The combination of low salinity and low temperature can limit the colonisation success of the non-native bivalve Rangia cuneata in brackish Baltic waters

Journal of Experimental Marine Biology and Ecology 524 (2020) 151228

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The combination of low salinity and low temperature can limit the colonisation success of the non-native bivalve Rangia cuneata in brackish Baltic waters

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Julia Tuszer-Kunca, , Monika Normant-Sarembaa, Agata Rychterb a University of Gdańsk, Faculty of Oceanography and Geography, Institute of Oceanography, Department of Experimental Ecology of Marine Organisms, Al. Marszałka J. Piłsudskiego 46, 81-378 Gdynia, Poland b State University of Applied Sciences in Elbląg, Institute of Technology, ul. Wojska Polskiego 1, 82-300 Elbląg, Poland

A R T I C LE I N FO

A B S T R A C T

Keywords: Atlantic Rangia Non-indigenous species Direct calorimetry Heat production Vistula Lagoon Baltic Sea

We studied the behavioural (burrowing and valves gaping) and metabolic responses of Atlantic rangia Rangia cuneata (G.B. Sowerby I, 1832) at different salinities and temperatures. Three salinities (S = 0.5, 7.0, 10.0) were tested at 20 °C, with an additional control of 2.4. The control salinity (S = 2.4) was also tested at 10 °C to examine responses specific to colder temperatures. Clams exposed to the lowest (S = 0.5, T = 20 °C) and the highest salinity (S = 10.0, T = 20 °C), as well as lower temperature (T = 10 °C, S = 2.4) significantly (p < .05) reduced the rate of routine metabolism. At the lowest salinity (S = 0.5, T = 20 °C) and temperature (T = 10 °C, S = 2.4), they were also more abundant on the surface of the sediment, however the differences in relation to control conditions (S = 2.4, T = 20 °C) were not significant (p > .05). Clams kept their shells open in the lowest salinity, but closed in the highest salinity and at lower temperature. The closure time was longer in salinity of 10.0 than at temperature of 10 °C, where the highest reduction in the metabolic rate occurred in relation to the control conditions. This latter fact, in combination with shell closure, may indicate the activation of an anaerobic pathway. Thus, in the long term, lower temperatures may adversely affect R. cuneata, especially at very low salinity (S = 0.5, 2.4), in which this hyper-osmoregulating species has to strongly pump water with ions and oxygen to compensate for energy demand. In salinity of 10.0, where the species is isosmotic to the environment, changes in behaviour (moving to the sediment surface and closing the shell) with a simultaneous reduction in the metabolic rate may be due to lower costs of osmoregulation. Our results may help explain the strong inter-annual fluctuations in the population size of this subtropical species in the Baltic coastal waters resulting from the mass mortality of adults after long winters.

1. Introduction Atlantic rangia Rangia cuneata (G.B. Sowerby I, 1832) is native to the Gulf of Mexico, from Laguna de Terminos, Campeche, Mexico in the east to northwest Florida in the north, where it is predominantly found in estuaries (e.g. Dall, 1894; LaSalle and de la Cruz, 1985). In the last decade, this species has been introduced to the Baltic Sea, where it has colonised many coastal regions (e.g. Bock et al., 2015; Wiese et al., 2016; Möller and Kotta, 2017; Solovjova et al., 2019). This bivalve was first recorded in the Vistula Lagoon (the southern Baltic Sea) in 2010, where it established a population (Ezhova, 2012; Rudinskaya and Gusev, 2012; Warzocha and Drgas, 2013). However, the abundance of this population strongly fluctuates throughout the year (Warzocha et al., 2016). Salinity and temperature are key factors determining the

