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L

Journal of Experimental Marine Biology and Ecology 245 (2000) 1–23

www.elsevier.nl / locate / jembe

Physiological variation related to shell colour polymorphism

in White Sea Littorina saxatilis

I.M. Sokolova , V.Ja. Berger

White Sea Biological Station, Zoological Institute of Russian Academy of Sciences, 199034 St. Petersburg, Universitetskaya nab., 1 Russia

Received 15 December 1998; received in revised form 3 June 1999; accepted 6 September 1999

Abstract

Responses to moderate and extreme salinity change were investigated in White Sea Littorina

saxatilis of different genetically determined shell colour morphs in order to test a hypothesis about

physiological selection as a driving force of the change of phenotypic structure of this species along a salinity gradient in White Sea estuaries. Some of the studied physiological responses did not differ in the snails with different shell coloration including oxygen consumption rate, rate of salt loss in extremely low salinity and rates of behavioural isolating and opening responses. However, snails with the brown tessellated unbanded shell (which are predominantly found in the estuaries) demonstrated better survivability under conditions of extremely low salinity and combination of low salinity and freezing temperatures as compared to the conspecifics of the purple tessellated unbanded morph which is more frequently found in the marine sites. Periwinkles with brown tessellated unbanded shells also tended to be more responsive to an unfavourable salinity change, so that relatively more animals of this morph isolated themselves inside the shell shortly after placement in low salinity. It is suggested that these physiological differences may provide selective advantage of the brown tessellated unbanded morph under extremely fluctuating salinity and temperature regime of the White Sea estuaries, and thus a considerable increase of the relative abundance of this morph towards the head of the White Sea estuaries may be a result of physiological selection on pheno- (geno-) typic structure of L. saxatilis populations.  2000 Elsevier Science B.V. All rights reserved.

Keywords: Shell colour polymorphism; Physiological selection; Salinity adaptations; Littorina saxatilis

*Corresponding author. Tel.:17-812-114-0097; fax:17-812-114-0444.

E-mail addresses: [email protected] (I.M. Sokolova), [email protected] (V.J. Berger)

Present address: Lab. Ecophysiology, Bio I, Alfred-Wegener Institute for Polar and Marine Research, Columbusstr. 30, 27568 Bremerhaven, Germany. E-mail: [email protected]

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1. Introduction

Shell colour polymorphism is a distinctive feature of the populations of many marine and terrestrial gastropods (e.g. Clarke, 1978; Raffaelli, 1982; Cain, 1983, 1988; Rehfeld, 1997). In all gastropod species studied so far in this respect, a direct genetic control of shell coloration has been demonstrated (e.g. Murray and Clarke, 1966, 1976a,b; Barker, 1968; Cain, 1983, 1984; de Matos, 1984; Kozminsky et al., 1995; Ekendahl and Johannesson, 1997). In many cases, variation in shell colour is related to environmental gradients such as climate (Currey and Cain, 1968; Cowie, 1990; Chang and Emlen, 1993; Honek, 1993), insolation (Heath, 1975; Heller and Volokita, 1981; Heller and Gadot, 1987; Etter, 1988; Berger et al., 1995), wave exposure (Etter, 1988) and salinity (Sergievsky, 1992; Sokolova et al., 1995, 1997). Such variation has proved to be stable and repetitive through time and space (Owen, 1963; Currey and Cain, 1968; Wolda, 1969; Cain, 1971; Goodhart, 1973; Murray and Clarke, 1978). This suggests an adaptive value to shell colour in gastropods and has stimulated numerous experimental works on the adaptive significance of shell coloration.

In gastropods, shell colour may have three functions: communication, crypsis and thermoregulation (Endler, 1978). For these functions, it is the shell coloration itself which is selectively important. Several cases of so-called physiological selection (Cain and Sheppard, 1961) on shell colour have also been documented, where the shell colour has been correlated with selectively valuable physiological traits including responses to temperature (Kavaliers, 1989), salinity (Sergievsky, 1992, 1995), metabolic rates (Steigen, 1979) and fecundity (Wolda, 1967). It is suggested that in these cases, correlation between individual physiology and shell colour polymorphism is a result of pleiotropic effects of genes responsible for the shell colour or a linkage between them and genes determining certain physiological features (Raffaelli, 1979, 1982).

L. saxatilis (Olivi) (Gastropoda: Prosobranchia) is an abundant species in the intertidal zone of North Atlantic, White and Barentz Seas (Reid, 1996). Extreme variability of shell coloration in L. saxatilis is well documented and partially responsible for the complicated synonymy of this species including 35 names of shell colour morphs (Reid, 1996). Shell colour is inherited in L. saxatilis (Kozminsky et al., 1995; Ekendahl and Johannesson, 1997) and hence, shell colour variability directly implies genetic variation in the population. In general, shell colour polymorphism was shown to be of adaptive value in L. saxatilis populations and related to crypsis (Atkinson and Warwick, 1983; Byers, 1990) and / or thermoregulation (Berger et al., 1995). Other studies (Sokolova et al., 1995, 1997; Sokolova, 1997) also suggested that physiological selection on shell colour may be the case in some populations of this species. Particularly, a clinal variation in the proportion of main shell colour morphs (phenotypes) of L. saxatilis was observed along the gradient of environmental salinity in White Sea estuaries (Sokolova et al., 1995, 1997; Sokolova, 1997). Towards the head of an estuary, the relative abundance of brown tessellated unbanded morphs increased 2 to 5–10-fold as compared to the adjacent marine sites, and the frequency of other colour phenotypes (mostly purple tessellated unbanded and plain purple unbanded) pro-portionally declined. This pattern was found in the three studied White Sea estuaries separated by a distance of over 100 km and was stable over time, during several years of

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the study (Sokolova, 1997). Previous studies (Sokolova et al., 1997) have suggested that the phenotypic differentiation along a salinity gradient cannot be explained by selective pressures imposed by the differences in crypsis and / or heating properties of the shell colour morphs involved and probably implies physiological selection.

