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Genetic and environmental regulation of HSP70 expression
Marine Environmenral Research 39 (1995) 18 l--l 84 01995 Elsevier Science Limited Printed in Great Britain. All rights reserved 0141-I 136/95/$09.50 ELSEVIER
0141-1136(94)00014-X
Genetic and Environmental Regulation of HSP70 Expression Drew C. Browqa Brian P. Bradley” & Michael Tedengrenb “Department of Biological Science, University of Maryland, Baltimore County, Baltimore, Maryland, USA ‘Department of Systems Ecology, University of Stockholm, S-106 91 Stockholm, Sweden
ABSTRACT North Sea Mytilis edulis dt#er genetically from M. edulis from lower salinity in the Baltic Sea. Gammarus duebeni can tolerate a broad range of salinities, while Gammarus oceanicus is more limited in its salinity range. These populations were exposed to cadmium and the level of expression of a stress protein, HSP70, was analysed on Western blots. HSP70 is one of a family of ubiquitous heat shock proteins, about 70 000 Da, that are induced by a wide variety of stressors. North Sea mussels had high levels and multiple forms of HSP70 and a low tissue concentration of cadmium. Baltic Sea mussels had low levels of HSP70 and accumulated three times the tissue concentration of cadmium as North Sea mussels. G. duebeni had varying levels of HSP70 and twice the tissue concentration of cadmium of G. oceanicus, which had no detectable HSP70 throughout the course of cadmium treatment. The quantity of HSP70 induced correlated well with survival in these four populations.
Populations that differ genetically may respond differently to pollution (Murphy & Belastock, 1980). The North Sea-Baltic Sea system presents a stable salinity gradient from 6 ppt in most of the Baltic proper to 30 ppt at the junction with the North Sea. Marine organisms have colonized areas of lower salinity over many generations and the system thus offers different populations that have evolved to live at different salinity regimes. We are particularly interested in separating the effects of pollution from the effects of naturally occurring environmental stressors such as salinity and temperature. We are studying the response of two ecotypes of the blue mussel, Mytilis edulis, and two species of amphipod to cadmium and other stressors. M. edulis is up to 12 cm long in the North Sea at full strength seawater, and 4.5 cm long in the Baltic Sea at 6 ppt salinity (Tedengren & Kautsky, 1986). Amphipods are found in rock pools and in the littoral zone along the North Sea coast. Gammarus duebeni survives in rock pools where the temperature, salinity, and oxygen saturation can vary dramatically in the space of a few hours, G. oceanicus does not 181
Drew C. Brown et al.
182
(Tedengren et al., 1988). The two mussel ecotypes are distinct genetically according to isozyme variability and seem to differ more than the two amphipod species (Johannesson et al., 1990). The protein criterion used to examine responses was based on heat shock, or stress, proteins, an evolutionarily conserved, rapid response to stress. These proteins are found in all organisms, are quite similar from bacteria to man, and respond to specific stresses, including heavy metals and organics (Lindquist & Craig, 1988). The most intensely studied stress protein is HSP70, which provides a general indicator of stress. They are induced by temperature, disease, and metals, among others (Nover, 199 1). Amphipods were collected from rock pools, acclimated (as shown by activity) in the laboratory to 20°C salinity, and exposed to increasing temperature (2G A
kDa
B
Heated aquarium
StdO 14192833
kDa
kDa
35 3741 445060 h
Heated aquarium
StdO14192833373941424444h
Control aquarium
Control aquarium kDa
Std 0 14 19 28 374144 60FK FK h
Std 0 14 19 28 37 4144 60FK FK h
Fig. 1. Antibody blots of temperature time courses for representative G. duebeni (A) and G. oceanicus (B). FK = field control. Amphipods were collected from rock pools and after 24 h acclimation without food were exposed to increasing temperature (12-32.7”C) for up to 60 h. Samples were taken at 1-14 h increments. Whole individuals were homogenized in phosphate buffered saline (pH 7.2) with protease inhibitor, and soluble protein (20 pg/lane) was electrophoresed on denaturing SDS-PAGE, transferred to nitrocellulose membrane, and probed with antiHSP70. The antibody was raised in rabbits to a 23 amino acid Cterminal consensus sequence conjugated to BSA. Bands were visualized by color reaction of alkaline phosphatase-conjugated secondary antibody (Bradley & Ward, 1990). Prestained molecular weight standards were used for immediate visibility. HSWO is induced in animals from heated aquaria but not from unheated control aquaria.