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overall fitness of aquatic organisms by affecting their physiology, including feeding, excretion, respiration as well as growth and reproduction (e.g. McLusky, 1973; Bayne et al., 1976; Almada-Villela, 1984; Bayne, 1998; Gosling, 2003; Sarà et al., 2008; Hiebenthal et al., 2012; Bertrand et al., 2017). As hypothesized by Kornijów et al. (2018), the mass mortality of R. cuneata observed in winter-spring months could be due to low temperatures (water temperature is approximately 10 °C) that weakened the clams' condition. High mortalities of R. cuneata have also been observed in the Curonian Lagoon (the southeastern Baltic Sea), which experiences low temperatures and salinities similar to these in the Vistula Lagoon (salinity even around 0.5 and temperature below 5 °C; Jakimavičius et al., 2018; Solovjova et al., 2019). In its native regions, R. cuneata occurs in estuaries and coastal lagoons where salinity ranges from 0 to 25 (e.g. Woodburn, 1962;

Corresponding author. E-mail address: [email protected] (J. Tuszer-Kunc).

https://doi.org/10.1016/j.jembe.2019.151228 Received 21 March 2019; Received in revised form 9 September 2019; Accepted 16 September 2019 0022-0981/ © 2019 Elsevier B.V. All rights reserved.

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2. Materials and methods

Hopkins et al., 1973; Swingle and Bland, 1974). However, in these habitats, it is sometimes also exposed to very low salinity, below 0.3, lasting up to several months (Hopkins and Andrews, 1970). Also in the Vistula Lagoon where R. cuneata occurs, the salinity varies from 0.5 to 6.5 (Chubarenko, 2008; Kruk, 2012). Salinity is -important for the osmoregulation process (Robertson, 1964) as well. R. cuneata is one of the few bivalve species that has two types of osmoregulation, i.e. isosmotic intracellular regulation and anisosmotic extracellular regulation (Gainey and Greenberg, 1977). This species is a hyper-osmoregulator up to salinity of 10.0 and an osmoconformist above this value (Bedford and Anderson, 1972; Gainey and Greenberg, 1977; Cooper, 1981). Therefore, when R. cuneata inhabits water near its isosmotic point (S = 10.0), the clams can expend less energy on osmotic adjustment. In turn, in the salinities lower than 10.0, the clams have to strongly pump ions from the water against the concentration gradient, which generates additional metabolic costs. Due to the fact that the metabolic rate is slower in lower temperatures (Kinne, 1971; Schmidt-Nielsen, 1997), this increased metabolic rate may not be sufficiently efficient to cover additional expenses associated with hyperosmoregulation in low salinity during cold months (Paul, 1980; Saucedo et al., 2004). The above considerations may indicate that, in terms of physiology and bioenergetics, less salty and cooler environments are more adverse to subtropical R. cuneata (LaSalle and de la Cruz, 1985). Higher energy demands in brackish water require clams to pump more water to extract more oxygen. This in turn may induce changes in their burial depth in the sediment or in their valve gaping behaviour, i.e. shell opening and closing (Rajagopal et al., 1997; Sparks and Strayer, 1998; Montagna and Ritter, 2006; Porter and Breitburg, 2016). Such changes could negatively affect this species, as moving to the sediment surface could cause them to lose their anchorage and increase their exposure to predators (de Goeij et al., 2001; Kornijów et al., 2018). On the other hand, clams on or near sediment surface would have better access to suspension feed and obtaining oxygen (Tallqvist, 2001; Long et al., 2008). In turn, closing the shell is a form of protection from unfavourable environmental conditions, but this behaviour makes feeding and oxygen uptake impossible, which, in the long run, can weaken bivalves' condition. Although R. cuneata is widespread in native and non-native regions, there is no information on its behaviour and metabolic rate under exposure to different salinities and temperatures. Therefore, we studied here the physiological functioning of this clam in colonised brackish water bodies located at higher latitudes, where the population size fluctuates considerably. The aim of the study was to determine how the salinity as well as change in temperature by 10 °C will affect behaviour and metabolic rate of R. cuneata. Due to the fact that this species is a hyper-osmoregulator up to salinity of 10.0 and an osmoconformist above this value, it was hypothesized that along with increased salinity its pumping activity (to extract more oxygen from water) as well as metabolic rate will decrease. In addition, taking into account that the metabolic rate rises with temperature, it was hypothesized that the decrease by 10 °C will also depress pumping activity and metabolic rate of R. cuneata. The obtained results could be used to make predictions concerning the likely distribution of R. cuneata in new environments, also in the context of the Baltic area climate change. Direct calorimetry was applied in our research, which is the most suitable technique to measure the heat dissipated in all chemical reactions and energy transformations taking place in the cells of living organisms (Gnaiger, 1979). This is particularly important in the studies of facultative anaerobes, like bivalves, which are able to use anaerobic pathways to obtain energy (Pamatmat, 1980). The advantage of direct calorimetry is the ability to record not only changes in the rate of metabolism, but also in the activity of a studied organism (Lamprecht, 1983).