In order to test the hypothesis about physiological selection as a driving force shaping pheno- (geno-)typic structure of L. saxatilis populations along a salinity gradient in White Sea estuaries, we investigated physiological responses of different shell colour morphs of L. saxatilis to salinity variation and also to the combination of low salinity and subzero temperatures which may be expected in White Sea estuaries in winter. Particular attention was given to the phenotypes which are most common in the studied populations of L. saxatilis and show the greatest and the most consistent difference in abundance between the marine and estuarine sites (purple tessellated unbanded and brown tessellated unbanded). Since it has been shown that the nature and the mechanisms of salinity adaptations depend on the degree of environmental disturbance (Kinne, 1964, 1971; Precht and Plett, 1979; Berger and Kharazova, 1997), we analysed responses of different shell colour morphs of L. saxatilis to moderate and extreme salinity changes. This allowed a comparison to assess the relationship between genetically determined shell colour polymorphism and physiological variation with respect to salinity in more detail.

2. Materials and methods

2.1. Study sites

Snails were collected in July–August, 1995 in Chupa, Keret and Kolvitsa Inlets of the Kandalaksha Bay of the White Sea (Fig. 1). In the estuaries, snails were collected close to the distribution limit of L. saxatilis in the head of the estuaries in the Keret (E-1) and the Kolvitsa (E-2) Inlets (Fig. 1). Measurements of the surface salinity in summer and autumn showed that salinity at low tides was usually below 10‰ in these sites, sometimes dropping down to 1–3‰. Overall, salinity was below 15‰ at all phases of the tidal cycle in these sites. In the Chupa Inlet, two populations from the marine sites were sampled (M-1 and M-2) (Fig. 1). Here surface salinity was relatively constant and varied between 24 and 26‰. This is the normal salinity of the surface waters in the White Sea in summer and autumn (Kuznetsov, 1960).

Most analyses were done in animals from sites M-1 and E-1. Animals from sites M-2 and E-2 were only used in one (exp. 3) or two experiments (exp. 2 and exp. 6), respectively, due to a limited amount of snails available.

2.2. Experimental methods

Snails were transferred to the laboratory within a few hours (populations M-1, M-2 and E-1) or 1 day (E-2) after collection. Prior to the experiments, they were acclimated for 2–5 days at 10618C and 24–25‰ in aquaria with well-aerated recirculating natural sea water. No food was allowed. The temperature was chosen to approximate the natural

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Fig. 1. Study sites in the Kandalaksha Bay (B) of the White Sea (A). Position of the two marine (M-1 and M-2) and two estuarine (E-1 and E-2) study sites is shown. Small boxes: C, Kolvitsa Inlet; D, Chupa and Keret Inlets.

conditions at the collection sites. It has been previously shown that acclimation to the optimum salinity (which is ca. 24–26‰ for the White Sea L. saxatilis) is very fast in intertidal gastropods and usually takes from several hours to a few days depending on the length of the preceding history at suboptimal salinity (Khlebovich and Berger, 1975). This suggests that an acclimation to laboratory conditions was fully accomplished in the studied periwinkles prior to the start of the experiments.

In each trial, two to seven shell colour variants were involved (Table 1). Shell colour was scored according to Sergievsky et al. (1995) using a combination of three traits — shell ground colour (C), presence / absence of tessellations of the colour different from the ground one (S) and number of longitudinal bands (B). Particular state of each trait was designated in a subscript or a superscript. In some snails, shell colour pattern was not distinguishable due to the heavy calcification of the shell upper layer. Such shells appear greyish-white (W ) or pure white (W ). In each experiment, two commonest shell2 3

p 1 colour morphs (purple tessellated and brown tessellated unbanded, C S B0 and

b 1

C S B ) were included, which account for the major differences in phenotypic structure0

between the estuarine and marine populations of L. saxatilis (Sokolova, 1997; Sokolova et al., 1997). Other shell colour variants were occasionally included in the experiments, when the amount of available animals was sufficient for the analysis.

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Table 1

Shell colour morphs of L. saxatilis used in the experiments

Abbreviation Calcification Ground Tessellations Bands Relative abundance of the

colour morphs (%) in populations:

M-1 M-2 E-1 E-2

p 1

C S B0 2 Dark purple 1 0 67.3 75.5 53.7 52.8

p 2

C S B0 2 Dark purple 2 0 8.2 11.2 4.9 NF

b 1

C S B0 2 Brown 1 0 16.8 9.4 40.7 47.2

o 1

C S B0 2 Orange 1 0 NF ,1.0 NF NF

W and W2 3 1 ? ? 0 NF 2.4 NF NF

W B3 2 1 ? ? 2 ,1.0 ,1.0 NF NF

Particular states of every trait are given in subscripts or superscripts, e.g. shell ground colour (C): p, purple; b, brown, o, orange; tessellations (S): 1, present; 2, absent; longitudinal bands (B): 0, absent; 2, two-banded morphs; degree of calcification (W): 2, moderate; 3, heavy. Calcification of shell upper layer:1, present; 2, absent. Presence / absence of intensive calcification of the upper shell layer is presumably determined by a separate locus in White Sea L. saxatilis (Kozminsky et al., 1995). This calcification is superimposed over other shell colour variants, therefore it is impossible to distinguish ground colour and tessellation pattern in the white morphs (W , W and W B ). Shell colour morphs W and W are intergradable,2 3 3 2 2 3

though normally they can be distinguished by a shade of the white (W is greyish-white, and W is pure white).2 3

NF, shell colour variant was not found.

natural filtered fresh water from the nearby Krivoye Lake and controlled with a densitometer in order to keep deviation from the intended salinity value below 0.3‰.

