Aquaculture Research, 2001, 32, 645±655
Response of ®sh membranes to environmental temperature T Farkas1, E Fodor1, K Kitajka1 & J E Halver2 1
Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, H-6701 Szeged, Hungary
2
School of Fisheries, University of Washington, Seattle, WA, USA
Correspondence: Tibor Farkas, Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, H-6701 Szeged, Hungary. E-mail:
[email protected]
Abstract The effect of temperature on ¯uidity, fatty acid and molecular species composition of liver and brain phospholipids in ®sh adapted or exposed to extreme temperatures was investigated. Membranes from cold-adapted ®sh were more ¯uid than those from warm-adapted ®sh. Ability to control membrane ¯uidity according to temperature appears in early ontogenesis and is ®rst evident in swim-up fry of carp. Red blood cells as well as neurons of adult carp can continuously adjust the ¯uidity of their external membranes to changing temperatures. Segregation of choline and ethanolamine phosphoglycerides from livers of ®sh adapted to a cold/warm environment showed an accumulation of molecular species containing a monoenic fatty acid in position sn-1 and a polyenic fatty acid in position sn-2 of the molecule in cold conditions. Model experiments using mixtures of synthetic 18:1/22:6 phoshatidylethanolamines and 16:0/18:1 phosphatidylcholines demonstrated the involvement of these molecular species in rendering the membranes less packed (more ¯uid) during adaptation to reduced temperatures. Keywords: brain, temperature, ¯uidity, adaptation, membranes.
phospholipids,
Introduction Temperature is the most important environmental factor affecting the activity of poikilothermic ã 2001 Blackwell Science Ltd
animals such as ®sh. Membranes are the ®rst targets affected by change of temperature and their lipid components respond immediately to this challenge. Expectedly, a decrease in temperature results in a decrease in motional freedom of acyl chains of constituent phospholipids and vice versa, with resulting alteration in membrane ¯uidity. In order to maintain structural and functional integrity of these structures, poikilotherms must counteract this effect of temperature and maintain proper ¯uidity in the new thermal environment. It can be hypothesized that organisms unable to adjust membrane physicochemical properties to temperature are not able to survive in a changing thermal environment. The response to temperature changes is expected to be rapid, reversible and should not depend on the thermal history of the species. The so-called `homeoviscous adaptation' of membrane ¯uidity was ®rst described by Sinensky (1974) for Escherichia coli and later extended to other species including ®sh (Behan-Martin, Jones, Bowler & Cossins 1993). Fish use several techniques to reach this goal, such as (i) altering the unsaturation of the constituent fatty acids; (ii) altering the polar head group composition of the constituent phospholipids; and (iii) altering the sterol to phospholipid ratio. Indeed, several studies show: 1 an accumulation of unsaturated fatty acids (Hazel & Prosser 1974; Williams & Hazel 1992; Wallrecht & Babin 1994); 2 an accumulation of wedge-shaped phospholipids such as phosphatidylethanolamine (Hazel & Landrey 1988; Hazel & Carpenter 1985); 645
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Aquaculture Research, 2001, 32, 645±655
Table 1 List of investigated species Species
Origin
Water temperature (°C)
Acerina cernua Catla catla Coregonus loverostris Cyprinus carpio Labeo rohita Oncorhynchus tshawytscha Pomadysis hasta Scorpaenichtis maromoratus
Finland (M) India (FW) Finland (FW) Hungary (FW) India (FW) USA (FW) South Chinese Sea (M) North Atlantic (M)
5 25 5±7 5, 22 22 10±15 27 5±10
M, marine; FW, freshwater.