2.1. Collection, maintenance and survival of animals Individuals of R. cuneata (mean shell length of ca. 2 cm) were collected in August 2017 from the western part of the Vistula Lagoon (54°18.559′N 19°28.710′E), from a research vessel by using a bottom dredge. Then, clams were transported in cool-boxes to the University of Gdańsk, where they were acclimatised in a laboratory for a week in tanks filled with aerated water (collected in situ, S = 2.4, T = 20 °C) and 3 cm of sand on the bottom. After acclimatisation, the clams were placed in artificially prepared water of the same temperature and salinity as in the environment. Subsequently, groups of 33 individuals were placed separately in four smaller tanks (with 3 cm of sand on the bottom) and gradually (2 salinity units per day) acclimated from ambient (control) conditions (S = 2.4, T = 20 °C) to experimental salinities of 0.5 (the lowest, which R. cuneata can experience in the Vistula Lagoon), 7.0 (medium, which is typical of the neighbouring Gulf of Gdansk, where R. cuneata has been also found) and 10.0 (in which R. cuneata is isosmotic with external environment). The control salinity (S = 2.4) was also tested at a temperature of 10 °C (which is the mean spring temperature-, when the mortality of R. cuneata is higher), and the clams were gradually acclimated (2 °C per day). After the salinity reached the experimental level, the clams were acclimated for at least two days to establish a steady state. In the laboratory, the animals were fed daily with a mixture of green microalgae obtained from the Culture Collection of Baltic Algae of the Institute of Oceanography. During the whole period of laboratory maintenance, daily survival of clams was monitored. Dead clams (on the sediment surface with wide open valves, not responsive to touch) were removed from the tanks immediately. 2.2. Metabolic rate and behaviour The total metabolic rate was determined based on heat dissipation measurements conducted in an isothermal twin calorimeter of the Calvet type with 24 mL vessels (measuring and reference), equipped with a flow-through system as described by Normant et al. (2007) and Jakubowska et al. (2013). The measuring procedure given by Jakubowska and Normant-Saremba (2015) was applied. Before measurements, the calorimeter was calibrated using electric heaters (1000 Ω) placed inside the vessels. The calibration factor K reached 108.15 ± 0.15 μV mW−1 at T = 20 °C and 104.79 ± 0.69 μV mW−1 at T = 10 °C. Measuring and reference vessels were filled with 7 g of sterilised (100 °C) sand and experimental water, which was continuously pumped (0.2 mL min−1) from a 1000 mL bottle through a low pulsation peristaltic pump (Ecoline VC-MS/CA REGLO Digital; Ismatec, Switzerland). In the first step, the baseline, i.e. the thermal signal of vessel without the animal was recorded within 12 h. Next, a single clam was placed inside the measuring vessel and acclimatised for 2 h, after which the metabolic rate was measured over a period of next 9 to 25 h (depending on the shell closure time – it was longer for clams which closed their valves). Clams from three salinities (S = 0.5, 7.0, 10.0) were tested at 20 °C, with an additional control of 2.4. The control salinity (S = 2.4) was also tested at 10 °C. After the measurements, each clam in the vessel was photographed to determine its final burrowing position – whether the individuals were on the sediment surface (Fig. 1A) or buried in it (Fig. 1B). After removal from the vessel, they were frozen (−20 °C) for further analyses of shell length ( ± 0.01 mm) and dry tissue weight (dw; ± 0.001 g). At each experimental treatment, 8–12 individuals were studied (mean shell length: 21.12 ± 1.08 mm, mean dry tissue weight 0.030 ± 0.008 g, N = 47). Power-time curves from calorimetric measurements were used to determine the activity of R. cuneata resulting from gaping behaviour as described by Jakubowska and Normant (2015) and Jakubowska and Normant-Saremba (2015). The peaks of varying heights and durations 2

