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TMAO and other organic osmolytes in the muscles of amphipods (Crustacea) from shallow and deep water of Lake Baikal
Comparative Biochemistry and Physiology, Part A 142 (2005) 58 – 64 www.elsevier.com/locate/cbpa
TMAO and other organic osmolytes in the muscles of amphipods (Crustacea) from shallow and deep water of Lake Baikal Irene Zerbst-Boroffka a,*, Ravil M. Kamaltynow b, Stefan Harjes c, Evamaria Kinne-Saffran c,T, Ju¨rgen Gross a,1 a
Institut fu¨r Biologie der Freien Universita¨t Berlin, Stoffwechsel-und Systemphysiologie Grunewaldstr. 34 D-12165 Berlin, Germany b Limnological Institute Irkutsk, Russia c Max Planck Institut fu¨r molekulare Physiologie, Dortmund, Germany Received 25 April 2005; received in revised form 23 July 2005; accepted 24 July 2005
Abstract Concentrations of trimethylamine oxide (TMAO) and other Fcompatible_ osmolytes were analyzed in the muscle tissue of Lake Baikal amphipods (Crustacea) in relation to water depth of the freshwater Lake Baikal. Using HPLC and mass spectrometry, glycerophosphoryl choline (GPC), betaine, S-methyl-cysteine, sarcosine, and taurine were detected for the first time in freshwater amphipods. These osmolytes were frequently found in the five species studied but mixtures were too complex to be quantified. The pattern of these osmolytes did not change with respect to water depth. The TMAO concentration, however, was significantly higher in the muscle tissue of amphipods living in deep water than of those living in shallow water, which supports the hypothesis that TMAO acts as a protective osmolyte at increased hydrostatic pressure. We propose that eurybathic amphipods, exposed to raised hydrostatic pressure in the extremely deep freshwater Lake Baikal, have elevated TMAO levels to counteract the adverse effect of high pressure on protein structure. The elevated intracellular osmotic pressure is balanced by upregulating the extracellular hemolymph NaCl concentration. D 2005 Elsevier Inc. All rights reserved. Keywords: Abyss; Amphipoda; Compatible osmolytes; Crustacea; Hydrostatic pressure; Lake Baikal; TMAO
1. Introduction Lake Baikal has a rich fauna of amphipods comprising more than 257 species, most of them endemic. They have colonized all regions and habitats in Lake Baikal down to 1600 m of water depth (Kozhova and Izmest’eva, 1998). Below 200 m, the temperature remains constant at 3.8 -C. Seasonal temperature differences do not exceed 4 -C in water depths of 50 –200 m (Kozhova and Izmest’eva, 1998). * Corresponding author. Tel.: +49 30 83853989/8324305; fax: +49 30 83853886. E-mail address: [email protected] (I. Zerbst-Boroffka). 1 Current address: Biologische Bundesanstalt fu¨r Land-und Forstwirtschaft, Institut fu¨r Pflanzenschutz im Obstbau, Dossenheim, Germany. T Deceased December 6, 2002. 1095-6433/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2005.07.008
The freshwater lake is characterized by full oxygen saturation (Martin et al., 1993) and extremely low electrolyte concentrations along the entire water column (Falkner et al., 1991, 1997). Thus, Lake Baikal provides the unique chance for field studies on biochemical adaptations to deep water without the additional complication of salinity zonation. The osmotic hemolymph concentrations in Baikalian amphipods living in shallow water are 270– 330 mosmol/kg H2O, which is in the range of palaearctic freshwater species (Zerbst-Boroffka, 1999). For some eurybathic species, however, it was demonstrated that hemolymph osmolalities and NaCl concentrations were related to water depth when analyzed immediately after capture from shallower (50 – 200 m) or deep water (950 – 1200 m). In Ceratogammarus dybowskii, Acanthogammarus reicherti, and Parapallasea
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lagowskii the hemolymph osmolality measured by freezing point depression was significantly higher ( p < 0.01) when compared between specimens from deep and from shallow water (C. dybowskii + 30 mosmol/kg H2O, A. reicherti +40 mosmol/kg H2O, and P. lagowskii + 50 mosmol/kg H2O) (Zerbst-Boroffka et al., 2000). The same study showed that these differences in osmolality match differences in the NaCl concentration. We argued that upregulation of the hemolymph osmolality is adaptive for abyssal species and populations. In freshwater environments, maintenance of hemolymph osmolality at a higher level than in shallow water costs more metabolic energy, raising the question of physiological cause and benefit of this investment. Field studies using hemolymph or plasma of freshly collected marine animals from different water depths are rare. The few data available indicate higher osmotic and inorganic ion concentrations in the hemolymph of species collected in deep than in shallow water. This was shown in osmoconforming Crustacea where higher osmolalities were attributed to higher medium salinity at greater water depth (Forward and Fyhn, 1983). It was also observed in hypoosmotically regulating teleostean fishes (Shelton et al., 1985; Gillett et al., 1997; Treberg and Driedzic, 2002). Organic osmolytes involved in cellular volume and osmotic regulation such as urea, neutral amino acids, methylamines, polyols and sugars were studied in muscles of marine animals captured in deep and shallow water, especially in teleosts and elasmobranchs (Gillett et al., 1997; Raymond and DeVries, 1998; Kelly and Yancey, 1999; Yancey et al., 2001; Treberg and Driedzic, 2002; Yancey et al., 2002) but also in crustaceans (Kelly and Yancey, 1999). Deep-water species and populations have higher TMAO (trimethylamine oxide) levels than those of shallower water. This holds true not only for muscles but also for other organs in deep-sea fish, although quantitatively less (Treberg and Driedzic, 2002). In teleosts the increase of molar TMAO concentration in the muscle corresponds to the rise in the blood osmolality. The higher concentration of TMAO found in the tissue of shrimps and crabs living in deep water, is accompanied by a decrease of other organic osmolytes, especially glycine in shrimps and betaine in crabs. This decrease, however, is not sufficient to compensate for the increased TMAO concentration (Kelly and Yancey, 1999). TMAO is known to counteract adverse effects by temperature, salinity, high urea and hydrostatic pressure (Yancey et al., 1982, 2001, 2002; Somero, 1992; Yancey, 1994; Gilles, 1997; Yancey and Siebenaller, 1999; Se´bert et al., 1997). The protein-stabilizing capability of TMAO is proposed as its main functional role in deep-sea animals as well as in the osmoconforming crustaceans and elasmobranchs as in hyporegulating teleosts (Gillett et al., 1997; Kelly and Yancey, 1999; Yancey et al., 2001, 2002). In marine field studies, the effect of increased hydrostatic pressure and salinity with water depth on physiology cannot be studied separately. In Lake Baikal, however, it is possible to investigate the relation of TMAO levels to hydrostatic
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pressure apart from salinity in the field and, important, in strong hyperregulators. Thus, we determined the concentrations of TMAO in amphipods collected from deep and shallower water of Lake Baikal. We also determined some other organic osmotic effectors commonly involved in cell volume and osmotic regulation and known as Fcompatible osmolytes_ (Gilles, 1997). Our goal was to explore whether the higher extracellular NaCl concentrations of deep-water amphipod species and populations (Zerbst-Boroffka et al., 2000) correlate with higher tissue TMAO levels and whether other intracellular organic osmolytes are affected.