3 a decrease in sterol to phosphlipid ratio in membranes of ®sh adapted to a cold environment (Wodtke 1978). However, an increase in the number of double bonds alone does not explain the increased ¯uidity in cold temperatures because biophysical characteristics (surface area, thermotropic phase transition) of phospholipids containing unsaturated fatty acids are rather close (Coolebar, Beroe & Keough 1983; Evans, Williams & Tinoco 1987), and phosphatidylethanolamine contracts the head group region of membranes (Michaelson, Horwitz & Klein 1974). In this paper we will show that alteration in molecular composition of the major phospholipids could be one of the factors responsible for proper ¯uidity relationships during thermal adaptation; this response is rather rapid and appears in the early stage of ontogenesis. The data presented here are based on investigations carried out and published from this laboratory. Materials and methods Animals Table 1 gives the data of ®sh species including location of sampling and the water temperature. Livers and brains were removed immediately after ®sh were captured and were placed either in liquid nitrogen or chloroform±methanol (2:1 v/v).
processing. Phospholipids and neutral lipids were separated on silicic acid column chromatograpy using chloroform to elute the neutral lipids and methanol to obtain the polar lipids (phospholipids). Phospholipids were further segregated on thin-layer chromatographic (TLC) plates (E. Merck, Darmstadt, Germany) according to the polar head group using the technique of Fine & Spercher (1982). Choline and ethanolamine phosphoglycerides were removed from the plates into chloroform±methanol±water (49:49:1 v/v). The extract was stored in benzene at ±70 °C as descirbed above. Fatty acid methyl esters were obtained after transesteri®cation in absolute methanol containing 5% HCl at 80 °C for 3 h. They were segregated on a capillary column (Nukol, 30 m 3 0.25 mm; Supelco, Bellefonte, PA, USA) at 180 °C using a Hewlett Packard Model 6890 Gas chromatograph. Separation of nerve cells Nerve cells were separated according to Tocher, Mourente & Sargent (1992). Freshly removed brains were collected into Hanks' balanced solution and chopped. The chopped cells were sieved through sterile nylon gauzes (100 and 30 mm) and the cell suspension was centrifuged and the cells were collected into sterile physiological solution containing streptomycin and penicillin. Germination of ®sh eggs
Extraction and separation of lipids Lipids were extracted according to Folch, Lees & Sloane-Stanley (1957). The lipid extract was transferred into benzene containing 0.01% butylated hydroxytoluene and stored at ±70 °C until 646
Carp (Cyprinius carpio) and salmon (Oncorhynchus tshawytscha) eggs were fertilized at 20 °C and 10 °C, respectively, and germinated at two extreme temperatures (26 °C and 18 °C for carp and 8 °C and 15 °C for salmon). Embryos were sampled at the ã 2001 Blackwell Science Ltd, Aquaculture Research, 32, 645±655
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four- to eight-cell stage, at the appearance of heart beat, eye pigmentation, free moving larvae and resorption of yolk sac for carp, and at the four- to eight-cell stage stage for salmon. Molecular species composition Molecular species composition of choline and ethanolamine phosphoglycerides was determined according to Takamura, Narita, Urade & Kito (1986) after digestion by phospholipase C of Bacillus cereus (Sigma, St Louis, MO, USA). The puri®ed diradylglycerols were anthroxylated and further separated onto subclasses (diacyl, alkylacyl, alkenylacyl) on TLC plates (10 3 10, E. Merck). Segregation into individual molecular species took place using a Hitachi-Merck high-performance chromatograph and an LC-18 column (1 mm 3 30 cm, Supelco, Bellefonte, PA, USA) and acetonytrile±isopropylalcohol (80:20 v/v). To quantitate derivatized 12:0/12:0, diglyceride was added to the spots of the phospholipid subclasses. Identi®cation was made using a mixture of synthetic phospholipids as described elsewhere (Fodor, Jones, Buda, Kitajka, Dey & Farkas 1995) and also using the Rf data of Bell & Dick (1991). Membrane ¯uidity Membrane ¯uidity was determined from phospholipid vesicles as well as from isolated membranes or cells. 1,6-Diphenyl-1,3,5-hexatriene (DPH), 3-[p(6-phenyl-1,3,5-hexatriene)phenyl]-proprionic acid (DPH-PA), 2-(N-9-anthroyloxy)stearic acid (2-AS), 12-(N-anthroyloxy)stearic acid (12-AS) and 16(Nanthroyloxy)stearic)acid (16-AP) (Molecular Probes Inc., Eugene, OR, USA) in tetrahydrofurane were added to the membranes and the temperature dependence of steady-state ¯uorescence anizotropy parameter, RSS, was determined using a computercontrolled Hitachi MPF 2 A spectrophoto¯uorimeter in heating mode (1 °C min±1). Steady-state ¯uorescence anisotropy parameter was calculated according to the equation: RSS = (I(vv) ± ZI(vh)/I(vv) + 2ZIvh) where I(vv) and I(vh) are, respectively, the vertically and horizontally polarized emission light intensities when exciting with vertically polarized light and Z = 0.92. Electron spin resonance spectroscopy Electron spin resonance spectroscopy on nerve cells was carried out using a computerized ECS-106 ã 2001 Blackwell Science Ltd, Aquaculture Research, 32, 645±655
Fish Membranes and temperature T Farkas et al.