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Fig. 1. R. cuneata burrowing behaviour patterns. Individuals on the sediment surface (A), and buried in the sediment (B).

of the power-time curves corresponded to periods when the shell valves were open (an active clam), while a smooth line corresponded to those when the shell was closed (resting clam). The total time of shell closure (min) was determined for individuals exposed to different experimental conditions. Routine (mean value calculated for the last 8 h of measurement), active (the highest heat production, shell open; mean value calculated for peaks of the maximum activity) and resting (the lowest heat production, shell closed; mean value calculated for the periods of varying lengths between the activity peaks) levels of the metabolic rate were calculated using the formula:

Q = ((U2 –U1)·K−1)·m−1 where U1 and U2 are calorimetric signals of the empty vessel and the vessel with a clam (μV), K is the above-mentioned calibration factor and m is the dry weight of clam's tissue (g). In cases when clams were continuously active, only the routine metabolic rate was determined. This led to a decrease in the number of analysed clams as compared to the number of studied individuals (measurements). The first 2 h of measurements were not used in the calculations as this time was needed to reach the steady state of the calorimeter and to allow the animal to acclimatise to the vessel conditions. Values of the metabolic rate were expressed as the mean ± SD in mW g−1 clam dw. Based on the resting and routine metabolic levels for R. cuneata at 10 and 20 °C (S = 2.4) the temperature coefficient Q10 was calculated. This value defined the decrease in the metabolic rate as the temperature declined by ten degrees (Newell, 1973; Schmidt-Nielsen, 1997). 2.3. Statistical analysis Differences in the independent proportions of individuals on the sediment surface and partially buried as well as closure frequency were tested using the two-proportion test at the 95% significance level, while significant differences in the metabolic rate were verified using oneway analysis of variance (ANOVA) followed by Tukey's post-hoc test for unequal sample size. Statistical analyses were carried out using STATISTICA 13.1 (StatSoft, Poland). 3. Results

Fig. 2. Examples of the power-time curves for R. cuneata individuals (dry tissue weights: 0.031, 0.049 and 0.026 g) showing different activity levels during exposure to control conditions of S = 2.4 and T = 20 °C (A), experimental salinity of 10.0 (B) and lower temperature of 10 °C (C).

3.1. Survival and behaviour During the study, R. cuneata clams were characterised by high survival (100%) across all salinity and temperature combinations after 19 days. 3

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significant (Tukey's post-hoc test for unequal sample size: p < .05) reduction (Q10 = 8.5) in the rate of resting metabolism (0.07 ± 0.05 mW g−1 dw, N = 5) compared to the control group (0.56 ± 0.24 mW g−1 dw, N = 3). The ratio of active to resting metabolic rates increased significantly (p < .05) to 32.3 ± 14.1 (N = 5) in 10 °C. In the salinity of 0.5 and 7.0, the level of resting metabolism was determined for only one and two individuals (respectively), which in turn made it impossible to compare the result with the control conditions.

