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J Comp Physiol B (2000) 170: 91±103 Ó Springer-Verlag 2000

ORIGINAL PAPER

I. M. Sokolova á C. Bock á H.-O. PoÈ rtner Resistance to freshwater exposure in White Sea Littorina spp. I: Anaerobic metabolism and energetics

Accepted: 4 October 1999

Abstract Anaerobic metabolism and changes in the anaerobic capacity of Littorina spp. are discussed in osmotic concentration of extravisceral ¯uid were studied relation to their vertical distribution, size and ecology. in the White Sea periwinkles (Littorina littorea, Littorina saxatilis and Littorina obtusata) during freshwater Key words Salinity stress á Resistance adaptations á exposure. Resistance to hypoosmotic stress increased in Anaerobic metabolism á ATP turnover á Littorina spp. the order: L. obtusata < L. saxatilis < L. littorea. Our data suggest that osmotic shock is not a primary reason Abbreviations ADP adenosine-5¢-diphosphate á AEC for mortality of the periwinkles under these conditions. adenylate energy charge á AMP adenosine-5¢- During environmental anaerobiosis, considerable monophosphate á ATP adenosine-5¢-triphosphate á Arg )1 succinate accumulation (up to 10±19 lmol g wet L-arginine á AWI Alfred-Wegener-Institute for Polar weight), and depletion of phosphagen and ATP pools and Marine Research á dG/dn Gibbs free energy were found in the studied species. Other metabolic end change á EDTA ethylenediaminetetraacetic acid á EF products (alanopine, strombine, lactate, acetate or pro- extravisceral ¯uid á HPLC High Performance Liquid pionate) were not detected. Succinate accumulation and Chromatography á HSD honestly signi®cant _ net ATP breakdown were the fastest in the least resistant di€erence á MATP ATP turnover rate á NTA species, L. obtusata, and slowest in the most resistant, nitrilotriacetic acid á PCA perchloric acid á Pi inorganic L. littorea. Rate of ATP turnover decreased during phosphate á PLA phospho-L-arginine á RPLA relative

freshwater exposure in L. littorea and L. saxatilis, but amount of phosphagen á RST standardised respiration not in L. obtusata. Our data suggest that di€erential rate resistance of three studied Littorina spp. to extreme hypoosmotic stress may be related to their di€erent abilities to reduce metabolic rate and ATP turnover Introduction during sustained anoxia. Species-speci®c variations in Salinity is an important environmental factor in¯uenc- ing various life characteristics of marine animals, including molluscs. Adaptation of marine molluscs to changes in salinity is achieved through two di€erent Communicated by G. Heldmaier systems which are used alternatively depending on the I. M. Sokolova degree of the environmental disturbance (e.g. Kinne White Sea Biological Station, 1964; Berger 1986; Berger and Kharasova 1997). If Zoological Institute of Russian Academy of Sciences, salinity changes moderately, so-called capacity adapta- Universitetskaya nab., 1, tions play a major part in adjusting to the new osmotic 199034 St. Petersburg, Russia e-mail: [email protected] conditions. In marine molluscs, these adaptations in- Tel.: +7-812-1140097; Fax: +7-812-1140444 volve isoosmotic cell volume regulation through, for C. Bock á H.-O. PoÈ rtner example, shifts of intracellular free amino acid and Alfred-Wegener-Institute for Polar and Marine Research, inorganic ion concentrations (e.g. Lockwood 1976; Ecology and Ecophysiology, Natochin et al. 1979; Taylor and Andrews 1988; Columbusstrasse 30, Hawkins and Hilbish 1992), and changes of oxygen 27568, Bremerhaven, Germany e-mail: [email protected], consumption rates during salinity acclimation (Kinne [email protected] 1971; Berger 1986). Capacity adaptations typically result Fax: +49-0471-4831149 in adjustments which ensure performance of the most 92 important life functions and potentially unlimited sur- allows correlation of changes in anaerobic metabolic vival in the new osmotic environment. In contrast, if rate and energy status with di€erential survival during environmental salinity strongly deviates from the opti- prolonged periods of hypoosmotic exposure and to mum, resistance adaptations are evoked. In marine suggest the primary mechanisms of mortality (and/or shelled gastropods these are so called escape responses resistance) under such conditions. (see review in Kinne 1971; Lockwood 1976; Berger The aim of our study was to investigate anaerobic 1986). Under conditions of extremely low or high metabolism in the White Sea periwinkles L. littorea, salinity, snails withdraw into the shell and isolate L. saxatilis and L. obtusata during extreme hypoosmotic themselves inside by tight closure of the shell aperture stress. We followed the time courses of mortality, an- with their operculum. This behavioural response greatly aerobic end-product accumulation and changes in the reduces water and salt exchange between the internal parameters of energy status and also analysed changes in medium of the animal and the unfavourable environ- the osmotic concentration of the extravisceral ¯uid (EF) ment (Avens and Sleigh 1965; Rumsey 1973; Berger in L. littorea during prolonged freshwater exposure. 