(Bruker, Billerica, MA, USA) instrument and a spin-labelled phospholipid, 14-doxyl-phosphatidylglycerol spin label (14-PGSL). The rate of motion was quantitated according to Kivelson (1972) and the effective order parameters according to Jost, Libertini, Herbert & Grif®th (1971). Separation of subcellular fractions Tissues were homogenized in 0.25 mol L±1 sucrose using a Potter homogenizer. After sedimenting the cell debris at 900 g, mitochondria were obtained by centrifugation at 9000 g. The supernatant was further separated into plasma membrane and microsomal fractions by sucrose density centrifugation according Koizumi et al. (1981). Labelling of liver slices Liver slices, obtained from carp livers, were incubated at two extreme temperatures in the simultaneous presence of [1-14C]-oleic acid and [3H]-palmitic acid (equal speci®c radioactivity and radioactive concentration) for various lengths of times. Phosphatidylcholines and phosphatidylethanolamines were separated from the tissues and subjected to phospholipase A2 digestion by Crotalus adamenteus (Sigma). The produced free fatty acids and lyso compounds were separated using thin-layer chromatogaphy, and counted. Results Structural order of membranes and sensitivity adaptation of ¯uidity to temperature Experiments on brain cells are shown here (Buda, Dey, Balogh, HorvaÂth, Maderspach, JuhaÂsz, Yeo & Farkas 1994). Brain was selected for this purpose because its lipid composition is independent of ingested food; thus, any change in its compositional and biophysical characteristics can be attributed to the temperature. Carps, both summer (25 °C) and winter (5 °C) adapted, were collected, their brains removed and neurons prepared as described above. Other groups of ®sh were kept in aquaria and the water temperature was raised or decreased by 0.5 °C h±1. The rotational correlation time of 14PGSL was determined at the beginning and end of the experiment (14-PGSL is trapped in the plasma membrane). As expected, the membranes of cold647
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Figure 1 (a) Temperature dependency of 14-PGSL embedded in plasma membranes of carp brain cells adapted to summer and winter temperatures or shifted to the opposite temperature. d, Warmadapted; m, cold-adapted; s, shift down; m, shift up. (b) Effect of temperature shift in vitro on ¯uorescence anisotropy of DPH-PA embedded in brain plasma membranes of carp adapted to 22 °C. Inset: Change in ¯uorescence anisotropy of DPH-PA in brain cells of carp adapted to 5 °C and shifted to 22 °C.