The power-time curves for R. cuneata, both in control and experimental conditions, showed periods of high and low (resting) activity inside the calorimetric vessel. The peaks of different height and duration (Fig. 2A) represented the spontaneous activity resulting from shell opening (and siphon stretching), whereas smooth signals (Fig. 2B and C) indicated resting periods caused by shell closure (and siphon contracting). However, the power-time curves (behaviour) differed between the experimental treatments. Under the control conditions (S = 2.4, T = 20 °C), as many as 67% of all individuals (N = 9) had their shells open throughout the measurement period and showed high activity levels (Fig. 2A). Exposure to the lowest salinity (S = 0.5) caused a statistically insignificant increase (p > .05) in the proportion of individuals with open shells during the measurement period, representing as much as 90% of all (N = 10) individuals. The reverse situation was observed in the highest experimental salinity (S = 10.0) – the number of individuals closing their shells for a certain time during the measurement period increased significantly (p < .05) and accounted for 92% of all (N = 12) individuals. In the salinity of 10.0, the valve closing time ranged from 48 min to 16 h 24 min, with an average of 9 h 23 min ± 4 h 44 min (N = 11). Only 33% of individuals closed their shells during the measurement period in the salinity of 2.4 (control), with an average closing time of 2 h 18 min ± 31 min (N = 3). Only two out of eight clams closed their shells at any point in the salinity of 7.0, and the closing time varied considerably – from 30 min to 24 h 30 min. Power-time curves also indicated the reduced activity of clams (lower peak height) in the salinity of 10.0 in relation to control conditions (Fig. 2B). At a lower temperature (T = 10 °C), 63% of individuals closed their shells as compared to control conditions (33%), however, the difference was not statistically significant (p > .05). The mean time of shell closure was similar to the control group (2 h 18 min ± 31 min; N = 3) and amounted to 2 h 27 min ± 1 h 53 min (N = 5). At the same time, power-time curves indicated much lower activity (peaks of much lower height) of individuals at a temperature of 10 °C compared to the control conditions (Fig. 2C). Photos taken after the calorimetric measurements showed that the number of clams on the sediment surface was the largest (40%; N = 8) in the salinity of 0.5 and decreased with salinity increase (33% (N = 9) in salinity of 2.4 and 25% (N = 8) in 7.0). Thus the largest number of partially buried individuals (83%; N = 12) was in the salinity of 10.0. The number of individuals present on the sediment surface also increased in the lower temperature (10 °C) compared to 20 °C – from 33% (N = 9) to 50% (N = 8).

4. Discussion Both of the studied environmental factors (temperature and salinity) directly affect the physiology of animals (Somero, 2012). Based on the analysis of behavioural changes and metabolic rates, which are nonspecific biomarkers of environmental stress, we demonstrated that both low salinity and low temperature could adversely affect R. cuneata. The reduction of salinity to 0.5 and temperature to 10 °C resulted in a behavioural change, manifesting itself in the emergence of the bivalve molluscs onto the surface of the sediment. In the lowest salinity the individuals on the sediment surface opened their shells, while in the low temperature their shells were closed, resulting in a significant reduction in both the rate of routine metabolism and resting metabolism by as much as 88%. Closure of the shell along with such a large reduction in the routine metabolic rate may indicate that energy was obtained through anaerobic metabolism during these periods of high stress. Migration to the surface of the sediment requires bivalve molluscs to lose their anchorage, making them more vulnerable to water currents. Reducing the depth of burial or staying on the sediment surface increases their mortality rate due to increased interactions with predators (de Goeij et al., 2001; Kornijów et al., 2018). The behaviour of R. cuneata observed in our study (moving to the sediment surface) has been noted for individuals of the same species in the native range and probably results from increased oxygen demand and hypersaline regulation of the blood at low salinities (Henry and Mangum, 1980). However, considering other molluscs, such as Mesodesma mactroides, which buries itself deep under the sediment surface as a response to low salinity (Carvalho et al., 2015), such behaviour is species specific. The closing of shells is a typical response of bivalve molluscs to isolate themselves from adverse environmental conditions, but the length of closure depends on both biotic and abiotic factors. The complete closure of the shell during the experiment observed at 10 °C indicates that this temperature was unfavourable for R. cuneata, which comes from the Gulf of Mexico, i.e. a warmer climate zone (e.g. Dall, 1894; Hopkins and Andrews, 1970). Hopkins and Andrews (1970) found that at higher temperatures, even 32 °C, this species can survive up to several months, while at lower temperatures, e.g. at 3 °C, its survival is reduced to a few hours. At a temperature of 10 °C, individuals of R. cuneata closed their shells for a maximum of 6 h although this species can stay closed much longer- up to 2 weeks (at T = 22 °C, S = not given; Hopkins et al., 1973). However, such isolation means that they have no access to oxygen or food, which, in the long run, may weaken their condition. In contrast, bivalve molluscs remaining on the surface of the sediment with open shells can uptake oxygen and food, which to some extent enables them to compensate for energy expenses, e.g. adjustment to salinity of 0.5. Due to the fact that the largest difference between the osmotic pressure of the haemolymph and the surrounding medium occurs in the salinity of 0.5 (Henry and Mangum, 1980; Bedford and Anderson, 1972), it was assumed that the intensive uptake of larger amounts of ions by R. cuneata would be reflected in the highest rate of routine metabolism, as seen in other hyperosmoregulators (Glazier and Sparks, 1997). However, the energy metabolism of R. cuneata in the salinity of 0.5 was lower than that in 2.4 (control), which may have been due to exceeding a certain threshold value and a lack of compensatory mechanisms. On the other hand, a