2. Materials and methods 2.1. Animals and their collection Six benthic amphipod species were collected during an expedition with the RV ‘‘Titov’’ from sublittoral/upper bathyal and abyssal water zones in early October 2001 at temperatures of 0 – 5 -C: Acanthogammarus albus (vertical distribution = 5 – 825 m), A. lappadeus lappadeus (= 11 – 130 m), A. grewingki (= 100 – 1380 m), A. reicherti (= 25 – 1371 m), Ceratogammarus dybowskii (= 82 – 1371 m), Parapallasea lagowskii (= 200 – 1350 m). A beam trawl (1.8 m) was used and trawling times were 10 and 20 min (6 km/h) up to a water depth of 200 m and 25 – 30 min for deeper zones. Ascending rate was about 0.5 m/s. All animals of the selected species were living after arrival at the surface and continued swimming when transferred into a bucket filled with cooled Lake Baikal-water (6 -C). Sixty minutes later, the water temperature did not exceed 8 -C. 2.2. Tissue preparation on board The tissue preparation started within minutes after arrival of the trawl at the water surface and took less than 2 min per specimen. The tissue preparation for all specimens was finished at the latest within 60 min. Animals were decapitated and opened ventrally. The dark colored intestine was discharged. The metasomal muscle tissue was removed, slightly blotted to reduce contaminating hemolymph, and transferred to pre-weighed test tubes on ice. The samples were stored in liquid nitrogen until processing at the Limnological Institute Irkutsk. 2.3. Processing of tissue samples Each tissue sample was prepared to analyze both (1) TMAO + TMA concentrations in TCA extracts and (2) other Fcompatible_ osmolyte concentrations in alcoholic extracts. Frozen tissue samples were brought to room temperature (3 min). Next, the fresh mass of the tissue sample was determined, then 100 AL of bi-distilled water were added. These samples were homogenized (1 –2 min) in the tube with a high-tensic steel homogenizer of adapted shape, boiled in
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a water bath (1 – 2 min) to stop any metabolic activity, weighed again to determine the unavoidable loss of homogenate (1.3 – 9%), and centrifuged (30 min) at 14,000 g. The supernatant was divided into two parts. One aliquot (50 AL) was deproteinized by adding 200 AL TCA (trichloroacetic acid, 5% wt/v), vortexed, centrifuged, and the protein free supernatant used for determination of TMAO + TMA. The second aliquot (30 – 50 AL) was transferred to a preweighed tube and the exact sample volume determined (assuming a density of 1g/mL), deproteinized with 50 AL ethanol, vortexed, and centrifuged. The TCA extracts and the alcoholic extracts were transferred to Berlin and Dortmund for chemical analysis. 2.4. Chemical analysis of TMAO (plus endogenous TMA) in the TCA extract Analysis was performed according to Wekell and Barnett (1991) but the method was adapted for small volumes. The small weight of the tissue samples did not allow to measure both total TMAO + TMA and TMA separately in one sample. TMAO was reduced by a mixture of FeSO4 plus EDTA (ethylene diamine tetraacetic acid) and the total TMA (trimethyl amine) extracted into toluene plus KOH. Adding 500 AL of the toluene layer and 1.5 mL picric acid yielded the picrate derivative of total TMA. Two aliquots of 500 AL were quantified colorimetrically at 410 nm in quartz cuvettes. The absorbance was in the same range as those of standard solutions, which were linearly related to concentrations of 0.125 – 3.000 Amol/mL TMAO. Standards were treated identical with each run. The measured TMAO concentration was used to calculate the TMAO (+ TMA) muscle content as micromole per gram fresh mass (fw). Since the endogenous TMA content in animals is low (Kelly and Yancey, 1999; Treberg and Driedzic, 2002) we refer to TMAO + TMA as TMAO in the following. 2.5. Chemical analysis of other organic osmolytes in the alcoholic extract The alcoholic extract was centrifuged (17,500 g, 15 min), the supernatant separated and evaporated in a vacuum centrifuge (Speedvac SC 110, Savant). The dry remainder was dissolved in 50 AL HPLC buffer (50 mg L 1 CaNa2EDTA (calcium disodium ethylene diamine tetraacetic acid) in bi-distilled water). Aliquots of 2 –30 AL were analyzed by HPLC (Waters, Eschborn, Germany) equipped with a Sugar-Pak 1 column (flow rate at 0.5 mL/min). The column combines gel filtration and ion exchange chromatography using calcium cations bound to the column matrix. The signal (mV) of a refractometer (Waters 410) was recorded versus time. Samples were compared with synthetic compounds. For mass spectrometry, aliquots of ca. 30 AL of the dissolved supernatant from 2 animal samples were analyzed by HPLC as described above and 1 mL fractions of the
eluate were collected. The fractions were evaporated and dissolved in a mixture of saturated 2-cyano-3-(4-hydroxyphenyl)-acrylic acid (a-cyano-4-hydroxycinnamic acid) in 0.1% trifluoroacetic acid and 50% acetonitrile. One microliter of each sample was spotted on a steel plate and airdried. MALDI-TOF spectra were recorded using a Voyager DE (Perspective Biosystems). 2.6. Statistics Values are expressed as mean T S.D. (standard deviation). Differences were considered statistically significant when p < 0.02 by a Mann –Whitney U-Test. The software Statistica 5.5 (StatSoft Inc. Hamburg, Germany) was used for analysis.