adapted neurons were more ¯uid than those of warm-adapted ones (Fig. 1a). Interestingly, up- or downshift of the water temperature of the aquarium resulted in rotation correlation times identical to those found for cold- and warm-adapted ®sh. From the heating and cooling rate it can be calculated that the experiment lasted 20 h, i.e. this time was quite suf®cient to allow membrane physical state to become adjusted temperature. However, more detailed experiments showed that this kind of response is even faster. When brain cells prelabelled with DPH-PA (trapped in the outer lea¯et of the membrane) from warm-adapted ®sh were added to an incubation medium at 5 °C there was an immediate decrease in the anisotropy parameter, and they reached maximum ¯uidity within 10 min. In contrast, up-shifting of the incubation temperature to 25 °C re-established the original ¯uidity of the plasma membrane of 648
neurons. In another series of experiments, prelabelled cells from cold-adapted ®sh were dropped in the cuvette of a spectrophoto¯uorimeter set to 25 °C. Rigidi®cation of membrane started immediately and reached maximum within 10 min (Fig. 1b). Similar results were obtained on red blood cells (Dey, Szegletes, Buda, NemcsoÂk & Farkas 1993). Williams & Hazel (1994) demonstrated that trout hepatocyte plasma membranes reacted to a drop in incubation temperature with an increase in ¯uidity within 6 h. Control of membrane ¯uidity by ®sh embryos Carp embryos were unable to adjust the ¯uidity of their mitochondria endoplasmic reticula and plasma membranes until absorption of yolk. In contrast, membranes of eggs germinating at 18 °C were more rigid than those germinating at 26 °C. Data on ã 2001 Blackwell Science Ltd, Aquaculture Research, 32, 645±655
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Fish Membranes and temperature T Farkas et al.
Figure 2 Temperature dependency of ¯uorescence anizotropy parameter of DPH embedded in mitochondria of developing ®sh embryos. (a) Carp embryos at the stage of heartbeat. j, 19 °C; h, 27 °C. (b) Salmon embryos at the 4±8-cell stage. j,15 °C; h, 9 °C.
mitochondrial membranes at the stage of start of heart beat are shown in Fig. 2a. In contrast, mitochondria of salmon embryos as early as the four- to eight-cell stage become more ¯uid at 9 °C than at 15 °C. To reach the heartbeat stage for carp took 23 h and 32 h at 18 °C and 27 °C (Fig. 2b) respectively, whereas for salmon, this stage took 8 h and 15 h at 9 °C and 15 °C respectively. Reorganization of phospholipids in response to temperature Alterations in membrane ¯uidity in response to changes in the ambient temperature (Farkas & Roy 1989) might be a result of adaptive changes of structural lipids caused by increase/decrease in ã 2001 Blackwell Science Ltd, Aquaculture Research, 32, 645±655
unsaturated fatty acids accompanied by a reorganization of molecular composition and hence molecular architecture of phospholipids. It has been demonstrated that introduction of a double bond into the molecule of a saturated fatty acid brings about a drastic change in surface area and melting point of phospholipids (Evans et al. 1987; Coolebar et al. 1983). To determine if such response takes place in ®sh livers, liver slices were incubated at two extreme temperatures in the presence of [3H]palmitic acid and [14C]-oleic acid and ratio of 14C to 3H was determined in position sn-1 and sn-2 of phosphatidylethanolamines and phosphatidylcholines. Figure 3 shows that liver slices preferentially incorporate oleic acid rather than palmitic acid into the sn-1 position of phosphatidylethanolamines and 649