In physiological experiments it is very important to standardise experimental animals as scrupulously as possible according to age, reproductive state, infection / disease etc. Therefore, in most experiments only adult sexually mature snails (5–8 mm shell diameter) were used. The only experiment in which a second age / size group (juveniles of 3–4 mm shell diameter), was experiment 6 as juveniles were not available in amounts sufficient for all analyses. To check for the trematode infection, dissection of the experimental animals is required. However, due to the time limitations this was not done. Instead, the pilot comparisons were performed in order: (1) to check for the differences in the tested physiological responses of infected and uninfected L. saxatilis irrespective of the shell colour, and (2) to compare infection prevalence in snails with different shell colour in the studied populations. It was found that trematode infection did not significantly influence oxygen consumption, rate of salt loss, mortality in fresh water or freeze resistance of L. saxatilis (Sokolova, unpublished data). This is in agreement with the results of previous studies which showed no differences in oxygen consumption, resistance to fresh water, and freezing tolerance between infected and uninfected Littorina spp. (Lyzen et al., 1992; Galaktionov, 1993; Sokolova, 1997). Furthermore, no differences in the trematode infection levels were detected between the

studied shell colour morphs in either studied population (x 50.09–0.36, df51, P50.45–0.77). Thus, the periwinkles were used in further experiments without special check for the trematode infection.

2.2.1. Experiment 1: oxygen consumption in low salinity

Oxygen consumption rate was determined in snails from the estuarine population E-1 and the marine site M-1 at 108C in salinity of the preliminary acclimation (24‰,

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controls) and after different exposure periods to low salinity (14‰). The latter value was chosen because it was the lower salinity limit at which all snails were able to maintain activity and did not withdraw into the shell. The same individuals were used to measure oxygen consumption in the control and after different exposure periods (1 day and 13 days in the population M-1 and 1 day and 7.5 days in the population E-1). Ten to nine

p 1 b 1

animals for each of the two main shell colour morphs (C S B and C S B ) were used.0 0

Oxygen consumption was measured using the method of closed respiration chambers. Snails were placed individually in the air-tight bottles (50–60 ml) half-filled with the air-saturated sea water of respective salinity at 108C and left for 30–60 min to reduce effect of handling. After this, water was carefully drained off using a plastic tubing in order to reduce disturbance to the snails, and the bottles were refilled. In each experimental set, 2–3 empty bottles (‘blanks’) were filled with the water of the same salinity and temperature. Bottles were sealed with air-tight corks and left for 8–10 h at 108C. Decrease in the oxygen concentration after this exposure period never exceeded 25–30% of the starting value. After the exposure, concentration of oxygen in the water was analytically determined by the Winkler method (Golterman, 1983). Snails were removed from the bottles, blotted dry and weighted to the nearest 1 mg. Wet weight of the snails ranged between 110 and 240 mg (population E-1), and 60 and 160 mg (population M-1).

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Oxygen consumption rate (MO ) was calculated as mg O h2 2 g fresh weight. In ~

the only case when MO2 was found to depend significantly on the weight of experimental animals (in the snails from the population M-1 after 13 days of exposure in 14‰), respiration rates were standardised to the average weight in the experimental group according to the formula (1):

b Wi ]

Rst5Ri3

S D

_¯ (1)

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where R is a standardised respiration rate (mg O hst 2 g ), R is the respiration rate ofi

21 21 _¯

ith animal (mg O h2 g ), W is the fresh (live) weight of ith animal (g), W is thei

average fresh weight in the experimental group (g), and b is a power coefficient in the b

equation: R5aW . This coefficient was obtained by a calculation of the linear regression relating log W to log R by the least square method (Sokal and Rohlf, 1995).

In final calculations, MO was expressed in relative units (percent from the control level2

in the same individual). For the control animals, respiration rates were expressed as percent of the respective mean control values.

2.2.2. Experiment 2: activity in low salinity

Snails from the populations E-1, E-2 and M-1 were used in the experiment. Animals were placed into 1–2 large Petri dishes with the water of 8 or 10‰ at 108C and covered with a glass lid. Salinity values of 8 and 10‰ were chosen in order: (1) to imitate brackish water conditions in the estuarine habitats of White Sea L. saxatilis and (2) to evoke differentiated response in the studied periwinkles, as only some individuals were able to maintain activity under these conditions while others ceased activity and isolated themselves inside the shells. At salinities higher than 10‰ or lower than 8‰, 100% or 0% of active animals, respectively, were observed at least in some of the studied

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populations. After 1, 2, 4 and 6 h of exposure, active and non-active (withdrawn into the shell and isolated by the operculum) periwinkles were counted. At each scoring period, water in the Petri dishes was changed. Care was taken not to disturb snails during this

p 1 b 1

procedure. We used 50–60 and 25–60 animals of C S B0 and C S B0 morphs, respectively.

2.2.3. Experiment 3: rates of isolating and opening responses

Rates of the behavioural response to fast and abrupt salinity change were tested in the animals from the populations M-1, M-2 and E-1. Experiments were carried out at 17–198C. Snails were placed individually into small vials (10 ml), fixed with the aperture upwards and covered with the natural sea water for 10–15 min. Then the water was carefully removed. Under these conditions, snails protrude the foot trying to recover the normal position. When the foot was fully extended, the vial was quickly filled with fresh water and time to close (from the first contact with fresh water to the closure of the operculum) was measured to the nearest 1 s. After the measurements, snails were left for 45 min in fresh water, then fresh water was carefully removed and the vial quickly filled with natural sea water. Time to open (from the first contact with sea water to the emergence of tips of the tentacles) was measured to the nearest 0.2 s. Different time scales were used for measuring opening and closing times because it normally took much longer in the periwinkles to close than to open the shell. N was 6–12 for each shell colour morph.

2.2.4. Experiment 4: rate of salt loss in fresh water

Snails from the populations E-1 and M-1 were rinsed in plenty of deionised water to remove salts from the shell surfaces and placed individually in 100-ml glass vials. Each vial was filled with 50 ml of NaCl solution in deionised water (3.5–4.0 mg / l) at

118–208C. The snails were left in the NaCl solution for 5 min, which was then well mixed. Its conductivity was measured using the reochord bridge according to the method described in Khlebovich and Berger (1965). After 2 h of exposure, conductivity of the solution was measured again.