3.2. Metabolic rate Exposure to the lowest (S = 0.5) and medium (S = 7.0) salinities tested led to a significant (p < .01) reduction in the routine metabolic rate of R. cuneata as compared to control salinity (S = 2.4), i.e. from the control of 7.52 ± 1.33 mW g−1 dw (N = 9) to 3.58 ± 1.20 (S = 0.5; N = 10) and 3.64 ± 1.36 mW g−1 dw (S = 7.0; N = 8; Fig. 3). On the other hand, the mean value in the highest experimental salinity (S = 10.0) was 5 times lower than in the control (1.57 ± 1.08 mW g−1 dw; N = 12; Tukey's post-hoc test for unequal sample size: p < .001). In the reduced temperature (T = 10 °C), the rate of routine metabolism in R. cuneata decreased significantly, i.e. as much as 6.5 times in relation to control conditions, reaching the value of 1.15 ± 0.5 mW g−1 dw (N = 8; Tukey's post-hoc test for unequal sample size: p < .001). No statistically significant differences were found (p > .05) in the rate of resting metabolism of R. cuneata between the control salinity (S = 2.4) and in the highest experimental salinity (S = 10.0). Mean values were 0.56 ± 0.24 mW g−1 dw (N = 3) and 0.43 ± 0.18 mW g−1 dw (N = 5), respectively. Also the ratio between the active and resting metabolic rates did not differ significantly (p > .05) between the control salinity and the highest experimental salinity and amounted to 16.3 ± 4.1 (N = 3) and 14.4 ± 5.4 (N = 11), respectively. Lowering the temperature to 10 °C resulted in a 4

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Fig. 3. Routine metabolic rates of R. cuneata (mean ± SD) in different salinity and temperature treatments during the last 8 h of measurements. The numbers in bars show the sample size (N). Different numbers of stars show statistically significant (p < .05) differences in routine metabolic rate between experimental variants.

control temperature (T = 20 °C) and as much as 32 times lower at 10 °C. The recorded ratios between the active and resting metabolic rate (so-called scope for activity) was high in R. cuneata in comparison to values observed in other invertebrates, which usually reached from 2:1 to 10:1 (e.g. Newell, 1973; Schmidt-Nielsen, 1997; Jakubowska and Normant, 2015). However, in the case of bivalves that may significantly reduce the metabolic rate when closing the shells and switching to anaerobic pathway, values > 10:1 are also recorded (Hammen, 1980; Jakubowska and Normant, 2015). Since its introduction to the Vistula Lagoon, the range of R. cuneata has extended in this shallow water body where salinity and temperature fluctuate both spatially and seasonally (Kruk, 2012; Warzocha et al., 2016; Matciak and Chyła, 2018). In the case of the former parameter, values may drop below 2.0, sometimes even 0.5 (Rychter, unpublished), whereas in the case of the latter below 1.5 °C, sometimes reaching even 0.5 °C (Grekow et al., 1975; Matciak and Chyła, 2018). From the physiological viewpoint, the most favourable salinity is that close to species' isoosmotic point. Our results show that for R. cuneata the most advantageous conditions seem to be salinity close to 10 and a temperature of about 20 °C. Based on our study, we hypothesize that the combination of low salinity and low temperature can limit colonisation success of R. cuneata in brackish Baltic waters. This environmental combination most likely occurs in winter-spring months, when the availability of food is also lower, which contributes to the reduction in individual condition. This may result in a significant reduction of the population size of this species observed in spring (Kornijów et al., 2018; Solovjova et al., 2019). In Europe, the northernmost site of R. cuneata is Pärnu Bay in Estonia (Baltic Sea), which indicates that its invasive potential to the north is likely to be limited by a thermal barrier (Möller and Kotta, 2017). Based on climate change projections, it can be assumed that similarly to another bivalve mollusc, Mytiliopsis leucophaeta from the Gulf of Mexico, climate warming (especially surface temperature rise), together with the potentially high phenotypic plasticity, may enable R. cuneata to adapt to conditions prevailing in the shallow waters of the Baltic Sea (Meier et al., 2012; Holopainen et al., 2016).