1986). A similar response is typical for bivalves (Kinne Additionally, we compared the routine metabolic rates 1971; Lockwood 1976). However, the resistance re- under normoxic control conditions in the three studied sponse provides only limited survival of the organism, species. and if the period of extreme salinity stress persists, This investigation intended to answer the following considerable mortality is observed. questions: Reasons for the mortality of shelled gastropods and 1. Could osmotic shock alone account for the mortality bivalves under conditions of extreme salinity have long onset in periwinkles during freshwater exposure? been debated in the literature (e.g. Akberali et al. 1977; 2. Are there any species-speci®c di€erences in the time Berger 1986). Principally, two di€erent causes of mor- courses of anaerobic end-product accumulation and tality have been suggested: (1) osmotic shock due to the changes of intracellular energy status of the peri- gradual gain or loss of salts and/or water in a strongly winkles during prolonged freshwater exposure? hyper- or hypoosmotic medium, and (2) unfavourable 3. Do anaerobic and aerobic metabolic rates (the latter changes of the acid-base and/or energy status of the as assessed by routine oxygen consumption) di€er in animal due to sustained environmental anaerobiosis in the studied Littorina spp.? the isolated state. There are some data suggesting that 4. Does di€erential resistance to fresh water correlate the ®rst mechanism is of less (if any) importance in some with variations in the rate of anaerobic metabolism intertidal molluscs (Littorina saxatilis, Mytilus edulis, (i.e. rate of end-product accumulation) and ATP Berger 1980) and the freshwater bivalve Anodonta turnover in the periwinkles? grandis (Byrne and McMahon 1991), whereas in Mya arenaria and Macoma baltica the osmotic shock due to marked dilution of the internal milieu was named among the probable causes of the mortality onset (Golikov and Materials and methods Smirnova 1974; Khlebovich 1974; Berger 1980). At the same time, there is no evidence for or against the second Sampling of the periwinkles hypothesis, as there are no data on changes of the acid- Sampling was performed in July 1996 and August 1997 in the base and/or energy status of the organism during Chupa Inlet of Kandalaksha Bay in the White Sea (66°20¢N, exposure to extreme salinities. 33°40¢E). Water temperature in the ®eld was 10±15 °C, salinity 25± Three closely related gastropod species ± Littorina 27&. L. saxatilis and L. obtusata were collected from the macro- saxatilis, Littorina obtusata and Littorina littorea ± are phyte belt and gravel patches in the middle and lower intertidal common inhabitants of White Sea intertidal and upper zone, and L. littorea was sampled from stones and pebbles in the lower intertidal to upper subtidal zones. Prior to incubation all subtidal zones. The periwinkles are convenient objects snails were kept for 2±5 days in recirculating aquaria with natural for the study of resistance adaptations to extremely low sea water (salinity 26±27&) at 7.7 ‹ 0.6 °C. Aquaria were equip- salinities. L. saxatilis inhabits the high intertidal zone ped with gravel bottom ®lters and air-pumps. Only adult snails were where it spends prolonged periods of air exposure in a used for the experiments. Shell diameters varied between 7±10 mm in L. saxatilis, 8±11 mm in L. obtusata and 22±26 mm in L. littorea. state of dormancy (McMahon 1990), L. obtusata lives in the middle and low littoral, mostly in the brown mac- rophyte (Fucus vesiculosus and Ascophyllum nodosum) Respiration rate canopy, and L. littorea occupies lower intertidal and subtidal horizons. In the White Sea, Littorina spp. may Measurements of routine respiration rate were performed by closed system respirometry (Lyzen et al. 1990). Animals were taken out of experience prolonged periods of extreme hypoosmotic the aquaria, placed individually into air-tight glass bottles half- stress, especially during spring ice-melting when surface ®lled with natural sea water (salinity 26±27&, temperature 7.7 ‹ water salinity can drop drastically (down to 2&) for as 0.6 °C) and left for 30 min to reduce the e€ect of handling. After long as a fortnight (Babkov and Lukanin 1985). These this, the bottles were carefully drained using a thin plastic tube, species exhibit rather high, species-speci®c di€erences in re®lled with sea water of the same salinity and temperature and tightly closed. Additionally, 2±3 control bottles of the same size the capability to survive prolonged periods in an (without snails) were ®lled with sea water. Bottles were exposed at extremely hypoosmotic medium (Berger 1986). This unchanged temperature for 8 h (L. saxatilis and L. obtusata)or4h 93