3. Results The fresh mass (fw) of the 81 tissue samples collected for this study depended mainly on size of the specimens and varied from 29.4 – 321.2 mg. Eleven samples were < 50 mg and 12 samples > 200 mg. Most samples (ca. 75%) ranged between 50 and 200 mg fw. TMAO was analyzed in 74 tissue samples (Table 1). Concentrations were neither correlated with sex, body size, visible parasites, nor with the time spent at the surface until tissue preparation. Thus, all values were combined to calculate average values. No specimens with eggs in the marsupium were observed. Samples of three species collected in shallower water (Acanthogammarus albus, A. lappadeus lappadeus, A. grewingki) had TMAO concentrations between 6– 18.1 Amol/g fw, while in four species (Acanthogammarus reicherti, A. grewingki, Ceratogammarus dybowskii, Parapallasea lagowskii) collected from deep water concentrations between 28.4 –47.3 Amol/g fw were measured. Specimens of A. grewingki collected in deeper water (930 m) had higher TMAO concentrations in their muscle tissue than conspecimens from shallow water (170 m). These differences are statistically significant ( p < 0.02) (Table 1). C. dybowskii collected in water depths of 930 and 1170 m showed identically high TMAO concentrations.
Table 1 Concentrations of TMAO (+TMA) in the metasomal muscle of Baikalian amphipods collected from deep and shallower water Water depth (m)
Species
Concentration (Amol/g fw)
50 170 200 930
A. lappadeus lappadeus A. grewingki A. albus A. grewingki A. reicherti C. dybowskii C. dybowskii P. lagowskii
6.0 T 2.8 (5) 18.1 T 3.0 (16) 16.1 T 4.9 (13) 28.4 T 9.7 (7) 31.6 T 3.0 (5) 47.3 T 8.0 (11) 43.3 T 14.3 (8) 32.0 T 4.4 (9)
1170
Values are means T S.D. Number of animals in parentheses.
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Comparing the TMAO concentrations of all individuals of the different species collected in deep water (930 – 1170 m) and those collected in shallower water (50 – 200 m) the TMAO concentrations were significantly higher ( p < 0.001) in the deep-water specimens (Table 1). HPLC analyses of 54 tissue samples were performed. In all chromatograms a large peak appeared followed by several smaller peaks (Fig. 1). A comparison of the chromatograms of two specimens of P. lagowskii and one of A. grewingki in Fig. 1 demonstrates intraspecific and interspecific variability
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but also interspecific similarities of the peak patterns. Five of the smaller but frequently appearing peaks had the same retention time as taurine, glycerophosphoryl choline (GPC), betaine, S-methyl-cysteine, and sarcosine, respectively (Fig. 1, lower panel). Adding of these standard substances to the diluted alcoholic hemolymph extracts enlarged the corresponding peak areas and yielded a rough calculation of the concentration of the compounds in some samples (1– 6 mmol/kg H2O). GPC, betaine, S-methyl-cysteine, and sarcosine were detected by MALDI-TOF in the correspond-
Fig. 1. HPLC-chromatograms of tissue extracts of two Baikalian amphipod species and five standard solutions. Ordinate = detector signal (relative units), abscissa = migration time (minutes), monitoring started just after injection of the sample. Upper panels = runs of three specimens collected in deep water. The amplitude of the first big peak was set to 100%. Lower panel = runs of five individually measured standard solutions projected overlapping on one baseline.
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ing 1 mL eluates of two alcoholic tissue extracts. Other organic osmolytes commonly separated by the Sugarpak I column such as urea, polyols and glycine could not have been identified in the tissue extracts because their concentrations were probably lower than 1 mmol/kg fw. The identified substances were normalized to 1 mg fw to compare the osmolyte concentrations between specimens, between species and between deep and shallow water. Individual differences of the peak areas/mg fw of the identified substances were high and many animal samples did not contain detectable amounts of all of these osmolytes (Fig. 1). No significant differences with respect to water depth could be demonstrated.