To transform conductivity into concentration units, calibration solutions of NaCl were used. Relationship between conductivity and salt concentration closely followed the

power function (R 597–99.9%). Power regression equations were used to calculate salt concentration in the solution at the start and after 2 h exposure. After the experiment, snails were taken out of the vials, blotted dry and weighed to the nearest 1 mg. Rate of

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salt loss was expressed as mg NaCl h g live weight. N was 8–12 for each shell colour morph.

2.2.5. Experiment 5: mortality in fresh water

Animals from the populations M-1 and E-1 were placed into the trays filled with 700–800 ml of fresh water at 108C. After specified exposure periods (7, 12 and 19 days), a portion of snails was removed from the tray, placed in sea water and allowed to recover for 2 h at 108C. The longest exposure period was chosen to produce ,80% of mortality and was 12 days for snails from the population M-1 and 19 days for individuals from the site E-1. After the recovery period, the number of dead and alive

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snails were counted. Those snails which failed to respond by contracture to being poked by the needle were considered dead. Mortality was expressed as percent of dead individuals. N was 458–543 for the purple tessellated unbanded morph and 92–98 for the brown tessellated unbanded morph.

2.2.6. Experiment 6: resistance to low salinity and subzero temperatures

Snails from the population E-2 were used to determine resistance to freezing at

29.08C and 8‰. This combination of temperature and salinity was chosen to approximate extreme conditions in White Sea estuaries which may be experienced by the intertidal snails during low tides in late autumn. During this period, the ice cover over the intertidal which in winter shields the substrate against freezing, is not yet formed, but frosty days with air temperature down to 29–108C are frequent (Savoskin, 1967).

Animals were placed in small plastic cylinders, covered with 15–20 ml of diluted sea water and slowly cooled down and frozen at the rate of 1.5 h at 3–48C, 1.5 h at 218C and then 2 h at 298C (temperature range 29.0–9.58C). Temperature control showed that it took 90 min to reduce the temperature from 218C down to 298C, so the actual exposure time at 298C was 30 min. After the exposure snails were slowly thawed at 3–48C for 3 h and at 15–178C for 2 h until ice crystals totally disappeared from the water, placed into natural sea water (24–25‰) at 15–178C and allowed to recover for 14 h. This period was found to be sufficient for all alive snails to resume activity. After the recovery period the number of dead snails was determined. N was 30 (adults) or 80 (juveniles) for each shell colour morph.

2.3. Statistics

Rates of oxygen consumption, salt loss and rate of opening and closing of the aperture were compared using standard ANOVA procedures (Sokal and Rohlf, 1995). Prior to the analysis, data were checked for the fit to normal distribution and for the heterogeneity of variances by chi-square or Kolmogorov–Smirnov tests and Cochran test, respectively. Assumptions of the normal distribution and homogeneity of variances was violated in neither of the studied parameters except the time to close the aperture upon transfer to fresh water (Table 3). For this variable, a log-transformation was used which resulted in a significant improvement of homogeneity of the variances.

To compare mortality / survivability and relative activity estimates, log-linear analysis followed by chi-square test or exact Fisher test (if the expected frequencies were less than 5) for pairwise comparisons were used (Sokal and Rohlf, 1995). To choose the best-fitted model in log-linear analysis, an iterative procedure was performed (Sokal and Rohlf, 1995). Model was considered to fit the data if the probability level for the model (P(Model)) exceeded 0.10. An effect of factor interactions was included if the improvement in the model fit (i.e. an increase in G-values of goodness-of-fit) was significant at the 5% level.

Results are expressed as percentages or means6standard errors if not mentioned otherwise.

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3. Results

3.1. Responses of L. saxatilis to moderate salinity change 3.1.1. Experiment 1: oxygen consumption at low salinity

Exposure to low salinity (14‰) for 24 h resulted in a significant reduction of the oxygen consumption rate (down to 22% of the control level) in L. saxatilis from the marine population M-1 (Fig. 2). However, after prolonged (13 days) acclimation at 14‰

respiration rate increased again, so that a marked overshoot of MO over the control2

values was detected. In snails from the estuarine site E-1, MO2 was similar in the

Fig. 2. Experiment 1: rates of oxygen consumption in L. saxatilis with different shell colour in control and after different exposures to low salinity. Phenotypes: Purp Tess, purple tessellated unbanded; Brow Tess,

brown tessellated unbanded. Populations: M-1 (marine), E-1 (estuarine). MO2 is given as percent of the respective control level. Vertical bars represent standard errors.

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control and after the different exposure periods in 14‰ (Fig. 2). Two-way mixed model ANOVAs were used to test for the effects of the factors ‘exposure duration’ (random) and ‘shell colour’ (fixed) on oxygen consumption rate in each of the two studied populations. Two-factor interactions were non-significant (M-1: F2,5150.93, P.0.40; E-1: F2,6650.69, P.0.50) suggesting that different shell colour morphs responded similarly to exposure at 14‰. Exposure duration had highly significant effect on oxygen consumption rate of the periwinkles from the population M-1 (F2,51572.23, P,0.0001), but not in the animals from the site E-1 (F2,6651.24, P.0.28). No significant

differences in MO2 were found between snails with different shell colours in either studied population (M-1: F1,252.11, P.0.28; E-1: F1,250.24, P.0.67).