Regulation of HSP70 expression
183
32.7”C) (see Fig. 1 caption for procedure). Mussels from the North Sea (29 ppt salinity) and from the Baltic Sea (6 ppt salinity) were exposed to 10 pg/liter cadmium, a concentration found in some polluted parts of the Baltic Sea, for up to 14 days (see Fig. 2 caption for procedure). Mortality and cadmium body burden (whole organisms) were determined for all organisms; energy expense, oxygen consumption, nitrogen excretion, and 0:N were also determined for mussels (n = 9 for mussels, 10 for amphipods) (Gilek et al., 1992). HSP70 levels were analysed (n = 5) from the soluble protein fraction on western blots, total protein profiles from duplicate silver stained gels. In the increasing temperature experiment, G. oceanicus died earlier and at lower temperatures, from 37 h (27.5T) to 44 h (32.4”Q than G. duebeni, which died from 44 h (32.4”C) to 60 h (32.7”C). In the total protein profiles, there was an increase in high molecular weight proteins ( > 120 kDa), in a 20 kDa, 25 kDa, and 45 kDa protein for G. oceanicus at high temperatures; no differences were observed with treatment for G. duebeni. High levels of HSP70 occurred on the westerns for G. oceanicus between 39 and 44 h and for G. duebeni between 41 and 60 h (Fig. 1). Production of HSP70 was therefore correlated as expected with raised temperature for both species, and appeared before any mortality occurred for G. duebeni, but after G. oceanicus had started dying. In the cadmium exposure experiment, the time course went to 24 h, at which time no mortality was observed. G. duebeni accumulated a higher body burden of cadmium-7.4 pg/g vs 4.4 pg/g for G. oceanicus. A little HSP70 was apparent on
BS
NS
Std
Oh
NS
BS IOh
NS 24
BS h
NS
BS 7d
Fig. 2. AntiHSP70 blot of time course of representative North Sea (NS) and Baltic Sea (BS) mussels exposed to 10 pg/liter cadmium. Non-reproductive mussels from the North Sea (29 ppt salinity) and from the Baltic Sea (6ppt salnity) were collected at 10°C in June and acclimated (with feeding) for 14 days at 12°C in a static system to 15 ppt salinity, a salinity commonly encountered by North Sea mussels and one which improves the physiological status of Baltic Sea mussels; they were then exposed to 10 pg/liter cadmium, a concentration that is found in some polluted parts of the Baltic Sea, for up to 14 days. Samples were taken at 5 h, 10 h, 1,2,4 and 7 days. Mussel mantle tissue was processed as described in Fig. 1; prestained molecular weight standards and antibody are the same.
184
Drew C. Brown et al.
the western blots for G. duebeni, and none for G. oceanicus. Under cadmium challenge, North Sea mussels produced high levels of 2-3 forms of HSP70 by 5 h (Fig. 2). Mussels from the North Sea had lower mortality rates, oxygen consumption, nitrogen excretion, cadmium body burden, and energy expense in cadmium than did the Baltic mussels. Species and ecotypes differed predictably in response to stresses. G. duebeni survived elevated temperature longer than G. oceanicus. As a resident of a variable environment like a rock pool, G. duebeni has evolved adaptations to these variations. One adaptation may be an efficient HSP70 that is induced faster or at lower temperatures than that of G. oceanicus. The Baltic mussels have adapted to a low but constant salinity. This adaptation may have put them close to the edge of survival. Additional stresses reduce the vigor of this ecotype, or coping with salinity stress confounds the ability to cope with another stress. It is possible that the mussel populations have not been separated long enough (Tedengren, 1990) for selection to mold randomly occurring genetic change into true adaptation to lower salinity and estuarine resilience, as has happened with the amphipods. The North Sea mussels and G. duebeni are the hardier variants and also produce more HSP70. HSP70 in this system is a predicter of pollution (cadmium) and natural stress (temperature) tolerance. The genetic divergence that has occurred between the mussel ecotypes and between the amphipod species illustrates both opposing viewpoints of the tolerance of estuarine versus oceanic species. Mussels adapting to lower (but constant) salinity have decreased resistance to at least one stressor (cadmium), while amphipods adapting to variable rock pool habitats have increased resistance to some stressors.
REFERENCES Bradley, B. P. & Ward, J. B. (1990). Mar. Env. Res., 28,471-5. Gilek, M., Tedengren, M. & Kautsky, N. (1992). Neth. J. Sea Res., 30, 11-21. Johannesson, K, Kautsky, N. & Tedengren, M. (1990). Mar. Ecol. Prog. Ser., 59, 211-9. Lindquist, S. & Craig, E. A. (1988). Annu. Rev. Genet., 22, 631-77. Murphy, L. S. & Belastock, R. A. (1980). Limnol. Oceanogr., 25, 16&5. Nover, L. (ed.) (1991). In Stress Proteins. CRC Press, Boca Raton, FL (1991). Tedengren, M. (1990). Ecophysioiogy and Pollution Sensitivity of Baltic Sea Invertebrates. PhD Thesis, Stockholm University, Sweden. Tedengren, M. & Kautsky, N. (1986). Ophelia, 25, 147-55. Tedengren, M., Arner, M. & Kautsky, N. (1988). Mar. Ecol. Prog. Ser., 41, 107-16.