4. Discussion Intracellular osmotic effectors are not well studied in freshwater crustaceans. Free amino acids were reported to contribute substantially (approximately 40%) to the total osmotically active solute in muscle of freshwater crayfishes (Gilles, 1979; Clark, 1985; Dooley et al., 2000). Absolute concentrations range between 2.4 and 79.0 mmol/kg fresh weight (fw) (Claybrook, 1983). The amino acid pools of Orconectes limosus, Astacus astacus, A. leptodactylus, and Cherax destructor range from 13.71 to 79 mmol/kg fw (Siebers, 1972; Marrewijk and Ravenstein, 1974; Schoffeniels, 1976; Dooley et al., 2000) and depend on season and ambient osmotic concentration. Glycine is the most prominent free amino acid in freshwater as well as in marine crustaceans. Glycine and taurine concentrations, important compounds of cellular volume and osmoregulation (Clark, 1985), were extensively studied in the euryhaline crab Eriocheir sinensis (Gilles, 1979) but scarcely in freshwater decapods. Glycine, taurine, and betaine concentrations are known for some crayfish species. The concentration of the secondary amino acid taurine is lower than 8 mmol/kg fw (Marrewijk and Ravenstein, 1974). Betaine has been detected in one of three crustacean freshwater species studied so far (Beers, 1967). Neither TMAO nor GPC were detected in freshwater crustaceans. In the present study, the osmolytes TMAO, GPC, betaine, sarcosine, S-methyl-cysteine and taurine were determined for the first time in freshwater amphipods (Fig. 1). In contrast to TMAO, discussed in detail below, their contribution to the total intracellular osmolality is negligible and differences correlated with water depth were not detected. The TMAO concentrations in muscle tissue of amphipods captured from deep water were significantly higher than those of amphipods captured from shallow water (Table 1), complementing our earlier finding that hemolymph osmolality increases with increasing water depth (Zerbst-Boroffka et al., 2000). Trawling and decompression might change membrane integrity and fluidity as well as oxygen management. However, any increase of osmolality in hyperregulating animals, such as the freshwater amphipods, is an energy-
requiring process and certainly not a by-product of membrane dysfunction due to decompression. In freshwater, a disorder of membrane function would rather lead to a decrease of osmotic concentration than to an increase. As shown previously (Zerbst-Boroffka et al., 2000), hemolymph lactate concentration, a fast signal for hypoxia, was low in Baikalian amphipods immediately after capture both from deep and from shallow water. Furthermore, amphipods captured from deep water needed two days for acclimation to a new (lower) steady-state concentration of osmolality. We therefore assume that the measured concentrations probably come close to the true values when determined immediately (< 60 min) after capture. The marked increase of TMAO concentration with increased water depth points to a function for adaptation and/or acclimatization to deep water in Baikalian amphipods. There is no salinity gradient and differences of temperature are negligible between the deeper and shallower water zones in the Lake Baikal. Thus, the observed increase in TMAO concentrations of amphipods living in deep water are not related to salinity or temperature. However, a relation to hydrostatic pressure – as discussed for marine animals – is a reasonable assumption. In marine crustaceans, the accumulation of TMAO with increased water depth seems to be quantitatively more important. On the other hand, the TMAO concentrations are already higher in marine species even in shallow water. For example, in muscles of three species of caridean shrimps living in shallow water the TMAO concentration is 76 mmol/kg fw and increases to 215 mmol/kg fw at about 1900 m water depth and to 244 mmol/kg fw at 2850 m water depth (Kelly and Yancey, 1999). Our data show an overall lower
LAKE BAIKAL CONCENTRATION
< 2 mOsm
low pressure: Shallow water amphipod
Hemolymph: 150 mM inorganic ions
≈ 300 mOsm
Cell content:
300 mOsm
high pressure: Deep water amphipod
Hemolymph: 170 mM inorganic ions
≈ 340 mOsm
Cell content:
300 mOsm + 40 mM (TMAO)
Fig. 2. Model mechanism of biochemical adaptation of amphipods to great water depths in the freshwater Lake Baikal. Circle = intracellular compartment; rectangle = extracellular compartment.
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concentration of TMAO in the freshwater amphipods of Lake Baikal when compared to marine crustaceans. However, the TMAO concentration but not that of the other osmolytes changes with water depth. The strong correlation of TMAO concentration and water depth supports the hypothesis that TMAO protects from adverse effects of elevated hydrostatic pressure of deep water, as proposed by several other authors for marine species. A function of other osmolytes identified by us could not be shown. In hyperregulating freshwater species as in the amphipods from Lake Baikal, elevation of the intracellular TMAO concentration without a corresponding reduction of other intracellular osmolytes substantially increases the osmotic gradient across cell membranes and needs to be balanced extracellularly. Indeed, extracellular osmolality increased with water depth, most of which is attributed to an increase of the extracellular NaCl concentration (Zerbst-Boroffka et al., 2000). Hemolymph concentrations of amphipods living in deep water were about 15 – 20 mmol/l NaCl and 20 –50 mosmol/kg H2O higher when compared to amphipods of shallow water. These differences are in the same range as the observed change of TMAO concentrations (Table 1). Thus, we propose that the adaptation and/or acclimatization of eurybathic benthic amphipods to the higher hydrostatic pressure at great depth of the freshwater Lake Baikal rely on two mechanisms (Fig. 2). First, TMAO levels in the muscle tissue increase to counteract the adverse effect of high pressure on protein structure (this study). Second, the increased intracellular osmolality due to TMAO is balanced by a concomitant increase of extracellular NaCl (ZerbstBoroffka et al., 2000). The benefit of accumulating intracellular TMAO as an osmoprotectant prevails the raised energy costs necessary to maintain an even higher osmotic gradient when living in the abyss.
Acknowledgements The help of Prof. M. A. Grachev who continuously promoted the project and saved the expedition from serious organisational problems is greatly appreciated. We are indebted to the captain and crew of the research vessel FTitov_ for their support, especially in rough weather. Special thanks to Brita Bazin for the reliable determinations of TMAO. Prof. Dr. R.K.H. Kinne is acknowledged for experimental support and stimulating discussions. Supported by DFG (436 RUS 18/9/01), RFBR 01-04-48970, RFBR 01-04-97214-a, and RFBR 04-04-48945-a.