more than threefold reduction in the rate of routine metabolism in the lowest salinity (S = 0.5) compared to the control salinity of 2.4 may be due to low efficiency of aerobic metabolism and the activation of anaerobic metabolism (De Zwaan and Wijsman, 1976; Guppy and Withers, 1999). The highest rate of routine metabolism in R. cuneata was recorded in salinity of 2.4 (T = 20 °C), which was probably the result of high activity related to water pumping in order to uptake more ions, such as Na+, K+, Ca2+, Mg2+ and Cl− from the hypo-osmotic medium (Henry and Mangum, 1980). These ions are accumulated below the isosmotic point, which in this case is 10.0 (Henry and Mangum, 1980; Bedford and Anderson, 1972). In this salinity, the rate of routine metabolism was significantly reduced, i.e. by as much as 79% compared to the control group, while the rate of resting metabolism remained unchanged. Some individuals moved to the sediment surface, and their shells were closed. It is worth noting that R. cuneata closed their shells in the salinity of 10.0 for > 16 h, i.e. longer than in the lowest salinity (S = 0.5) and lower temperature (T = 10 °C). In this case, however, shell closure does not seem to have been caused by a need of isolation from adverse environmental conditions, but rather a reduction in activity (pumping water and food intake) due to lower energy expenditure on osmoregulation (Bedford and Anderson, 1972). By closing their shells, bivalve molluscs reduce energy costs associated with maintenance (Stanley, 1970), which does not always mean switching to anaerobic metabolism, though. According to Ortmann and Grieshaber (2003), even clams closed for a long time can obtain energy through aerobic metabolism. However, without an analysis of the concentrations of substrates and products in the tissues, it is difficult to clearly determine which type of metabolism was applied by closed R. cuneata. Exposure of Atlantic rangia to the lowest salinity (S = 0.5) and lower (than control) temperature (T = 10 °C) caused a reduction in the rate of routine and resting metabolism. The routine and resting metabolic rates were > 6 and 8.5 times lower, respectively, at 10 °C compared to the control temperature of 20 °C. This was probably due to valve closure and a likely transition to anaerobic metabolism. A similar metabolic response was observed in another bivalve mollusc – Corbicula fluminea, whose Q10 coefficient was, on average, 3.8 in the case of active and 8.5 in the case of resting metabolism (Ortmann and Grieshaber, 2003). Bivalve molluscs can reduce metabolic rates up to 90% to survive in unfavourable thermal conditions from which they cannot escape (Loosanoff, 1939; Ortmann and Grieshaber, 2003), but can also reduce metabolic rates in other, non-stressful conditions (e.g. Ortmann and Grieshaber, 2003; Jakubowska and Normant, 2015; Jakubowska and Normant-Saremba, 2015). Due to inter alia, or valve gaping behaviour, closed (resting) clams were characterised by a very low metabolic rate.compared to open (active) clams, which was 16 times lower at the

Acknowledgements This research was supported by the statutory funds of the Department of Experimental Ecology of Marine Organisms No. DS/530G220-D427-17. The Authors thank Dr. Amy Fowler from George Mason University, USA, as well as two anonymous reviewers, for constructive comments that helped improve the quality of the manuscript. M. Normant-Saremba dedicates this paper to Prof. Anna Szaniawska from University of Gdańsk on the occasion of her retirement.

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