0141-1136(94)00014-X
Genetic and Environmental Regulation of HSP70 Expression Drew C. Browqa Brian P. Bradley” & Michael Tedengrenb “Department of Biological Science, University of Maryland, Baltimore County, Baltimore, Maryland, USA ‘Department of Systems Ecology, University of Stockholm, S-106 91 Stockholm, Sweden
ABSTRACT North Sea Mytilis edulis dt#er genetically from M. edulis from lower salinity in the Baltic Sea. Gammarus duebeni can tolerate a broad range of salinities, while Gammarus oceanicus is more limited in its salinity range. These populations were exposed to cadmium and the level of expression of a stress protein, HSP70, was analysed on Western blots. HSP70 is one of a family of ubiquitous heat shock proteins, about 70 000 Da, that are induced by a wide variety of stressors. North Sea mussels had high levels and multiple forms of HSP70 and a low tissue concentration of cadmium. Baltic Sea mussels had low levels of HSP70 and accumulated three times the tissue concentration of cadmium as North Sea mussels. G. duebeni had varying levels of HSP70 and twice the tissue concentration of cadmium of G. oceanicus, which had no detectable HSP70 throughout the course of cadmium treatment. The quantity of HSP70 induced correlated well with survival in these four populations.
Populations that differ genetically may respond differently to pollution (Murphy & Belastock, 1980). The North Sea-Baltic Sea system presents a stable salinity gradient from 6 ppt in most of the Baltic proper to 30 ppt at the junction with the North Sea. Marine organisms have colonized areas of lower salinity over many generations and the system thus offers different populations that have evolved to live at different salinity regimes. We are particularly interested in separating the effects of pollution from the effects of naturally occurring environmental stressors such as salinity and temperature. We are studying the response of two ecotypes of the blue mussel, Mytilis edulis, and two species of amphipod to cadmium and other stressors. M. edulis is up to 12 cm long in the North Sea at full strength seawater, and 4.5 cm long in the Baltic Sea at 6 ppt salinity (Tedengren & Kautsky, 1986). Amphipods are found in rock pools and in the littoral zone along the North Sea coast. Gammarus duebeni survives in rock pools where the temperature, salinity, and oxygen saturation can vary dramatically in the space of a few hours, G. oceanicus does not 181
Drew C. Brown et al.
182
(Tedengren et al., 1988). The two mussel ecotypes are distinct genetically according to isozyme variability and seem to differ more than the two amphipod species (Johannesson et al., 1990). The protein criterion used to examine responses was based on heat shock, or stress, proteins, an evolutionarily conserved, rapid response to stress. These proteins are found in all organisms, are quite similar from bacteria to man, and respond to specific stresses, including heavy metals and organics (Lindquist & Craig, 1988). The most intensely studied stress protein is HSP70, which provides a general indicator of stress. They are induced by temperature, disease, and metals, among others (Nover, 199 1). Amphipods were collected from rock pools, acclimated (as shown by activity) in the laboratory to 20°C salinity, and exposed to increasing temperature (2G A
kDa
B
Heated aquarium
StdO 14192833
kDa
kDa
35 3741 445060 h
Heated aquarium
StdO14192833373941424444h
Control aquarium
Control aquarium kDa
Std 0 14 19 28 374144 60FK FK h
Std 0 14 19 28 37 4144 60FK FK h
Fig. 1. Antibody blots of temperature time courses for representative G. duebeni (A) and G. oceanicus (B). FK = field control. Amphipods were collected from rock pools and after 24 h acclimation without food were exposed to increasing temperature (12-32.7”C) for up to 60 h. Samples were taken at 1-14 h increments. Whole individuals were homogenized in phosphate buffered saline (pH 7.2) with protease inhibitor, and soluble protein (20 pg/lane) was electrophoresed on denaturing SDS-PAGE, transferred to nitrocellulose membrane, and probed with antiHSP70. The antibody was raised in rabbits to a 23 amino acid Cterminal consensus sequence conjugated to BSA. Bands were visualized by color reaction of alkaline phosphatase-conjugated secondary antibody (Bradley & Ward, 1990). Prestained molecular weight standards were used for immediate visibility. HSWO is induced in animals from heated aquaria but not from unheated control aquaria.