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TMAO and other organic osmolytes in the muscles of amphipods (Crustacea) from shallow and deep water of Lake Baikal Irene Zerbst-Boroffka a,*, Ravil M. Kamaltynow b, Stefan Harjes c, Evamaria Kinne-Saffran c,T, Ju¨rgen Gross a,1 a
Institut fu¨r Biologie der Freien Universita¨t Berlin, Stoffwechsel-und Systemphysiologie Grunewaldstr. 34 D-12165 Berlin, Germany b Limnological Institute Irkutsk, Russia c Max Planck Institut fu¨r molekulare Physiologie, Dortmund, Germany Received 25 April 2005; received in revised form 23 July 2005; accepted 24 July 2005
Abstract Concentrations of trimethylamine oxide (TMAO) and other Fcompatible_ osmolytes were analyzed in the muscle tissue of Lake Baikal amphipods (Crustacea) in relation to water depth of the freshwater Lake Baikal. Using HPLC and mass spectrometry, glycerophosphoryl choline (GPC), betaine, S-methyl-cysteine, sarcosine, and taurine were detected for the first time in freshwater amphipods. These osmolytes were frequently found in the five species studied but mixtures were too complex to be quantified. The pattern of these osmolytes did not change with respect to water depth. The TMAO concentration, however, was significantly higher in the muscle tissue of amphipods living in deep water than of those living in shallow water, which supports the hypothesis that TMAO acts as a protective osmolyte at increased hydrostatic pressure. We propose that eurybathic amphipods, exposed to raised hydrostatic pressure in the extremely deep freshwater Lake Baikal, have elevated TMAO levels to counteract the adverse effect of high pressure on protein structure. The elevated intracellular osmotic pressure is balanced by upregulating the extracellular hemolymph NaCl concentration. D 2005 Elsevier Inc. All rights reserved. Keywords: Abyss; Amphipoda; Compatible osmolytes; Crustacea; Hydrostatic pressure; Lake Baikal; TMAO
1. Introduction Lake Baikal has a rich fauna of amphipods comprising more than 257 species, most of them endemic. They have colonized all regions and habitats in Lake Baikal down to 1600 m of water depth (Kozhova and Izmest’eva, 1998). Below 200 m, the temperature remains constant at 3.8 -C. Seasonal temperature differences do not exceed 4 -C in water depths of 50 –200 m (Kozhova and Izmest’eva, 1998). * Corresponding author. Tel.: +49 30 83853989/8324305; fax: +49 30 83853886. E-mail address: [email protected] (I. Zerbst-Boroffka). 1 Current address: Biologische Bundesanstalt fu¨r Land-und Forstwirtschaft, Institut fu¨r Pflanzenschutz im Obstbau, Dossenheim, Germany. T Deceased December 6, 2002. 1095-6433/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2005.07.008
The freshwater lake is characterized by full oxygen saturation (Martin et al., 1993) and extremely low electrolyte concentrations along the entire water column (Falkner et al., 1991, 1997). Thus, Lake Baikal provides the unique chance for field studies on biochemical adaptations to deep water without the additional complication of salinity zonation. The osmotic hemolymph concentrations in Baikalian amphipods living in shallow water are 270– 330 mosmol/kg H2O, which is in the range of palaearctic freshwater species (Zerbst-Boroffka, 1999). For some eurybathic species, however, it was demonstrated that hemolymph osmolalities and NaCl concentrations were related to water depth when analyzed immediately after capture from shallower (50 – 200 m) or deep water (950 – 1200 m). In Ceratogammarus dybowskii, Acanthogammarus reicherti, and Parapallasea
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lagowskii the hemolymph osmolality measured by freezing point depression was significantly higher ( p < 0.01) when compared between specimens from deep and from shallow water (C. dybowskii + 30 mosmol/kg H2O, A. reicherti +40 mosmol/kg H2O, and P. lagowskii + 50 mosmol/kg H2O) (Zerbst-Boroffka et al., 2000). The same study showed that these differences in osmolality match differences in the NaCl concentration. We argued that upregulation of the hemolymph osmolality is adaptive for abyssal species and populations. In freshwater environments, maintenance of hemolymph osmolality at a higher level than in shallow water costs more metabolic energy, raising the question of physiological cause and benefit of this investment. Field studies using hemolymph or plasma of freshly collected marine animals from different water depths are rare. The few data available indicate higher osmotic and inorganic ion concentrations in the hemolymph of species collected in deep than in shallow water. This was shown in osmoconforming Crustacea where higher osmolalities were attributed to higher medium salinity at greater water depth (Forward and Fyhn, 1983). It was also observed in hypoosmotically regulating teleostean fishes (Shelton et al., 1985; Gillett et al., 1997; Treberg and Driedzic, 2002). Organic osmolytes involved in cellular volume and osmotic regulation such as urea, neutral amino acids, methylamines, polyols and sugars were studied in muscles of marine animals captured in deep and shallow water, especially in teleosts and elasmobranchs (Gillett et al., 1997; Raymond and DeVries, 1998; Kelly and Yancey, 1999; Yancey et al., 2001; Treberg and Driedzic, 2002; Yancey et al., 2002) but also in crustaceans (Kelly and Yancey, 1999). Deep-water species and populations have higher TMAO (trimethylamine oxide) levels than those of shallower water. This holds true not only for muscles but also for other organs in deep-sea fish, although quantitatively less (Treberg and Driedzic, 2002). In teleosts the increase of molar TMAO concentration in the muscle corresponds to the rise in the blood osmolality. The higher concentration of TMAO found in the tissue of shrimps and crabs living in deep water, is accompanied by a decrease of other organic osmolytes, especially glycine in shrimps and betaine in crabs. This decrease, however, is not sufficient to compensate for the increased TMAO concentration (Kelly and Yancey, 1999). TMAO is known to counteract adverse effects by temperature, salinity, high urea and hydrostatic pressure (Yancey et al., 1982, 2001, 2002; Somero, 1992; Yancey, 1994; Gilles, 1997; Yancey and Siebenaller, 1999; Se´bert et al., 1997). The protein-stabilizing capability of TMAO is proposed as its main functional role in deep-sea animals as well as in the osmoconforming crustaceans and elasmobranchs as in hyporegulating teleosts (Gillett et al., 1997; Kelly and Yancey, 1999; Yancey et al., 2001, 2002). In marine field studies, the effect of increased hydrostatic pressure and salinity with water depth on physiology cannot be studied separately. In Lake Baikal, however, it is possible to investigate the relation of TMAO levels to hydrostatic
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pressure apart from salinity in the field and, important, in strong hyperregulators. Thus, we determined the concentrations of TMAO in amphipods collected from deep and shallower water of Lake Baikal. We also determined some other organic osmotic effectors commonly involved in cell volume and osmotic regulation and known as Fcompatible osmolytes_ (Gilles, 1997). Our goal was to explore whether the higher extracellular NaCl concentrations of deep-water amphipod species and populations (Zerbst-Boroffka et al., 2000) correlate with higher tissue TMAO levels and whether other intracellular organic osmolytes are affected.