(L. littorea). After the speci®ed exposure periods, oxygen concen- 8±12 animals were pooled. Ca. 300 mg of tissue powder were added tration was measured in experimental and control bottles to the to an excess (5´) volume of pre-cooled 0.6 mol á l)1 perchloric acid nearest 0.01 mg l)1 using an OXY-320 oxygen meter (WTW, (PCA) and homogenised. Precipitated protein was removed by Germany). The oxygen content in the experimental bottles never centrifugation. The extract was neutralised with 5 mmol á l)1 po- fell by more than 25±30% below control levels. tassium hydroxide to pH 7.0±7.5. Precipitated potassium perchlo- After the measurements, snails were taken out of the bottles and ride was removed by a second centrifugation. Extracts were stored rapidly killed in boiling water. Soft tissues were removed, blotted at )80 °C. and weighed to the nearest 0.5 mg. The presence of trematodes In a second set of samples adenylate extraction by PCA was (microphallids of the ``pygmaeus'' group) was recorded under a maximised by the addition of EDTA (also see Results). Foot tissue binocular microscope. Respiration rates were calculated as lgO2 of L. littorea collected under control conditions and after 3 days of consumed by a snail per hour per gram wet tissue weight. Average freshwater exposure was ground with mortar and pestle under liquid wet tissue weight was 143.4 ‹ 7.3 mg in L. obtusata (n = 18), nitrogen. The tissue powder was divided into two approximately 138.9 ‹ 6.2 mg in L. saxatilis (n = 19) and 951.0 ‹ 41.4 mg in equal portions which were extracted with 0.6 mol á l)1 PCA or L. littorea (n = 19). 0.6 mol á l)1 PCA with 10 mmol á l)1 EDTA as described above. It has been demonstrated that infection by the microphallids of Concentrations of succinate, lactate, acetate and propionate in the ``pygmaeus'' group did not in¯uence respiration rate of PCA extracts were measured according to a method modi®ed from L. saxatilis and L. obtusata (Lyzen et al. 1990; Sokolova 1997). Hardewig et al. (1991). Fatty acids were separated on an ion Preliminary analysis showed that this was also the case with the exclusion column (Dionex ICE-AS 1) at a ¯ow rate of 1 ml min)1 studied periwinkles. Thus, we pooled data for infected and unin- and 40 °C with 0.125 mmol á l)1 hepta¯uorobutyric acid used as an fected snails for subsequent comparisons. eluent. Peaks were monitored with a conductivity detector. A micro membrane suppressor (Dionex AMMS-ICE) was used to decrease background conductivity. In some extracts, succinate and L-lactate Incubation procedure were also determined spectrophotometrically using enzymatic tests (Bergmeyer 1985). Concentrations of the metabolites obtained by Experimental incubation and tissue sampling were performed at the either method were in close agreement. White Sea Biological Station (Cape Kartesh, White Sea). Further Alanopine and strombine were measured by cation exchange analyses were carried out at the Alfred-Wegener-Institute for Polar chromatography. Separation was carried out at 45 °C on an In- and Marine Research (AWI, Bremerhaven, Germany). Incubation teraction ARH-601 column for aromatic acids (ICT-ASS-Chemi- temperature was 7.7 ‹ 0.6 °C. Control animals were incubated in cals, Bad Homburg, Germany); a solution of 1 mN H2SO4 was aquaria with well-aerated natural sea water of 26±27&. Experi- used as an eluent. A conductivity detector was utilised to monitor mental animals were placed in plastic trays and covered with plenty the peaks. Standard opine solutions were prepared according to a of natural fresh water from the nearby Krivoye Lake, a typical lake method modi®ed from Siegmund and Grieshaber (1983). for the Karelian shore of the White Sea characterised with very low Concentrations of AMP, ADP and ATP were measured using inorganic ion concentrations (Naumov, personal communication). an HPLC system (Beckman Instruments, USA) and Spherisorb The water in incubation trays was changed daily. After speci®ed C18 column (Latek, Germany) according to a method modi®ed exposure periods a number of snails were removed from the tray. from Fischer (1995). Separation was carried out at a ¯ow rate of The snails which responded by withdrawing the body deeper into 0.8 ml min)1 using the mobile phase of 2 mmol á l)1 11-amino- the shell after being poked by a needle inserted under the oper- undecanoic acid in 25 mmol á l)1 phosphate bu€er with 8% (v/v) culum, were scored as alive and used further for tissue and EF methanol (pH = 6). Peaks were monitored at 210 nm using a sampling. Animals were dissected and rapidly examined for the Diode Array Detector Module 168 (System Gold, Beckman In- presence of trematode parasites. Infected individuals were dis- struments, Munich, Germany). carded. In the uninfected snails the foot was quickly ectomised and Concentrations of phospho-L-arginine (PLA) and L-arginine immediately frozen in liquid nitrogen. Tissue samples were stored (Arg) were assayed spectrophotometrically using the enzymatic test in liquid nitrogen. described by Grieshaber et al. (1978). Octopine dehydrogenase for In L. littorea, a sample of EF (ca. 100 ll) was taken before these determinations was puri®ed from the adductor muscles of dissection, by means of a syringe inserted into the extravisceral Pecten maximus following the procedure described by GaÈ de and cavity between shell and body. Samples of the EF were centrifuged Carlsson (1984). Inorganic phosphate (Pi) concentrations were for 3±4 min at 13500 rpm, the supernatant collected and stored at measured enzymatically according to PoÈ rtner (1990). )20 °C. The remaining animals were taken out of the tray and placed into sea water (26±27&). After 2 h these sub-samples were examined for the presence of dead snails which did not resume Chemicals activity. All chemicals were purchased from Sigma Chemicals (St. Louis, Mo., USA) or Merck (Darmstadt, Germany). Measurement of EF osmolarity