3.1.2. Experiment 2: relative activity in low salinity

Exposure to 8 or 10‰ caused cessation in activity and isolation inside the shell in a considerable proportion of the snails from all the studied populations (Fig. 3). At all comparable exposure periods, percentage of active individuals was the lowest in the periwinkles from the marine site M-1 as compared to the snails from the estuarine populations E-1 and E-2. Moreover, patterns of the changes in relative number of active snails over time differed between the samples from the estuarine and marine sites. In the animals from the marine site M-1 a proportion of active snails declined drastically during the exposure at low salinity. While at the initial period of exposure at 8‰ 10–24% of the periwinkles were active in the sample from the population M-1, all of them ceased activity after 6 h. In 10‰ some snails were still active after 6 h of exposure, but their proportion decreased 4–5-fold as compared to the first hour of the exposure (Fig. 3). In contrast, percentage of the active snails changed only slightly in the samples from the estuarine sites E-1 and E-2 during 6 h of exposure to 10‰ or 8‰ (Fig. 3). Log-linear analysis involving linear combination of five factors (population of origin (P), salinity of exposure (S), exposure duration (E), shell colour (C), and activity state (A)) showed that two of the four-factor interactions were significant including the

interactions P3E3C3A (x 519.44 for marginal association, df56, P,0.004) and

P3S3C3A (x 58.58 for marginal association, df52, P,0.02) suggesting the interactive effects of respective factors on the relative amount of active snails and hence supporting the observation on the differences in degree and dynamics of response to low salinity in different populations and at different salinities. Therefore, effects of salinity, exposure duration and shell colour on the ability of the snails to retain activity in low salinity were further tested separately for each population.

Log-linear analysis showed that the relative activity of snails in low salinity was dependent on salinity and duration of exposure in all the studied populations (note significant E3A, S3A or S3E3A interactions in Table 2). For the animals from the populations M-1 and E-2, the best fitted log-linear models also included significant interactions between the shell colour and activity in low salinity (M-1: Table 2A) or the shell colour, exposure duration and activity (E-2: Table 2C) thus implying differences in the relative activity between the studied shell colour morphs. Unlike this, no significant effect of shell colour on the relative activity was found in the periwinkles from the population E-1 (Table 2B). Detailed pairwise comparisons at each combination of salinity and exposure showed that the relative amount of active snails was higher in the

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Fig. 3. Experiment 2: ability of L. saxatilis with different shell colour to retain activity in low salinity. Phenotypes: Purp Tess (filled symbols), Brow Tess (hollow symbols). Percentages of animals active after different exposure periods at 8‰ (triangles) and 10‰ (circles) are shown. Populations: M-1 (marine), E-1 and E-2 (estuarine). Vertical bars represent standard errors. Significant differences in the relative activity between the two shell colour morphs at each salinity and exposure are shown with asterisks (*P,0.05 and **P,0.01).

purple tessellated unbanded morphs as compared to the brown tessellated unbanded in the samples from the sites M-1 and E-2 (Fig. 3). These differences were significant at least at some exposures: after 2 and 4 h in 10‰ (Fisher’s exact test, P50.02 and 0.002,

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Table 2

Log-linear analysis: effects of the population of origin (P), salinity of exposure (S), exposure duration (E) and shell colour of the periwinkles (C) on the relative amount of active L. saxatilis (A) in low salinity (experiment

A. Population M-1

Best fitted model: df x P

S3A1E3A1C3A 20 21.28 0.38

2 2

Interactions df x (marginal P (marginal x (partial P (partial

association) association) association) association)

S3A 1 48.25 ,0.001 52.60 ,0.001

E3A 3 50.10 ,0.001 54.50 ,0.001

C3A 1 16.27 ,0.001 18.49 ,0.001

B. Population E-1

Best fitted model: df x P

S3A1E3A 22 13.45 0.92

2 2

Interactions: df x (marginal P (marginal x (partial P (partial

association) association) association) association)

S3A 1 133.71 ,0.001 135.39 ,0.001

E3A 3 9.89 0.020 11.42 0.010

C. Population E-2

Best fitted model: df x P

S3E3A1E3C3A1S3C 7 5.50 0.60

2 2

Interactions: df x (marginal P (marginal x (partial P (partial

association) association) association) association)

S3C 1 1.58 0.209 8.26 0.004

S3E3A 3 15.15 0.002 10.48 0.015

E3C3A 3 14.20 0.003 6.50 0.090

The best-fitted model was chosen by iterative procedure (Sokal and Rohlf, 1995) according to the following criteria: (1) a model was considered to fit the data if P(Model) exceeded 0.10; (2) an effect of factor interactions was included if the improvement in the model fit was significant at the 5% level. Significant interaction of any factor(s) with the factor A (activity state) can be interpreted as a statistically significant effect of the respective factor or interaction of factors on the relative amount of active snails.

respectively) and after 2 h in 8‰ (x 57.40, df51, P,0.01) in the population E-2 and

after 2 h of exposure at 10‰ in the population M-1 (x 514.01, df51, P,0.01). Though the snails with purple tessellated unbanded shells also tended to have higher relative activities at other exposure periods at 8 and 10‰ in the samples from the populations M-1 and E-1 and at 2–6 h of exposure at 8‰ in the animals from the population E-1, these differences were not statistically significant.

3.2. Responses of L. saxatilis to extreme salinity change 3.2.1. Experiment 3: rates of isolating and opening responses

Due to the unbalanced design of the experiment (different number of shell colour morphs available in each studied population), effects of the factors ‘population’ (random) and ‘shell colour’ (fixed) were analysed only for the brown tessellated and purple

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Table 3

Effects of the population of origin and shell colour on the rate of closing and opening responses to abrupt

salinity changes in White Sea L. saxatilis (experiment 3)

Time to open Time to close (log -transformed)10

df (effect, error) F P df (effect, error) F P

A. Two-way ANOVAs: effects of the population of origin and shell colour on time to open and to close the aperture. Only purple tessellated unbanded and brown tessellated unbanded morphs are included

Shell colour (S) 1, 2 1.55 0.339 1, 2 1.13 0.398

Population (P) 2, 49 2.60 0.084 2, 41 27.42 ,0.001

S3P interaction 2, 49 0.18 0.835 2, 41 1.80 0.178

B. One-way ANOVAs: effect of shell colour on times to open and close the shell (within populations). See Fig. 4 for shell colour morphs included in the analyses