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phosphatidylcholines at 5 °C. This indicates a rapid temperature-mediated restructuring of membrane phospholipids in ®sh livers. Relationship between molecular composition of phospholipids and adaptation temperature in ®sh liver and brain Phosphatidylcholines and phosphatidylethanolamines from brains and livers of ®sh, evolutionarily or seasonally adapted to two extreme temperatures, were subjected to analysis by high-performance liquid chromatography (HPLC) (Dey et al. 1993; Buda et al. 1994; Fodor et al. 1995). This technique resolved these phospholipids into 20±25 different molecular species differing in fatty acids in position sn-1 and sn-2 of the molecule. To gain a generalized picture, marine and freshwater ®sh were collected from different geographical locations (from Arctic to subtropic regions). In Tables 2 and 3, the most
Figure 3 Ratios of 14C:3H in position sn-1 and sn-2 of phosphatidylcholines and phosphatidylethanolamines of carp liver slices incubated at 5 °C and 20 °C. Black bars, phosphatidylcholine; open bars, phosphatidylethanolamine
important molecular species of liver phosphatidylcholines and brain phosphatidylethanolamines of ®sh adapted to extreme temperatures are given. Phosphatidylcholines followed the same trends. In Table 4 shows seasonal changes in molecular species composition of carp liver diacyl phosphatidylcholines and diacyl phosphatidylethanolamines. In all cases, independent of the location of sampling and type of ®sh (freshwater or marine), levels of certain sn-1 monoenic and sn-2 polyenic molecular species (18:1/20:5, 18:1/22:6, 18:1/20:4) were higher in cold environments. This would indicate that these phospholipid molecules play a speci®c role in thermoadaptation processes of ®sh. In addition, in all cases there was a reduction in levels of 16:0/18:1 phosphatidylcholine and phosphatidylethanolamine species in cold, thus resulting in increases in the and 18:1/22:6±16:0/18:1 ratios. Role of 18:1/22:6 phosphatidylethanolamine and phosphatidylcholine on determining the structural order of arti®cial membranes Figure 1a shows that brain cell membranes from cold-adapted ®sh are more ¯uid than those from warm-adapted ones. The question arises whether or not the accumulation of sn-1 monoenic and sn-2 polyenic phospholipid molecular species is casually related to this increase in ¯uidity. Theoretically, these kinds of molecules may affect the packing order of membranes because of the presence of a cis double bond instead of a saturated fatty acid in position sn-1. To answer the question, model experiments were carried out on phospholipid vesicles made of a mixture of either 16:0/18:1 phosphatidylethanolamine and 16:0/22:6 phosphatidylcholine in a ratio of 25% to 75% or 18:1/22:6
Table 2 Selected molecular species from liver phosphatidylcholines (% w/w)
18:1/20:5 18:1/22:6 16:0/22:6 18:0/22:6 18:1/18:1 16:0/18:1 16:0/16:0
L. rohita
P. hasta
C. loverostris
S. marmoratus
0.3 6.8 16.0 11.0 3.4 17.6 10.1
0.5 6.5 23.3 7.5 2.6 7.8 tr
8.7 25.2 19.7 10.3 0.6 5.8 3.2
3.6 20.4 21.3 2.5 tr 3.2 tr
tr, trace.
650
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Fish Membranes and temperature T Farkas et al.
Table 3 Selected molecular species in brain phosphatidylethanolamines (% w/w)
18:1/20:5 18:1/22:6 16:0/22:6 18:0/22:6 18:1/18:1 16:0/18:1 16:0/16:0
Table 4 Seasonal changes in molecular species composition of liver phosphatidylcholines and phosphatidylethanolamines of Cyprinus carpio
C. carpio 5 °C
C. carpio 5 °C
A. cernua 22 °C
C. catla 22 °C
0.5 14.1 12.9 24.8 6.6 4.0 0.3
2.0 14.4 26.1 32.4 2.1 2.2 0.2
0.2 5.1 13.8 39.1 3.7 5.5 0.6
0.5 2.5 27.3 49.1 0.4 3.6 0.4