The osmolarity of the EF (mOsmol kg)1) was measured at the AWI Derived indices by a Wescor vapour pressure osmometer (Wescor, Logan, Utah). For each sample, three to four measurements were made, and the Adenylate energy charge (AEC) was calculated using the tradi- mean value calculated. To transform osmolarity into salinity units, tional formula: six concentrations of arti®cial sea water (0, 10, 15, 20, 25 and 30&) ‰ATPŠ‡0:5 ‰ADPŠ were prepared using Milli-Q deionized water and arti®cial sea salt AEC ˆ 1† (Tropic Marin, Germany), and their osmolarity was measured. ‰ATPŠ‡‰ADPŠ‡‰AMPŠ Values of the EF osmolarity were transformed into salinity units by where [AMP], [ADP] and [ATP] are concentrations (lmol g)1 wet interpolation using the regression line for arti®cial sea water. weight) of the corresponding compounds. The relative amount of phosphagen (RPLA) was calculated Metabolite determination according to the formula: ‰PLAŠ R ˆ 2† For the determination of metabolite concentrations, foot tissue was PLA ‰PLAŠ‡‰ArgŠ powdered with mortar and pestle under liquid nitrogen. In the case of L. littorea, each extraction was made from the tissue of a single where [PLA] and [Arg] are tissue concentrations of PLA and specimen; for the smaller L. saxatilis and L. obtusata, tissues from Arg, respectively (lmol g)1 wet weight). 94

The rate of ATP turnover during anaerobiosis (amount of ATP consumed per gram wet weight per day) was calculated from end- product accumulation and ATP and PLA depletion as described in PoÈ rtner et al. (1984). An ATP equivalent of 2.75 lmol lmol)1 succinate was adopted (de Zwaan 1983). The aerobic ATP turnover rate was calculated from routine oxygen consumption rates as- )1 suming ATP production of 6 mol ATP mol O2. The Gibbs free energy change (dG/dn) of ATP hydrolysis was calculated according to the methodology described in detail by PoÈ rtner et al. (1996). Concentrations of ATP, PLA, Arg and Pi were adopted from this study, whereas pHi and concentrations of free magnesium ions were calculated from 31P-NMR spectra as described in our companion paper (Sokolova et al. 1999).

Statistics

Statistical treatment was performed using standard Model I AN- OVA after testing the assumptions of normal distribution and homogeneity of variance of the data (Sokal and Rohlf 1995). We used Tukey's honestly signi®cant di€erence (HSD) test for unequal n as a method of post-hoc comparisons. Parameters of linear re- gression were calculated according to Sokal and Rohlf (1995), and the ®tting of the data into the linear regression model was estimated by ANOVA. Pairwise comparison of slope and intercept of re- gression lines was performed after algorithm described in Urbakh (1964). The di€erences were considered signi®cant if the probability level of Type I error was less than 0.05. Results are expressed as percentages or mean values ‹ SE. Prior to statistical analysis, respiration rates (calculated as lgO2 consumed per hour per gram wet tissue weight) were standardised to the average wet tissue weight in each experimental group (L. saxatilis, L. obtusata or L. littorea) according to formula 3: Fig. 1 Mortality of White Sea periwinkles (A) and changes in the  osmotic concentration of extravisceral ¯uid of Littorina littorea (B) W b during freshwater exposure. A The number of snails scored for R ˆ R  3† st i W mortality varied between 20±90 in L. littorea and 500±1000 in the i other two species. B Vertical bars representSEofthemean,n is given )1 )1 where Rst is a standardised respiration rate (lgO2 h g ), Ri is a in brackets. Note the fairly stable osmolarity of extravisceral ¯uid )1 )1 respiration rate of i-th animal (lgO2 h g ), Wi is wet tissue (EF) between days 7 and 12 despite increasing mortality in L. littorea weight of i-th animal (g), W is the average wet tissue weight in the experimental group (g), and b is a power coecient in equation: b R=aW. This coecient was obtained by a calculation of the Mortality rates and osmotic concentration of EF linear regression relating log W to log R by the least square method (Sokal and Rohlf 1995). Mortality during freshwater exposure was di€erent in the three studied species. L. littorea was the most resis- Results tant species and L. obtusata the least resistant one (Fig. 1A). Onset of mortality occurred between the 7th Respiration rates under control conditions and 9th day of freshwater exposure in L. littorea, at the 3rd day of exposure in L. saxatilis, and apparently Standardised aquatic respiration rates were similar in before the 3rd day of exposure in L. obtusata. the three studied species and equalled 81.0 ‹ 3.8 lg Owing to the limited size of the other species, the )1 )1 )1 )1 O2 h g , 76.6 ‹ 3.3 lgO2 h g and 67.1 ‹ osmotic concentration of the EF was measured only in )1 )1 2.2 lgO2 h g in L. obtusata, L. littorea and L. littorea. During freshwater exposure, the osmolarity L. saxatilis, respectively. Tukey's HSD test showed that of EF was signi®cantly reduced (P < 0.05) from 696 the respiration rate was signi®cantly higher in L. obtusata mOsmol kg)1 (26.7&) to 517 mOsmol kg)1 (19.8&) and as compared to L. saxatilis (P < 0.01). levelled at approximately 20& during days 7±12 of the The allometric relationship between soft body weight experiment (Fig. 1B). and respiration rate was statistically signi®cant for L. saxatilis (b = )0.378, P < 0.05) and L. littorea (b = )0.826, P < 0.01), but not for L. obtusata Accumulation of anaerobic end products (b = 0.068, P > 0.80). However, our data do not sug- gest an allometric dependency of aerobic metabolism on The mean concentration of succinate in the foot tissue body size in a between-species comparison, as two spe- did not di€er signi®cantly between control animals of cies with similar body size (L. saxatilis and L. obtusata) either species and ranged between 0.2 lmol g)1 and had the most contrasting respiration rates, and the 0.8 lmol g)1 wet weight. During freshwater exposure, largest one, L. littorea, demonstrated an intermediate snails showed marked accumulation of this end product rate of oxygen consumption. (Fig. 2), and tissue succinate levels of the experimental 95

Fig. 2 Succinate accumulation in foot tissues of three species of White Sea periwinkles during freshwater exposure. Dots represent original data, and lines are linear regressions for succinate accumulation over time in Littorina littorea (L.l) (Suc = 1.37 + 1.65 à Exp), Littorina saxatilis (L.s.) (Suc = 1.39 + 1.82 à Exp) and Littorina obtusata (L.o) (Suc = 0.08 + 2.79 à Exp) where Suc is succinate concentration (lmol g)1 wet weight) and Exp is exposure duration (days). All regressions are statistically signi®cant (P < 0.05) animals were signi®cantly above control values in all studied species. The rate of increase in succinate con- centration was adequately approximated by the linear Fig. 3 Changes in the concentrations of phospho-L-arginine (PLA; A) regression model (Fig. 2). Intercepts did not di€er from and L-arginine (Arg; B) and relative amount of phospho-L-arginine, zero in either studied species. Pairwise between-species RPLA (C) in White Sea Littorina spp. during freshwater exposure. Vertical bars represent standard errors; n is 4±5. A, C ± all values for comparisons suggested that rates of succinate accumu- days 3±12 of exposure are signi®cantly di€erent from the respective lation (=slopes of the regression lines) were similar in controls; B ± values which are signi®cantly di€erent from the L. littorea and L. saxatilis (P > 0.05), but signi®cantly respective controls are marked with asterisks. The irregular pattern higher in L. obtusata (P < 0.05) during 0±5 days of of the changes in Arg content is probably due to the catabolism and/ freshwater exposure. or unregistered e‚ux of this metabolite Besides succinate, we tested for the following end products of anaerobic metabolism: opines (alanopine The concentrations of PLA were similar in the studied and strombine), lactate (D- and L-stereoisomeres not Littorina spp. for all comparable exposure periods being separated), acetate and propionate. None of these (P > 0.60). Phosphagen depletion was accompanied by metabolites were present in control animals nor accu- the concomitant increase in Arg content (Fig. 3B). mulated during the experiment in amounts detectable by However, the pattern of change in the concentrations of the methods applied. this metabolite was not as clear as for PLA. The relative amount of phosphagen in the total phosphagen/aphosphagen pool (RPLA) decreased sig- ni®cantly during freshwater exposure. The rate of de- High-energy phosphates and energy status cline was fastest in the least resistant species, L. obtusata, of the tissue and somewhat slower in L. littorea and L. saxatilis (Fig. 3C). However, due to the high inter-individual Phosphagen variability of this ratio, no statistically signi®cant dif- ferences were found between the studied species at either Mean values of PLA or Arg contents, as well as the of the comparable exposure periods. RPLA ratio did not di€er signi®cantly in control animals of either studied Littorina spp. During exposure to fresh water, the amount of PLA declined signi®cantly in all Adenylates studied species as compared to the respective control values (Fig. 3A). It is worth noting that the phosphagen In control animals, no signi®cant di€erences were found was nearly depleted already after 3 days of freshwater between the studied species in the levels of di€erent exposure. After that time no further decline in PLA adenylate compounds or the total concentration of content was detected in either studied species (Fig. 3A). adenylates. 96

Fig. 5 Adenylate energy charge (AEC; A) and Gibbs free energy (B) Fig. 4 Concentrations of adenine nucleotides in the foot tissues of of ATP hydrolysis in White Sea Littorina spp. during freshwater White Sea Littorina spp. during freshwater exposure. The total exposure. A Vertical bars represent SE. Number of samples for each concentration of adenylates is shown by closed circles connected with exposure period is the same as in Fig. 4. AEC values after 3±12 days a solid line. Vertical bars represent SE. The number of samples of exposure are signi®cantly di€erent from the respective controls in examined for each exposure period is given in brackets. Values which either of the studied species. B The Gibbs free energy change of ATP are signi®cantly di€erent from the respective control concentrations hydrolysis was calculated using average values of intracellular aremarkedwithanasterisk parameters (ATP, PLA, Pi,pHi, etc.) for each exposure period. Note a good correspondence between the pattern