I .M. Sokolova, V.J. Berger J. Exp. Mar. Biol. Ecol. 245 2000 1 –23
9
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 control
2
~ values was detected. In snails from the estuarine site E-1, MO
was similar in the
2
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. MO is given as percent of the
2
respective control level. Vertical bars represent standard errors.
10 I
.M. Sokolova, V.J. Berger J. Exp. Mar. Biol. Ecol. 245 2000 1 –23
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: F
50.93, P.0.40;
2,51
E-1: F 50.69, P.0.50 suggesting that different shell colour morphs responded
2,66
similarly to exposure at 14‰. Exposure duration had highly significant effect on oxygen consumption rate of the periwinkles from the population M-1 F
572.23, P,0.0001,
2,51
but not in the animals from the site E-1 F 51.24, P.0.28. No significant
2,66
~ differences in MO
were found between snails with different shell colours in either
2
studied population M-1: F 52.11, P.0.28; E-1: F
50.24, P.0.67.
1,2 1,2
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
2
interactions P3E3C3A x 519.44 for marginal association, df56, P,0.004 and
2
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
I .M. Sokolova, V.J. Berger J. Exp. Mar. Biol. Ecol. 245 2000 1 –23
11
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, P 50.02 and 0.002,
12 I
.M. Sokolova, V.J. Berger J. Exp. Mar. Biol. Ecol. 245 2000 1 –23 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
2 A. Population M-1
2
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
2
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
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
a
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 PModel 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 factors 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.
2
respectively and after 2 h in 8‰ x 57.40, df51, P,0.01 in the population E-2 and
2
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
I .M. Sokolova, V.J. Berger J. Exp. Mar. Biol. Ecol. 245 2000 1 –23
13 Table 3
Effects of the population of origin and shell colour on the rate of closing and opening responses to abrupt
a
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
a
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 F
51.55, P50.339 and
1,2
F 51.13, P50.398 for times to open and close, respectively. This finding was also
1,2
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: F
52.84, P.0.06; on time
2,99
to close: F 538.13, P,0.001. In general, the periwinkles from the marine site M-2
2,87
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: F
50.07,
2,21
P .0.92; E-1: F 52.31, P.0.14. As was shown by the mixed-model two-way
1,22 p
1 b
1
ANOVA involving snails of the two shell colour morphs C S B and C S B , the rate of salt loss in fresh water was significantly different in the periwinkles from the
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.M. Sokolova, V.J. Berger J. Exp. Mar. Biol. Ecol. 245 2000 1 –23
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 .
2 3
3 2
Populations: M-1, marine; E-1, estuarine. Vertical bars represent standard errors.
I .M. Sokolova, V.J. Berger J. Exp. Mar. Biol. Ecol. 245 2000 1 –23
15
estuarine population E-1 and the marine site M-1 F 514.69, P,0.001. Animals
1,36 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 F 50.001, P.0.97.
1,1
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, 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.
16 I
.M. Sokolova, V.J. Berger J. Exp. Mar. Biol. Ecol. 245 2000 1 –23 Table 4
Log-linear analysis: effects of the population of origin P and shell colour of the periwinkles C on the
a
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
2
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
a
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 PModel 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 factors 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 with
p 1
purple tessellated unbanded shells C S B Fig. 5C, for 12 days of freshwater
2
exposure see Table 4; for 19 days of exposure in the population E-1: x 58.78, df51,
P 50.003. 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
2
differences were only statistically significant in juveniles for juveniles: x 517.51,
2
df51, P ,0.0001; for adults: x 51.46, df51, P50.23.
4. Discussion