phosphatidylethanolamine and 16:0/22:6 phosphatidylcholine in the same ratio, and ¯uidity was determined at different membrane depths using speci®c ¯uorescent labels (2-AS, 12-AS, 16-AP). Figure 4a shows that 16:0/18:1 phosphatidylethanolamine rendered the vesicles more ordered in the upper membrane regions reported by 2-AS, whereas 18:1/22:6 had the opposite effect, even in the middle of the bilayer reported by 12-AS (Fig. 4b). This decrease in membrane packing was accompanied with a decrease in solid gel to liquid crystalline thermotropic phase transition temperature by about 10 °C (Fodor et al. 1995). Discussion The ®sh investigated in this study originate from different thermal environments and from different parts of the globe and consume foods of different fatty acid compositions. Despite these differences they gave essentially the same response to the ambient temperature even if it varied seasonally, i.e. accumulation of phospholipid molecules in a cold environment rich in species of sn-1 monoenic and sn-2 polyenic fatty acids. Dietary fatty acids did not determine the molecular composition of membrane phospholipids, and membrane ¯uidity follows our recent investigations on ¯uidity and molecular species composition of liver plasma membranes of four freshwater ®sh species of different feeding habits, adapted to winter temperatures. In every case the level of sn-1 monoenic and sn-2 polyenic phospholipid molecular species was equally high despite differences in gross fatty acid compositions (Roy, Fodor, Kitajka & Farkas 1999). Accumulation of these phospholipids at reduced temperatures ã 2001 Blackwell Science Ltd, Aquaculture Research, 32, 645±655
18:1//20:5 18:1/22:6 16:0/22:6 18:1/20:4 18:0/22:6 18:0/20:4 18:1/18:1 16:0/18:1 16:0/16:0
Phosphatidylcholines (% w/w)
Phosphatidylethanolamines (% w/w)
Summer
Winter
Summer
Winter
0.7 3.9 22.9 4.3 12.4 2.3 2.8 23.1 3.7
4.7 6.9 23.5 8.2 6.7 1.9 3.0 11.8 0.8
tr 5.4 23.1 4.8 17.0 23.5 tr 3.4 1.4
0.9 10.2 15.8 17.4 6.2 5.7 0.6 1.1 0.3
appears to be a general phenomenon. Tanaka, Ikita, Ashida, Motoyama, Yamaguchi & Satouchi (1996) reported the same for the nematode Caenorhabditis elegans, Farkas, Dey, Buda & Halver (1994) for the shrimp Pandulus borealis, and Lahdes, Balogh, Fodor & Farkas (2000) for Gammarus ssp., from the Baltic Sea. Although more direct investigations would be necessary to draw a more generalized picture, it can be hypothesized that these phospholipid molecular species play an important role in controlling structural and functional integrity of membranes of ®sh and other aquatic poikilotherms. Certain tissues may respond immediately to changes in temperature (Fig. 1a and b) whereas others delay, and this response may appear during embryonic life (Fig. 2a), although interspeci®c differences may exist. It is important to note that only species able to acclimatize to reduced temperatures can respond in the above way. A study in progress in this laboratory shows that frogs hibernating in winter cannot increase the ¯uidity of their liver phospholipids and accumulate phospholipid molecular species similar to ®sh adapted to cold although their phospholipids are relatively rich in long-chain polyunsaturated fatty acids. The molecular shape of phosphatidylethanolamines, in contrast to phosphatidylcholines, is conic, and it has been shown that conic-shaped phospholipids accumulate in the cold (Wieslander et al. 1980; Farkas, Nemecz & Csengeri 1984; Pruit 651
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Figure 4 Temperature dependency of ¯uorescence anisotropy of 2-AS, 12-AS and 16-AP embedded in (a) 100% 16:0/ 22:6 phosphatidylcholine (d, 2-AS; m, 12-AS; j, 16-AP) or in 75% 16:0/22:6 phosphatidylethanolamines (open symbols) or in (b): 100% phosphatidylcholine (closed symbols) or 75% 16:0/22:6 phosphatidylcholine (open symbols) and 25% 18:1/22:6 phosphatidylethanolamine.