Population

M-1 3, 20 0.62 0.607 3, 20 2.64 0.077

M-2 5, 48 0.23 0.949 5, 48 1.56 0.194

E-1 1, 22 0.46 0.506 1, 22 0.97 0.338

Log-transformation of time to close the aperture upon exposure to freshwater was performed to assure homogeneity of variances in this variable. Degrees of freedom (df) for the respective effect and error terms are separated by comma. Population of origin was treated as a random factor, and shell colour as a fixed one.

tessellated morphs (Table 3A). The rates of behavioural responses to abrupt salinity changes were not significantly affected by the shell colour (F1,251.55, P50.339 and F1,251.13, P50.398 for times to open and close, respectively). This finding was also supported by the set of one-way ANOVAs involving a wider range of the tested shell colour variants within each of the studied populations (Table 3B). The population of origin had a significant effect on the time to close the aperture upon transfer to fresh water, and a marginally significant effect on the rate to open the shell upon returning to sea water (Table 3A). In order to increase the power of the analysis, we pooled all shell colour morphs within each population. However, this affected but slightly the obtained results (effect of the population of origin on time to open: F2,9952.84, P.0.06; on time to close: F2,87538.13, P,0.001). In general, the periwinkles from the marine site M-2 were fastest to close in the fresh water, and it took longest to close in the snails from the estuarine population E-1 (Fig. 4A and C). Conversely, animals from the estuarine population E-1 tended to open the shell faster when placed into the sea water, while the rate of the opening response was somewhat lower in the snails from the marine sites (Fig. 4B and D).

3.2.2. Experiment 4: salt loss in fresh water

No differences in the rate of salt loss were detected between the different shell colour morphs of L. saxatilis within each of the studied populations (M-1: F2,2150.07, P.0.92; E-1: F1,2252.31, P.0.14). As was shown by the mixed-model two-way

p 1 b 1

ANOVA involving snails of the two shell colour morphs (C S B and C S B ), the0 0

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Fig. 4. Experiment 3: rates of isolation (A, C) and opening (B, D) of different shell colour morphs of L. saxatilis in response to fast and strong changes of environmental salinity. Average time to close the shell aperture upon contact with freshwater for different shell colour morphs (A) and for all animals irrespective of shell colour (C) is shown. Mean time required to respond to the placing into seawater by opening the aperture is also given for snails with different shell colour (B) and for the whole sample (D). Phenotypes: Purp Tess,

p 1 b 1 p 2 o 1

C S B ; Brow Tess, C S B ; Purp Plain, C S B ; Orange, C S B ; White, W and W ; White Band, W B .0 0 0 0 2 3 3 2

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estuarine population E-1 and the marine site M-1 (F1,36514.69, P,0.001). Animals 21 21

from the marine site lost salts at the rate of 0.3760.07 mg NaCl h g live weight, 21

whereas respective values for the estuarine periwinkles were 0.1560.02 mg NaCl h 21

g live weight (Fig. 5A and B). Again, no differences between the shell colour variants were found in this two-factor design (F1,150.001, P.0.97).

Fig. 5. Experiment 4: rates of salt loss (A, B) and mortality (C) of different shell colour morphs of L. saxatilis

p 1 b 1 p 2

in freshwater. Phenotypes: Purp Tess, C S B ; Brow Tess, C S B ; Purp Plain, C S B . Populations: M-1,0 0 0

marine; E-1, estuarine. Rate of salt loss is given for snails with different shell colour (A) and for the whole sample (B) in the two studied populations. (C) Mortality of different shell colour morphs in freshwater at 108C. Vertical bars represent standard errors of estimate.

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Table 4

Log-linear analysis: effects of the population of origin (P) and shell colour of the periwinkles (C) on the

mortality of White Sea L. saxatilis (M) during fresh water exposure at 108C (experiment 5) Populations M-1 and E-1, 12 days of freshwater exposure at 108C

Best fitted model: df x P

P3M1C3M 2 1.07 0.59

2 2

Interactions df x (marginal P (marginal x (partial P (partial

association) association) association) association)

P3M 1 130.50 ,0.001 126.82 ,0.001

C3M 1 26.80 ,0.001 23.12 ,0.001

The best-fitted model was chosen by iterative procedure (Sokal and Rohlf, 1995) according to the following criteria: (1) a model was considered to fit the data if P(Model) exceeded 0.10; (2) an effect of factor interactions was included if the improvement in the model fit was significant at the 5% level. Significant interaction of any factor(s) with the factor M (mortality) can be interpreted as a statistically significant effect of the respective factor or interaction of factors on mortality rate.

3.2.3. Experiment 5: mortality in fresh water at 108C

Due to the low expected frequencies of the dead snails after 7 days of freshwater exposure (Fig. 5C), mortality rates across populations and shell colour morphs were compared for the later exposures only. The best fitted log-linear model for the data on mortality rates after 12 days of freshwater exposure included two highly significant factor combinations: C3M (shell colour and mortality) and P3M (population of origin and mortality) (Table 4) suggesting that mortality was both dependent on shell colour of the periwinkles and on the population of origin. Snails from the estuarine population E-1 were more resistant to prolonged fresh water exposure at 108C than animals from the marine site M-1 (Fig. 5C, Table 4). In both estuarine and marine sites, the periwinkles

b 1

with brown tessellated unbanded shell (C S B ) exceeded in resistance the animals with0 p 1

purple tessellated unbanded shells (C S B ) (Fig. 5C, for 12 days of freshwater0 2

exposure see Table 4; for 19 days of exposure in the population E-1: x 58.78, df51, P50.003).

3.2.4. Experiment 6: freezing resistance at low salinity

Both juveniles and adult molluscs demonstrated good survivability after 30 min freezing at 298C, to up to 60–95% (Fig. 6). Log-linear model including linear combinations of three factors (age of the snails Ag, shell colour S and mortality M) suggested that the studied shell colour morphs responded differently to freezing in low salinity (note the significant C3M interaction in the best fitted model, Table 5). In general, snails of the brown tessellated unbanded morph demonstrated higher surviv-ability than animals with the purple tessellated unbanded shells (Fig. 6), though these

differences were only statistically significant in juveniles (for juveniles: x 517.51,

df51, P,0.0001; for adults: x 51.46, df51, P50.23).