of changes in AEC and ATP free energy change during the ®rst 5 days of freshwater exposure In all studied Littorina spp., the amount of AMP in the foot muscle increased during freshwater exposure (Fig. 4). However, the rates of AMP accumulation were The total amount of adenylates in the muscle tissue di€erent. The rate of increase in AMP content was also decreased during the experiment. Again, the rate of slowest in L. littorea. In this species, the average AMP this decline di€ered between the studied species. After 5 concentration equalled 0.18 lmol g)1 wet weight at day days of freshwater exposure, adenylates decreased by 5 of freshwater exposure, whereas the respective values 0.53 lmol g)1 and 0.94 lmol g)1 wet weight below in L. saxatilis and L. obtusata were 0.31 lmol g)1 and control levels in L. saxatilis and L. obtusata, respec- 0.33 lmol g)1 wet weight, respectively. The AMP con- tively. In L. littorea, no signi®cant change in the ade- tent in L. littorea muscles reached only 0.22 lmol g)1 nylates concentration of the foot muscle was observed wet weight at the end of the experiment which lasted for until after day 7 of experimentation. At this point, the 12 days. adenylate content declined signi®cantly by 0.63±0.80 ADP concentrations increased during freshwater ex- lmol g)1 wet weight. posure in L. littorea and L. saxatilis, but remained fairly The AEC in the muscle tissues of control animals was constant in L. obtusata (Fig. 4). In contrast, the con- very similar at 0.85±0.90. During freshwater exposure a centration of ATP showed a signi®cant decline during marked decrease of AEC was found (Fig. 5A). After 3 freshwater exposure in all three species. The decrease in days of exposure this decline was equally pronounced in the ATP content was fastest in L. obtusata, intermediate all studied species, but at day 5 the AEC of L. littorea in L. saxatilis and slowest in L. littorea. After 5 days of foot tissue was signi®cantly higher than in L. obtusata. freshwater exposure, the mean relative amount of ATP At this time the AEC of L. saxatilis tissues did not di€er equalled 0.47, 0.68 and 0.81 lmol g)1 wet weight in signi®cantly from either species. L. obtusata, L. saxatilis and L. littorea, respectively The dynamics of the free energy change of ATP hy- (Fig. 4). It is worth noting that ATP concentration drolysis followed a similar trend. Under control condi- dropped signi®cantly below control levels after 3 days of tions, dG/dn values were very similar in the three studied exposure in L. saxatilis and L. obtusata, and only after 7 species and varied between )55.7 kJ mol)1 and )56.3 kJ days in L. littorea. mol)1 (Fig. 5B). In freshwater-exposed Littorina spp. a 97 equivalents at this time derived from succinate produc- tion (78.5±78.7%). Later on, the relative amount of ATP equivalents derived from the utilisation of ATP and phosphagen stores greatly reduced to <0.01±0.5% (for ATP) and 0.1±3.1% (for phosphagen). The vast majority of ATP equivalents (96.9±99.7%) were derived from catabolism. It is worth noting that in our study we used a stan- dard method of PCA extraction that is widely applied for di€erent animal tissues (Bergmeyer 1985). However, as was pointed out by one of the reviewers of this paper, tissue adenylate levels may be underestimated by this approach in calcium-rich molluscan tissues due to the artifactual precipitation of adenylates. To test for this e€ect, we performed an additional series of experiments in L. littorea and carried out tissue extractions with PCA containing EDTA in order to trap the Ca2+ ions. It was found that addition of EDTA signi®cantly in- ¯uenced the measurable amount of ATP (ANOVA: F1,20 = 194.00, P < 0.0001), whereas ADP or AMP levels were not signi®cantly a€ected (ANOVA: F1,20 = 0.15, P = 0.70 and F1,20 = 1.04, P = 0.32 for _ Fig. 6A, B ATP turnover rates (MATP) in White Sea Littorina spp. ADP and AMP, respectively). Addition of EDTA in- during freshwater exposure. A Aerobic ATP turnover was calculated creased ATP levels by 20±25% in the tissues of the from the routine oxygen consumption rates of Littorina spp. assuming control and freshwater-exposed animals. ATP concen- _ formation of 6 mol ATP per mol of consumed O2. Anaerobic MATP tration in the tissue samples extracted with EDTA was was calculated from the change in succinate concentration assuming production of 2.75 lmol ATP mol)1 of accumulated succinate, as well higher by a factor of 1.32 ‹ 0.09 (n = 4) in controls as from ATP and phosphagen depletion over the respective exposure and by a factor of 1.25 ‹ 0.11 (n = 8) after 3 days of periods. B Relative amount of ATP equivalents derived from di€erent freshwater exposure. These factors were not signi®cantly sources during freshwater exposure in White Sea Littorina spp di€erent in control and freshwater-exposed animals (ANOVA: F1,10 = 0.172, P = 0.69) indicating that the ``drop'' (meaning a decrease in driving force) of the increase in ATP levels was proportional in these groups, Gibbs free energy of ATP hydrolysis was observed to despite the drastic di€erence in ATP levels (see below). between )48.1 kJ mol)1 and )49.2 kJ mol)1. It was This e€ect should be the same in all three studied Lit- most pronounced and occurred somewhat faster in the torina species, because of