2. Materials and methods 2.1. Animals and their collection Six benthic amphipod species were collected during an expedition with the RV ‘‘Titov’’ from sublittoral/upper bathyal and abyssal water zones in early October 2001 at temperatures of 0 – 5 -C: Acanthogammarus albus (vertical distribution = 5 – 825 m), A. lappadeus lappadeus (= 11 – 130 m), A. grewingki (= 100 – 1380 m), A. reicherti (= 25 – 1371 m), Ceratogammarus dybowskii (= 82 – 1371 m), Parapallasea lagowskii (= 200 – 1350 m). A beam trawl (1.8 m) was used and trawling times were 10 and 20 min (6 km/h) up to a water depth of 200 m and 25 – 30 min for deeper zones. Ascending rate was about 0.5 m/s. All animals of the selected species were living after arrival at the surface and continued swimming when transferred into a bucket filled with cooled Lake Baikal-water (6 -C). Sixty minutes later, the water temperature did not exceed 8 -C. 2.2. Tissue preparation on board The tissue preparation started within minutes after arrival of the trawl at the water surface and took less than 2 min per specimen. The tissue preparation for all specimens was finished at the latest within 60 min. Animals were decapitated and opened ventrally. The dark colored intestine was discharged. The metasomal muscle tissue was removed, slightly blotted to reduce contaminating hemolymph, and transferred to pre-weighed test tubes on ice. The samples were stored in liquid nitrogen until processing at the Limnological Institute Irkutsk. 2.3. Processing of tissue samples Each tissue sample was prepared to analyze both (1) TMAO + TMA concentrations in TCA extracts and (2) other Fcompatible_ osmolyte concentrations in alcoholic extracts. Frozen tissue samples were brought to room temperature (3 min). Next, the fresh mass of the tissue sample was determined, then 100 AL of bi-distilled water were added. These samples were homogenized (1 –2 min) in the tube with a high-tensic steel homogenizer of adapted shape, boiled in
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a water bath (1 – 2 min) to stop any metabolic activity, weighed again to determine the unavoidable loss of homogenate (1.3 – 9%), and centrifuged (30 min) at 14,000 g. The supernatant was divided into two parts. One aliquot (50 AL) was deproteinized by adding 200 AL TCA (trichloroacetic acid, 5% wt/v), vortexed, centrifuged, and the protein free supernatant used for determination of TMAO + TMA. The second aliquot (30 – 50 AL) was transferred to a preweighed tube and the exact sample volume determined (assuming a density of 1g/mL), deproteinized with 50 AL ethanol, vortexed, and centrifuged. The TCA extracts and the alcoholic extracts were transferred to Berlin and Dortmund for chemical analysis. 2.4. Chemical analysis of TMAO (plus endogenous TMA) in the TCA extract Analysis was performed according to Wekell and Barnett (1991) but the method was adapted for small volumes. The small weight of the tissue samples did not allow to measure both total TMAO + TMA and TMA separately in one sample. TMAO was reduced by a mixture of FeSO4 plus EDTA (ethylene diamine tetraacetic acid) and the total TMA (trimethyl amine) extracted into toluene plus KOH. Adding 500 AL of the toluene layer and 1.5 mL picric acid yielded the picrate derivative of total TMA. Two aliquots of 500 AL were quantified colorimetrically at 410 nm in quartz cuvettes. The absorbance was in the same range as those of standard solutions, which were linearly related to concentrations of 0.125 – 3.000 Amol/mL TMAO. Standards were treated identical with each run. The measured TMAO concentration was used to calculate the TMAO (+ TMA) muscle content as micromole per gram fresh mass (fw). Since the endogenous TMA content in animals is low (Kelly and Yancey, 1999; Treberg and Driedzic, 2002) we refer to TMAO + TMA as TMAO in the following. 2.5. Chemical analysis of other organic osmolytes in the alcoholic extract The alcoholic extract was centrifuged (17,500 g, 15 min), the supernatant separated and evaporated in a vacuum centrifuge (Speedvac SC 110, Savant). The dry remainder was dissolved in 50 AL HPLC buffer (50 mg L 1 CaNa2EDTA (calcium disodium ethylene diamine tetraacetic acid) in bi-distilled water). Aliquots of 2 –30 AL were analyzed by HPLC (Waters, Eschborn, Germany) equipped with a Sugar-Pak 1 column (flow rate at 0.5 mL/min). The column combines gel filtration and ion exchange chromatography using calcium cations bound to the column matrix. The signal (mV) of a refractometer (Waters 410) was recorded versus time. Samples were compared with synthetic compounds. For mass spectrometry, aliquots of ca. 30 AL of the dissolved supernatant from 2 animal samples were analyzed by HPLC as described above and 1 mL fractions of the
eluate were collected. The fractions were evaporated and dissolved in a mixture of saturated 2-cyano-3-(4-hydroxyphenyl)-acrylic acid (a-cyano-4-hydroxycinnamic acid) in 0.1% trifluoroacetic acid and 50% acetonitrile. One microliter of each sample was spotted on a steel plate and airdried. MALDI-TOF spectra were recorded using a Voyager DE (Perspective Biosystems). 2.6. Statistics Values are expressed as mean T S.D. (standard deviation). Differences were considered statistically significant when p < 0.02 by a Mann –Whitney U-Test. The software Statistica 5.5 (StatSoft Inc. Hamburg, Germany) was used for analysis.
