TMAO and other organic osmolytes in the muscles of amphipods (Crustacea) from shallow and deep water of Lake Baikal

Abstract

Concentrations of trimethylamine oxide (TMAO) and other ‘compatible’ 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.

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). 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, Falkner et al., 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 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, Yancey et al., 2001, Yancey et al., 2002, Somero, 1992, Yancey, 1994, Gilles, 1997, Yancey and Siebenaller, 1999, Sébert 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, Yancey et al., 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 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 ‘compatible 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.