Regulation of HSP70 expression
183
32.7”C) (see Fig. 1 caption for procedure). Mussels from the North Sea (29 ppt salinity) and from the Baltic Sea (6 ppt salinity) were exposed to 10 pg/liter cadmium, a concentration found in some polluted parts of the Baltic Sea, for up to 14 days (see Fig. 2 caption for procedure). Mortality and cadmium body burden (whole organisms) were determined for all organisms; energy expense, oxygen consumption, nitrogen excretion, and 0:N were also determined for mussels (n = 9 for mussels, 10 for amphipods) (Gilek et al., 1992). HSP70 levels were analysed (n = 5) from the soluble protein fraction on western blots, total protein profiles from duplicate silver stained gels. In the increasing temperature experiment, G. oceanicus died earlier and at lower temperatures, from 37 h (27.5T) to 44 h (32.4”Q than G. duebeni, which died from 44 h (32.4”C) to 60 h (32.7”C). In the total protein profiles, there was an increase in high molecular weight proteins ( > 120 kDa), in a 20 kDa, 25 kDa, and 45 kDa protein for G. oceanicus at high temperatures; no differences were observed with treatment for G. duebeni. High levels of HSP70 occurred on the westerns for G. oceanicus between 39 and 44 h and for G. duebeni between 41 and 60 h (Fig. 1). Production of HSP70 was therefore correlated as expected with raised temperature for both species, and appeared before any mortality occurred for G. duebeni, but after G. oceanicus had started dying. In the cadmium exposure experiment, the time course went to 24 h, at which time no mortality was observed. G. duebeni accumulated a higher body burden of cadmium-7.4 pg/g vs 4.4 pg/g for G. oceanicus. A little HSP70 was apparent on
BS
NS
Std
Oh
NS
BS IOh
NS 24
BS h
NS
BS 7d
Fig. 2. AntiHSP70 blot of time course of representative North Sea (NS) and Baltic Sea (BS) mussels exposed to 10 pg/liter cadmium. Non-reproductive mussels from the North Sea (29 ppt salinity) and from the Baltic Sea (6ppt salnity) were collected at 10°C in June and acclimated (with feeding) for 14 days at 12°C in a static system to 15 ppt salinity, a salinity commonly encountered by North Sea mussels and one which improves the physiological status of Baltic Sea mussels; they were then exposed to 10 pg/liter cadmium, a concentration that is found in some polluted parts of the Baltic Sea, for up to 14 days. Samples were taken at 5 h, 10 h, 1,2,4 and 7 days. Mussel mantle tissue was processed as described in Fig. 1; prestained molecular weight standards and antibody are the same.
184
Drew C. Brown et al.
the western blots for G. duebeni, and none for G. oceanicus. Under cadmium challenge, North Sea mussels produced high levels of 2-3 forms of HSP70 by 5 h (Fig. 2). Mussels from the North Sea had lower mortality rates, oxygen consumption, nitrogen excretion, cadmium body burden, and energy expense in cadmium than did the Baltic mussels. Species and ecotypes differed predictably in response to stresses. G. duebeni survived elevated temperature longer than G. oceanicus. As a resident of a variable environment like a rock pool, G. duebeni has evolved adaptations to these variations. One adaptation may be an efficient HSP70 that is induced faster or at lower temperatures than that of G. oceanicus. The Baltic mussels have adapted to a low but constant salinity. This adaptation may have put them close to the edge of survival. Additional stresses reduce the vigor of this ecotype, or coping with salinity stress confounds the ability to cope with another stress. It is possible that the mussel populations have not been separated long enough (Tedengren, 1990) for selection to mold randomly occurring genetic change into true adaptation to lower salinity and estuarine resilience, as has happened with the amphipods. The North Sea mussels and G. duebeni are the hardier variants and also produce more HSP70. HSP70 in this system is a predicter of pollution (cadmium) and natural stress (temperature) tolerance. The genetic divergence that has occurred between the mussel ecotypes and between the amphipod species illustrates both opposing viewpoints of the tolerance of estuarine versus oceanic species. Mussels adapting to lower (but constant) salinity have decreased resistance to at least one stressor (cadmium), while amphipods adapting to variable rock pool habitats have increased resistance to some stressors.
REFERENCES Bradley, B. P. & Ward, J. B. (1990). Mar. Env. Res., 28,471-5. Gilek, M., Tedengren, M. & Kautsky, N. (1992). Neth. J. Sea Res., 30, 11-21. Johannesson, K, Kautsky, N. & Tedengren, M. (1990). Mar. Ecol. Prog. Ser., 59, 211-9. Lindquist, S. & Craig, E. A. (1988). Annu. Rev. Genet., 22, 631-77. Murphy, L. S. & Belastock, R. A. (1980). Limnol. Oceanogr., 25, 16&5. Nover, L. (ed.) (1991). In Stress Proteins. CRC Press, Boca Raton, FL (1991). Tedengren, M. (1990). Ecophysioiogy and Pollution Sensitivity of Baltic Sea Invertebrates. PhD Thesis, Stockholm University, Sweden. Tedengren, M. & Kautsky, N. (1986). Ophelia, 25, 147-55. Tedengren, M., Arner, M. & Kautsky, N. (1988). Mar. Ecol. Prog. Ser., 41, 107-16.