1988; Hazel & Carpenter 1985; Hazel & Landrey (1988). Wieslander et al. (1980) suggested that conic-shaped molecules contribute to bilayer stability in cold conditions. Evidently, the sn-1 monoenic and sn-2 polyenic phosphatidylethanolamines (and also phosphatidylcholines) are more conic than their sn-1 saturated, sn-2 polyunsaturated homologues. Computer modelling of different phospholipid molecules has shown that the surface area of 18:1/22:6 phosphatidylcholine is roughly 30% larger than that of 18:0/22:6 phosphatidylcholine (Zabelinski, Brovstina, Cheboterova, Gorbunova & Krivschenko 1995). The presence of a cis double bond in the sn-1 position of phosphatidylethanolamine not only decreases packing order in membranes but also prevents molecular interactions in head group region of membranes. Figure 4a and b shows that, although 16:0/18:1 phosphatidylethanolamine increased the rigidity in the upper segment of the bilayer, 18:1/22:6 phosphatidylethanolamine decreased it. Indeed, this is not caused by differences in molecular shape of unsaturated fatty acids in position sn-2 of the molecule. Molecular modelling of different phospholipids showed that 22:6, like 16:0, adopts an extended rod-like con®guration (Applegate & Glomset 1991). Thus, it appears that the best solution by ®sh and some other poikilotherms to control membrane ¯uidity under changing thermal environment is to manipulate the content of molecular species with a monoenic fatty acid in 652
position sn-1 and a polyenic fatty acid in position sn-2 of phospholipids. In discussing the role these molecular species play in membrane processes, their propensity to form non-bilayer phase should also be considered. The presence of non-bilayer-forming lipids is required for the activity of some enzymes, such as phospholipase A2 (Zidovetzki 1997) or Ca2+-ATPase (Yang & Hwang 1996). Phosphatidylethanolamine (18:1/ 22:6) has been found to exhibit a very low bilayerto-non bilayer transition temperature (Giorgione, Epand, Buda & Farkas 1995), and this may contribute to the maintenance of normal enzymatic activities in ®sh during long- or short-term exposure to cold. It has also been demonstrated that this molecule increases the activity of protein kinase C (Giorgione et al. 1995). Formation of molecular species such as 18:1/ 22:6 requires concerted biochemical processes such as the production of oleic acid from its precursor and esteri®cation of oleic acid to position sn-1 after deacylation. It has been demonstrated that the induction of delta-9 desaturase in carp liver takes about 12 h (Wodtke & Cossins 1991). Also, Tiku, Gracey, Macartney, Beynon, & Cossins (1996) demonstrated that detectable accumulation of mRNAs in ®sh livers takes place within 24 h and is at a maximum after 5 days. Differences in ¯uidity of embryos at the four- to eight-cell stage of carp and salmon might be caused by insuf®cient time for induction of the delta-9 desaturase. However, other ã 2001 Blackwell Science Ltd, Aquaculture Research, 32, 645±655
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reaction routes, such as desaturation of the existing 18:0/22:6±18:1/22:6, deacylation of 18:1/18:1 phosphatidylethanolamine in position sn-2 and reacylation with 22:6, should also be considered. Although more direct investigations are required to support this idea, it must be mentioned that in phosphatidylethanolamines of cold-adapted freshwater and marine ®sh, the level of 18:1/18:1 species is lower than in their warm-adapted counter partners (Table 2). The advantage of this reaction would be that no involvement of delta-9 desaturase is required, only the activation of phospholipase A2 and an acyltransferase placing 22:6 in the position sn-2. This hypothesis is supported by Neas & Hazel (1985), who showed that the speci®c activity of phospholipase A2 is higher in the microsomes of rainbow trout acclimatized to 5 °C than in those acclimatized to 20 °C. The alternative route could also be possible: deacylation of 18 0/22:6 phosphatidylethanolamine at position sn-1 and reacylation with 18:1. This route, however, requires the involvement of phospholipase A1, which was demonstrated from carp liver (Farkas & Roy 1989). It is possible that different ®sh species follow different routes to achieve the main goal: to increase levels of sn-1-unsaurated, sn-2-polyunsaturated phospholipid molecular species to maintain structural and functional integrity of their cellular membranes in the cold. This assumption is justi®ed by observations that there are differences in abilities to convert linolenic acid to long-chain polyunsaturated fatty acids: in contrast to marine ®sh (Tocher & Dick 1990; Mourente & Tocher 1993), freshwater ®sh have high capacity for chain elongation and desaturation (Farkas, Csengeri, Majoros & OlaÂh 1980; Kayama, Hirata & Hisai 1986; Olsen, Henderson & McAndrew 1990; Muci, Vonghia & Gnoni 1992). Thus, freshwater ®sh rely on their own capacity to obtain 22:6 for formation of phospholipids, whereas marine ®sh must rely on their diet! However, the question has to be answered whether ®sh adapted to a given temperature and exposed only to minor changes of their thermal environment (like subtropical ®sh) can respond in the same way as described here or cannot tolerate the temperature change and would die under such stress conditions. References Applegate K.R. & Glomset G.A. (1991) Effect of acyl-chain unsaturation on the conformation of model
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