similar calcium levels found in least resistant species, L. obtusata, whereas the least and the tissue homogenates of these species (Sokolova et al. a slower decrease in the driving force of ATPases was 1999). Hence, the correction for potential calcium found in the most resistant L. littorea. However, values precipitation of ATP would have no e€ect on the be- of dG/dn were quite close in the three studied species tween-species comparisons of the dynamics of these throughout the exposure periods, the between-species parameters during freshwater exposure or on the dG/dn variation comprising less than 2 kJ mol)1. of ATP hydrolysis. Nonetheless, we corrected ATP, The comparison of aerobic and anaerobic ATP adenylate and AEC levels in Figs. 4±6 indusive for this _ turnover rates (MATP) suggests the onset of a dramatic e€ect. As expected, this correction did neither change metabolic rate depression in all three species during the overall picture of dynamics of adenylate concen- freshwater exposure. During the ®rst 3 days of fresh- trations and energy related parameters, nor the statis- _ )1 water exposure, MATP varied between 7.3 lmol g tical signi®cance of the above-described contrasts (data day)1 and 8.3 lmol g)1 day)1 which is equivalent to 2± not shown). _ 3% of the aerobic MATP of the studied species (Fig. 6A). _ Subsequently, MATP during freshwater exposure decreased considerably in L. saxatilis and L. littorea (to Inorganic phosphate _ down to 1±1.5% of aerobic MATP), but not in L. obtusata (Fig. 6A). Changes in Pi concentration during freshwater exposure The relative amount of ATP equivalents derived from showed variable patterns among studied species di€erent sources was similar in the studied species and (Table 1). In L. littorea, changes in Pi content between changed considerably in the course of freshwater expo- control and freshwater-exposed animals were not sta- sure (Fig. 6B). During the ®rst 3 days of exposure, 5.5± tistically signi®cant. In contrast, a signi®cant increase of 1.2% of total ATP turnover was due to the breakdown Pi concentration was found in L. saxatilis and L. obtu- of ATP stored in the tissue. Utilisation of phosphagen sata after 3 or 5 days of freshwater exposure as com- comprised 16±20% of the ATP turnover. Most ATP pared to the respective control levels. 98

Table 1 Concentration of in- organic phosphate (lmol g)1 Exposure Littorina littorea n Littorina saxatilis n Littornia obtusata n wet weight) in the foot tissues of White Sea periwinkles during 0 2.64 ‹ 0.54 6 1.90 ‹ 0.36 5 2.22 ‹ 0.15 5 freshwater exposure 3 4.33 ‹ 0.85 6 4.12 ‹ 0.72* 4 4.09 ‹ 0.49* 7 5 3.09 ‹ 0.70 5 3.75 ‹ 0.40* 6 2.85 ‹ 0.62 6 12 2.11 ‹ 0.87 4 ± ± ± ± * indicates values that are signi®cantly di€erent from the respective control (P < 0.05)

the time courses of anaerobic product accumulation and Discussion changes in parameters of energy status of the snails and compared these to the mortality rates of the three Freshwater resistance and osmotic changes Littorina spp. in order to ®nd out which is the crucial of the internal medium parameter for survival under conditions of freshwater exposure. Presumably, the limiting parameter should Exposure to long periods of extremely low salinity is not reach the same value in all species at the time of the onset an unrealistic experience for White Sea Littorina spp. of mortality. Marked seasonal salinity variations are a well known Our study has for the ®rst time clearly demonstrated feature of the White Sea (Kuznetsov 1960). In open that freshwater exposure leads to the onset of anaero- parts of the studied area of Kandalaksha Bay, surface biosis in these marine gastropods. Onset of anaerobic water salinity can drop down to 2& for as long as a energy metabolism in freshwater-exposed periwinkles is fortnight during spring ice-melting (Babkov and Luka- clearly indicated by signi®cant succinate accumulation. nin 1985). This salinity is well below levels tolerated by This metabolite is regarded as an ideal signal of anaer- the periwinkles and causes behavioural escape response obiosis within the mitochondrial compartment of the (isolation). In estuaries such periods of low salinity may cells (Grieshaber et al. 1994). The rate of succinate be even longer and more pronounced (Berger 1986). The accumulation which is indicative of anaerobic metabolic three studied species of periwinkles demonstrated dif- rate, is the highest in the least resistant species, L. obtu- ferent levels of resistance to freshwater exposure. Sur- sata, and fairly similar in the other two species with vival rates in fresh water decrease in the order L. littorea intermediate and high freshwater resistance. Mortality ADP>AMP which is typical for un- by di€erent rates of anaerobic metabolism. Osmotic stressed animals (Beis and Newsholme 1975). During shock due to the gradual dilution of the internal medium freshwater exposure ATP levels declined signi®cantly in seems to be of minor (if any) importance for the survival all studied species, however, at di€erent rates. ATP of the White Sea periwinkles under conditions of hy- breakdown was fastest in the least resistant L. obtusata poosmotic exposure. Minimal resistance of L. obtusata and slowest in the most resistant species, L. littorea. to anoxic exposure correlates