3. Results The fresh mass (fw) of the 81 tissue samples collected for this study depended mainly on size of the specimens and varied from 29.4 – 321.2 mg. Eleven samples were < 50 mg and 12 samples > 200 mg. Most samples (ca. 75%) ranged between 50 and 200 mg fw. TMAO was analyzed in 74 tissue samples (Table 1). Concentrations were neither correlated with sex, body size, visible parasites, nor with the time spent at the surface until tissue preparation. Thus, all values were combined to calculate average values. No specimens with eggs in the marsupium were observed. Samples of three species collected in shallower water (Acanthogammarus albus, A. lappadeus lappadeus, A. grewingki) had TMAO concentrations between 6– 18.1 Amol/g fw, while in four species (Acanthogammarus reicherti, A. grewingki, Ceratogammarus dybowskii, Parapallasea lagowskii) collected from deep water concentrations between 28.4 –47.3 Amol/g fw were measured. Specimens of A. grewingki collected in deeper water (930 m) had higher TMAO concentrations in their muscle tissue than conspecimens from shallow water (170 m). These differences are statistically significant ( p < 0.02) (Table 1). C. dybowskii collected in water depths of 930 and 1170 m showed identically high TMAO concentrations.
Table 1 Concentrations of TMAO (+TMA) in the metasomal muscle of Baikalian amphipods collected from deep and shallower water Water depth (m)
Species
Concentration (Amol/g fw)
50 170 200 930
A. lappadeus lappadeus A. grewingki A. albus A. grewingki A. reicherti C. dybowskii C. dybowskii P. lagowskii
6.0 T 2.8 (5) 18.1 T 3.0 (16) 16.1 T 4.9 (13) 28.4 T 9.7 (7) 31.6 T 3.0 (5) 47.3 T 8.0 (11) 43.3 T 14.3 (8) 32.0 T 4.4 (9)
1170
Values are means T S.D. Number of animals in parentheses.
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Comparing the TMAO concentrations of all individuals of the different species collected in deep water (930 – 1170 m) and those collected in shallower water (50 – 200 m) the TMAO concentrations were significantly higher ( p < 0.001) in the deep-water specimens (Table 1). HPLC analyses of 54 tissue samples were performed. In all chromatograms a large peak appeared followed by several smaller peaks (Fig. 1). A comparison of the chromatograms of two specimens of P. lagowskii and one of A. grewingki in Fig. 1 demonstrates intraspecific and interspecific variability
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but also interspecific similarities of the peak patterns. Five of the smaller but frequently appearing peaks had the same retention time as taurine, glycerophosphoryl choline (GPC), betaine, S-methyl-cysteine, and sarcosine, respectively (Fig. 1, lower panel). Adding of these standard substances to the diluted alcoholic hemolymph extracts enlarged the corresponding peak areas and yielded a rough calculation of the concentration of the compounds in some samples (1– 6 mmol/kg H2O). GPC, betaine, S-methyl-cysteine, and sarcosine were detected by MALDI-TOF in the correspond-
Fig. 1. HPLC-chromatograms of tissue extracts of two Baikalian amphipod species and five standard solutions. Ordinate = detector signal (relative units), abscissa = migration time (minutes), monitoring started just after injection of the sample. Upper panels = runs of three specimens collected in deep water. The amplitude of the first big peak was set to 100%. Lower panel = runs of five individually measured standard solutions projected overlapping on one baseline.
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ing 1 mL eluates of two alcoholic tissue extracts. Other organic osmolytes commonly separated by the Sugarpak I column such as urea, polyols and glycine could not have been identified in the tissue extracts because their concentrations were probably lower than 1 mmol/kg fw. The identified substances were normalized to 1 mg fw to compare the osmolyte concentrations between specimens, between species and between deep and shallow water. Individual differences of the peak areas/mg fw of the identified substances were high and many animal samples did not contain detectable amounts of all of these osmolytes (Fig. 1). No significant differences with respect to water depth could be demonstrated.