4. Discussion

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Fig. 6. Experiment 6: survival of L. saxatilis with different shell colour after exposure at298C and 8‰.

p 1

Juvenile (juv) and adult (ad) animals from the estuarine site E-2 were used. Phenotypes: Purp Tess, C S B ;0 b 1

Brow Tess, C S B . Vertical bars represent standard errors of estimate.0

varied independently of shell colour of L. saxatilis, several important parameters related to capacity and resistance salinity adaptations differed considerably between the two

b 1 p 1

most abundant shell colour morphs, ‘estuarine’ C S B and ‘marine’ C S B .0 0

Oxygen consumption rate is an integral measure of metabolic rate of an individual. This parameter was frequently and successfully used as a measure of metabolic disturbance imposed by a salinity stress and also as a measure of the rate and completeness of subsequent salinity acclimation (Kinne, 1971; Khlebovich and Berger, 1975; Berger and Kharazova, 1997). Our data showed that during the early stage of acclimation to low salinity (after 24 h of exposure to 14‰), oxygen consumption rate declined markedly in the snails from the marine population, while aerobic metabolism appeared to be only slightly and non-significantly disturbed in the estuarine periwinkles. Table 5

Log-linear analysis: effects of the age (Ag) and shell colour of the periwinkles (C) on the mortality of White

Sea L. saxatilis (M) after freezing at298C and 8‰ (experiment 6) Population E-2

Best-fitted model: df x P

Ag1C3M 3 4.89 0.18

2 2

Interactions df x (marginal P (marginal x (partial P (partial

association) association) association) association)

Ag 1 44.79 ,0.001 44.79 ,0.001

C3M 1 18.17 ,0.001 18.57 ,0.001

The best-fitted model was chosen by iterative procedure (Sokal and Rohlf, 1995) according to the following criteria: (1) a model was considered to fit the data if P(Model) exceeded 0.10; (2) an effect of factor interactions was included if the improvement in the model fit was significant at the 5% level. Significant interaction of any factor(s) with the factor M (mortality) can be interpreted as a statistically significant effect of the respective factor or interaction of factors on mortality rate.

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Nevertheless, snails from either studied population were able to successfully acclimate to ~

salinity of 14‰ and to restore nearly control levels of MO2 after 1–2 weeks of ~

acclimation. There is no evidence of the difference in MO between the different shell2

colour morphs thus suggesting that acclimation rates and ability to maintain a stable level of aerobic metabolism during a moderate salinity change are probably unrelated to shell colour in White Sea L. saxatilis.

Relative activity of L. saxatilis in low salinity demonstrated significant within- and among-population variation related to the habitat and the shell colour, respectively. In general, relatively more snails from the estuarine sites were capable of maintaining activity in low salinity as compared to the animals from the marine site. This agrees with the results of a previous study on seven populations of L. saxatilis from the White Sea which demonstrated that optimal salinity range where activity can be maintained is shifted towards lower salinities in animals from estuarine habitats as compared to intermediate and especially marine sites (Sokolova, 1997). The present study has also shown that prolonged exposure to sub-optimal salinities has differential effect on the number of the active individuals from the estuarine and marine population. During prolonged exposure to 10 and especially 8‰, progressively more snails from the marine site ceased activity, while in animals from the estuarine sites this reduction was much slower (at 8‰) or practically absent (at 10‰). Enhanced ability to tolerate suboptimal salinities without cessation of activity may be advantageous for the periwinkles under conditions of fast and frequent salinity fluctuations in the estuary, increasing a potential period of activity when feeding, reproduction and other life-important functions can be performed. This also agrees with a finding of this study that estuarine snails were significantly slower to close when placed in extremely hypoosmotic medium and tended to open faster upon returning to sea water as compared to the periwinkles from the marine sites.

Interestingly, the ability to maintain activity in low salinity demonstrated significant intrapopulational differentiation related to the shell colour in two of the three studied populations. In contrast to what might be expected, relative activity of the ‘estuarine’

b 1

morph C S B in low salinity was lower than in the animals of the ‘marine’ morph0 p 1

C S B0 in the populations M-1 and E-2. Similar tendency, though statistically insignificant, could be observed in snails from the estuarine site E-1 at 8‰. This suggests that the animals of the ‘estuarine’ shell colour morph are less capable to tolerate moderate salinity decrease and have thus a relatively limited salinity range where their capacity adaptations are effective.

On the contrary, the differential resistance of the two commonest shell colour morphs of L. saxatilis to extreme hypoosmotic stress and to the combined effect of low salinity / low temperature stress is in agreement with the expectations based on their relative abundance in estuarine and marine sites. It is worth noting that survivability in fresh water was generally higher in the animals from the estuarine site as compared to those from the marine one thus suggesting that a shift (either acclimatory or genetic) towards higher resistance to extreme salinity stress might have occurred in all shell colour morphs in the estuarine habitat. Similar increase in fresh water resistance has been previously reported for the littorinids from estuarine and brackish water habitats as comparison to the conspecifics from the marine sites in the White and Barentz Seas

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(Golikov and Smirnova, 1974; Berger, 1986). Nevertheless, in the studied populations from either estuarine or marine habitat, the ‘estuarine’ brown tessellated unbanded morph was found to be more resistant to extreme hypoosmotic stress as compared to the ‘marine’ purple tessellated unbanded morph. The between-morph differences in fresh water resistance are obviously not related to the efficiency of insulation from unfavour-able environment, as there was no evidence for the differences in rate of salt loss between snails of different shell colour phenotypes. More probably, differential ability to survive extreme hypoosmotic stress in isolated state is related to anaerobic capacity as was shown for three White Sea Littorina spp. including L. saxatilis (Sokolova et al.,

b 1

1999). Moreover, snails of the ‘estuarine’ shell colour phenotype C S B demonstrated0

higher resistance to combined low salinity / low temperature stress as compared to the ‘marine’ purple tessellated unbanded morph. These differences were more pronounced in juveniles, whereas in adults a shift towards generally higher resistance was accompanied by levelling of the resistance levels of different shell colour morphs. Our data are not sufficient to decide whether this would be a result of individual acclimation, or an adaptation on the population level.

