only in ciliary activity in the fresh water mussel Anodonta.

whole animals, takes 2 or more days in time and is J. Therm. Biol. 3, 153-157.

probably associated with homeoviscous acclimation Steinert S. A. and Pickwell G. V. (1988) Expression of heat

of the cell membrane fluidity. Heat shock response is shock proteins and metallothionein in mussels exposed to heat stress and metal ion challenge. Mar. Emiron. Res. 24,

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48 K. Y. H. LAGERSPETZ et nl.

Veldhuizen-Tsoerkan M. B., Holwerda D. A., van der Mast Veldhuizen-Tsoerkan M. B., Holwerda D. A., van der Mast C. A. and Zandee D. I. (1990) Effects of cadmium C. A. and Zandee D. I. (1991) Synthesis of stress proteins exposure and heat shock on protein synthesis in gill tissue under normal and heat shock conditions in gill tissue of of the sea mussel Mytiius edulis L. Camp. Biochem. sea mussels (Mytilus edulis) after chronic exposure to Physiol. 96C, 419-426. cadmium. Camp. Biochem. Physiol. lOOC, 699-706.