4. Discussion Intracellular osmotic effectors are not well studied in freshwater crustaceans. Free amino acids were reported to contribute substantially (approximately 40%) to the total osmotically active solute in muscle of freshwater crayfishes (Gilles, 1979; Clark, 1985; Dooley et al., 2000). Absolute concentrations range between 2.4 and 79.0 mmol/kg fresh weight (fw) (Claybrook, 1983). The amino acid pools of Orconectes limosus, Astacus astacus, A. leptodactylus, and Cherax destructor range from 13.71 to 79 mmol/kg fw (Siebers, 1972; Marrewijk and Ravenstein, 1974; Schoffeniels, 1976; Dooley et al., 2000) and depend on season and ambient osmotic concentration. Glycine is the most prominent free amino acid in freshwater as well as in marine crustaceans. Glycine and taurine concentrations, important compounds of cellular volume and osmoregulation (Clark, 1985), were extensively studied in the euryhaline crab Eriocheir sinensis (Gilles, 1979) but scarcely in freshwater decapods. Glycine, taurine, and betaine concentrations are known for some crayfish species. The concentration of the secondary amino acid taurine is lower than 8 mmol/kg fw (Marrewijk and Ravenstein, 1974). Betaine has been detected in one of three crustacean freshwater species studied so far (Beers, 1967). Neither TMAO nor GPC were detected in freshwater crustaceans. In the present study, the osmolytes TMAO, GPC, betaine, sarcosine, S-methyl-cysteine and taurine were determined for the first time in freshwater amphipods (Fig. 1). In contrast to TMAO, discussed in detail below, their contribution to the total intracellular osmolality is negligible and differences correlated with water depth were not detected. The TMAO concentrations in muscle tissue of amphipods captured from deep water were significantly higher than those of amphipods captured from shallow water (Table 1), complementing our earlier finding that hemolymph osmolality increases with increasing water depth (Zerbst-Boroffka et al., 2000). Trawling and decompression might change membrane integrity and fluidity as well as oxygen management. However, any increase of osmolality in hyperregulating animals, such as the freshwater amphipods, is an energy-
requiring process and certainly not a by-product of membrane dysfunction due to decompression. In freshwater, a disorder of membrane function would rather lead to a decrease of osmotic concentration than to an increase. As shown previously (Zerbst-Boroffka et al., 2000), hemolymph lactate concentration, a fast signal for hypoxia, was low in Baikalian amphipods immediately after capture both from deep and from shallow water. Furthermore, amphipods captured from deep water needed two days for acclimation to a new (lower) steady-state concentration of osmolality. We therefore assume that the measured concentrations probably come close to the true values when determined immediately (< 60 min) after capture. The marked increase of TMAO concentration with increased water depth points to a function for adaptation and/or acclimatization to deep water in Baikalian amphipods. There is no salinity gradient and differences of temperature are negligible between the deeper and shallower water zones in the Lake Baikal. Thus, the observed increase in TMAO concentrations of amphipods living in deep water are not related to salinity or temperature. However, a relation to hydrostatic pressure – as discussed for marine animals – is a reasonable assumption. In marine crustaceans, the accumulation of TMAO with increased water depth seems to be quantitatively more important. On the other hand, the TMAO concentrations are already higher in marine species even in shallow water. For example, in muscles of three species of caridean shrimps living in shallow water the TMAO concentration is 76 mmol/kg fw and increases to 215 mmol/kg fw at about 1900 m water depth and to 244 mmol/kg fw at 2850 m water depth (Kelly and Yancey, 1999). Our data show an overall lower
LAKE BAIKAL CONCENTRATION
< 2 mOsm
low pressure: Shallow water amphipod
Hemolymph: 150 mM inorganic ions
≈ 300 mOsm
Cell content:
300 mOsm
high pressure: Deep water amphipod
Hemolymph: 170 mM inorganic ions
≈ 340 mOsm
Cell content:
300 mOsm + 40 mM (TMAO)
Fig. 2. Model mechanism of biochemical adaptation of amphipods to great water depths in the freshwater Lake Baikal. Circle = intracellular compartment; rectangle = extracellular compartment.
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concentration of TMAO in the freshwater amphipods of Lake Baikal when compared to marine crustaceans. However, the TMAO concentration but not that of the other osmolytes changes with water depth. The strong correlation of TMAO concentration and water depth supports the hypothesis that TMAO protects from adverse effects of elevated hydrostatic pressure of deep water, as proposed by several other authors for marine species. A function of other osmolytes identified by us could not be shown. In hyperregulating freshwater species as in the amphipods from Lake Baikal, elevation of the intracellular TMAO concentration without a corresponding reduction of other intracellular osmolytes substantially increases the osmotic gradient across cell membranes and needs to be balanced extracellularly. Indeed, extracellular osmolality increased with water depth, most of which is attributed to an increase of the extracellular NaCl concentration (Zerbst-Boroffka et al., 2000). Hemolymph concentrations of amphipods living in deep water were about 15 – 20 mmol/l NaCl and 20 –50 mosmol/kg H2O higher when compared to amphipods of shallow water. These differences are in the same range as the observed change of TMAO concentrations (Table 1). Thus, we propose that the adaptation and/or acclimatization of eurybathic benthic amphipods to the higher hydrostatic pressure at great depth of the freshwater Lake Baikal rely on two mechanisms (Fig. 2). First, TMAO levels in the muscle tissue increase to counteract the adverse effect of high pressure on protein structure (this study). Second, the increased intracellular osmolality due to TMAO is balanced by a concomitant increase of extracellular NaCl (ZerbstBoroffka et al., 2000). The benefit of accumulating intracellular TMAO as an osmoprotectant prevails the raised energy costs necessary to maintain an even higher osmotic gradient when living in the abyss.
Acknowledgements The help of Prof. M. A. Grachev who continuously promoted the project and saved the expedition from serious organisational problems is greatly appreciated. We are indebted to the captain and crew of the research vessel FTitov_ for their support, especially in rough weather. Special thanks to Brita Bazin for the reliable determinations of TMAO. Prof. Dr. R.K.H. Kinne is acknowledged for experimental support and stimulating discussions. Supported by DFG (436 RUS 18/9/01), RFBR 01-04-48970, RFBR 01-04-97214-a, and RFBR 04-04-48945-a.
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