It is worth noting that exposure to long periods of extremely low salinity is not an unrealistic experience for the White Sea Littorina spp. In White Sea estuaries environmental salinity undergoes rapid and drastic circatidal variation. For example, in summer–autumn period, salinity of the near-shore surface water fluctuates between 10 and 13‰ at high tide and 1 and 5‰ at ebbing and low tide at the E-1 site, while in the marine sites M-1 and M-2 a stable salinity of 24–25‰ was found throughout the tidal cycle (Sokolova, 1997; Sokolova, unpubl. data). Besides, seasonal salinity variation is well pronounced in the White Sea (Kuznetsov, 1960). In open parts of the studied area of Kandalaksha Bay, surface water salinity can drop down to 2‰ for as long as a fortnight during spring ice-melting (Babkov and Lukanin, 1985). This salinity is well below levels tolerated by the periwinkles and causes behavioural escape response (isolation within the shell). In estuaries such periods of low salinity may be even longer and more pronounced (Berger, 1986). During late autumn and early winter low salinity may combine with subzero temperatures in the littoral zone of White Sea estuaries (Savoskin, 1967) thus imposing additional stress on intertidal inhabitants. Hence, long periods of extremely suboptimal salinity (down to levels causing total cessation of activity of the periwinkles), which in some seasons is also combined with extremely low temperatures, are among major factors influencing the periwinkles in White Sea estuaries and may be expected to exert strong selection pressure on their populations.

Our data show that physiological differences in response to low salinity between the studied shell colour morphs of L. saxatilis are consistent with the hypothesis of physiological selection on shell colour polymorphism imposed by extreme salinity conditions of White Sea estuaries. Towards the head of an estuary, relative abundance of

b 1

the morph (C S B ) which has the highest resistance to extreme salinity and to low0

salinity / low temperature stress increases while frequency of the less resistant phenotype

p 1

C S B0 declines (Sokolova et al., 1995, 1997; this study). Interestingly, elevated frequency of the brown shells has been also found in L. rudis (5L. saxatilis) populations from the salt marshes in the North Atlantic which are usually characterised by a very unstable salinity regime (Raffaelli, 1979). At the same time, no consistent differences

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were found between the studied colour morphs of L. saxatilis in response to moderate salinity changes or in behavioural responses to salinity fluctuations. Similar results were

p 2

reported for White Sea L. obtusata where the ‘estuarine’ morph C S B0 was characterised by higher resistance to extremely low salinity but similar or even lower tolerance of moderate salinity changes as compared to the shell colour morphs predominantly found in the marine sites (Sergievsky and Berger, 1984; Berger and Sergievsky, 1986; Sergievsky, 1992, 1995). Probably, this may be partially attributed to the specific selective conditions in White Sea estuaries which not only involve an overall lowered salinity but also (and especially so) prolonged periods of extremely low salinity which is far beyond acclimation potential of this species. Moreover, most capacity responses to a moderate salinity change are physiologically very plastic and subject to fast acclimatory changes to match requirements of the estuarine habitat (Khlebovich and Kondratenkov, 1973; Khlebovich and Berger, 1975; Berger, 1986; Sokolova, 1997). It’s only the conditions which exceed the potential of physiological plasticity of the individual which result in selection upon the population. This may explain why particularly the morph with better abilities to survive periods of extreme salinity and temperature stress may have an advantage in White Sea estuaries.

To summarise, the observed changes of pheno- (geno-)typic structure of L. saxatilis in White Sea estuaries may be classified as a case of so-called physiological selection implying genetic correlates between shell colour and selectively important traits (in this case, resistance to extreme salinity and / or extreme salinity / temperature combinations) probably due to pleiotropic effects or gene linkage. A similar result has been previously reported for White Sea L. obtusata populations arrayed along a salinity gradient in White Sea estuaries (Sergievsky and Berger, 1984; Berger and Sergievsky, 1986; Sergievsky, 1992; Sokolova et al., 1997). This together with previous findings of the selective importance of shell coloration of Littorina spp. for crypsis and thermoregulation, strongly emphasise an adaptive value of shell colour polymorphism in populations of the periwinkles. However, further study is necessary in order to reveal fine mechanisms maintaining shell colour polymorphism within populations of Littorina spp. and possible physiological and / or populational trade-offs between high resistance to extremely low salinity and low salinity / low temperature stresses and other fitness-related traits preventing any morph from becoming fixed in a given habitat.

Acknowledgements

This work was partially supported by the Russian Foundation for Basic Research, grants N 96-04-48394 (BVJ) and 98-04-49977 (SIM). [SS]

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Nevertheless, snails from either studied population were able to successfully acclimate to

salinity of 14‰ and to restore nearly control levels of MO2 after 1–2 weeks of

acclimation. There is no evidence of the difference in MO between the different shell2

b 1

morph C S B in low salinity was lower than in the animals of the ‘marine’ morph0 p 1

(2)

b 1

1999). Moreover, snails of the ‘estuarine’ shell colour phenotype C S B demonstrated0

b 1

the morph (C S B ) which has the highest resistance to extreme salinity and to low0

salinity / low temperature stress increases while frequency of the less resistant phenotype

p 1

(3)

were found between the studied colour morphs of L. saxatilis in response to moderate salinity changes or in behavioural responses to salinity fluctuations. Similar results were

p 2

Acknowledgements

This work was partially supported by the Russian Foundation for Basic Research, grants N 96-04-48394 (BVJ) and 98-04-49977 (SIM). [SS]

References

(4)

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Berger, V.Ja., Sergievsky, S.O., 1986. Differences in adaptive responses to salinity changes of the gastropod

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