with a higher anaerobic Decrease in ATP content was linked to a parallel in- metabolic rate (re¯ected in the higher rate of succinate crease in ADP and especially AMP concentrations, accumulation), faster net ATP breakdown and the in- however, the rise in ADP levels was not always signi®- ability to reduce ATP turnover rate during prolonged cant. Total adenylate content declined during freshwater anaerobiosis. This may probably indicate less ecient exposure in the three studied Littorina spp. which was metabolic arrest under conditions of anoxia in L. obtu- probably a result of AMP degradation (Atkinson 1977; sata as compared to the other two species. Inability of PoÈ rtner 1987a). anaerobic energy production to keep pace with energy Despite signi®cant net breakdown of phosphagen and demand leads to the observed impairment of energy ATP, enzymatically determined inorganic phosphate status which is again most pronounced in the least re- showed only a slight (if any) increase during freshwater sistant species L. obtusata. To the best of our knowledge, exposure. This is an unexpected ®nding because phos- this is a ®rst study, so far, which clearly indicates the phagen and ATP depletion are assumed to lead to a crucial role of energy balance in supporting cell function stoichiometric increase in free Pi levels (PoÈ rtner 1987b and integrity under conditions of extreme hypoosmotic and references therein). Moreover, studies of functional stress. anaerobiosis during increased exercise in the squid Illex illecebrosus and Lolliguncula brevis showed that accu- mulated Pi does not leave the muscle cells during rather Ecological and evolutionary implications short term stress (PoÈ rtner et al. 1996). Long term release from the cells appears possible (PoÈ rtner et al. 1991). Behavioural isolation inside the shell in response to Alternatively, there might have been a measurement extreme salinities, typical as it is for the marine 2+ artefact owing to Pi precipitation by Ca derived from conchiferous molluscs, appears not to be a speci®c the calcium carbonate stores of the foot muscle (Mason salinity adaptation. Ability to survive prolonged periods et al. 1984; see also Sokolova et al. 1999). of anoxia caused by such isolation is typical for many The decrease in high-energy phosphate concentra- intertidal invertebrates and has probably evolved as an tions during prolonged freshwater exposure led to an adaptation to life in the intertidal zone. Di€erences in impaired energy status of the tissues in White Sea Lit- the ability to withstand prolonged anoxia correlate to a torina spp. re¯ected in a decline of the adenylate energy certain extent with the ecological peculiarities of charge and the free energy change of ATP hydrolysis. the studied species, in particular with their vertical The driving force by ATP hydrolysis (absolute value of distribution. L. saxatilis which possesses a rather high the dG/dn) decreased from ca. 56 kJ mol)1 under con- capability to survive anoxia is a typical eulittoral fringe trol conditions to a minimum of 48±49 kJ mol)1 after 3± species which normally inhabits upper intertidal 5 days of freshwater exposure. Although our data give horizons (McMahon 1988, 1990). There the rough 101 periwinkles have to face extended periods of limited water/anoxia resistance may possess a certain degree of oxygen availibility at low tide, when they isolate them- evolutionary conservatism and further emphasises the selves inside the shell not only to escape freshwater ex- need to account for evolutionary constraints when dis- posure during rain or ice melt but also to save water cussing physiological adaptations of a species in relation during air exposure. The least anoxia-resistant L. obtu- to its ecology. sata is a typical mid- and low-shore dweller and expe- riences comparative short periods of aerial exposure. Acknowledgements Inna Sokolova is grateful to the Otto Kinne Moreover, the ¯at periwinkles maintain activity and Foundation for granting the OKF Fellowship which served for thus remain aerobic during most of the air exposure partial ®nancial support of her studies in 1996 and to Alfred-We- period, as the problem of water conservation is not so gener Institute for Polar and Marine Research (Bremerhaven, acute in this species (McMahon 1988, 1990). Taken to- Germany) for supporting her work as a guest scientist in 1996± 1998. Field work was in part supported by the Russian Foundation gether, the peculiarities of vertical distribution and be- of Basic Research (grant N 98-04-49977 to IS). We are greatly haviour of L. obtusata results in that much shorter indebted to Dr. Angela Sommer, Mr. Timo Hirse and Mr. Boris periods of oxygen limitation are normally experienced Klein for considerable help during the experimental work and to by this species at low tide. two anonymous reviewers for helpful comments and constructive criticism. All experiments complied with the current laws of the The most resistant species, L. littorea, seems to be an countries (Russia and Germany) where they were performed. exception from this pattern. In the White Sea the com- mon periwinkles typically inhabit low intertidal and upper subtidal zones and thus endure the shortest or no exposure to air. 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