Broad plasticity in the salinity tolerance of a marine copepod species, Acartia longiremis, in the Baltic Sea

Broad plasticity in the salinity tolerance of a marine copepod species, Acartia longiremis, in... Abstract We assessed feeding, reproduction, survival and respiration in the boreal–polar copepod Acartia longiremis over the salinity range of 3–16 in the Baltic Sea. Feeding and egg production were not affected at a salinity of 7–16, representing the range in which the species naturally occurs, but decreased significantly at a lower salinity. Survival experiments showed a broad physiological plasticity with no increase in mortality upon immediate exposure to salinities of 16–7. Acclimation of females to low salinity extended the survival range to a salinity of 5. While the response in vital rates was characteristic of a tolerant, brackish water species, unusually high respiration rates at a salinity of 7–16 indicated that the species experienced osmotic stress, and that the mechanism maintaining physiological integrity was energetically expensive. Divergent responses of an increase in respiration rate and a decrease in feeding rate at a salinity below 7 indicated a disruption of the energetic balance under which the osmotic stress could not be counteracted. Our results show that A. longiremis persists close to its physiological limit in the Baltic Sea, which makes the species vulnerable to small changes in future salinity. INTRODUCTION Physical oceanographic factors have a central role in determining the distribution and abundance of pelagic organisms in aquatic environments. In estuarine and coastal ecosystems, salinity gradients, together with the seasonal variation in temperature, food availability and predators, strongly influence the composition and spatial succession of zooplankton communities (Collins and Williams, 1982; Ambler et al., 1985; Kimmel and Roman, 2004; Kayfetz and Kimmerer, 2017). Physiological studies on copepods have demonstrated large differences in the ability of different species to adjust the osmolyte content of the hemolymph (Lance, 1965; Brand and Bayly,1971; Lee et al., 2012; Svetlichny and Hubareva, 2014) and to maintain rates of feeding (Lance,1964b; Calliari et al., 2006), reproductive success (Chinnery and Williams, 2004; Calliari et al., 2006; Beyrend-Dur et al., 2009; Hemraj et al., 2018) or survival and development at changing salinities (Lee et al., 2007; Devreker et al. 2007). The distribution of copepods in their original habitat often reflects these species-specific tolerance levels, showing a spatial succession from stenohaline marine to true estuarine species along salinity gradients (Collins and Williams, 1982; Sotaert and van Rijswijk, 1993; Islam et al., 2006). A large proportion of euryhaline marine species characterizes the zooplankton of the brackish Baltic Sea. Temora longicornis, Centropages hamatus, Acartia longiremis and Pseudocalanus acuspes, among others, are often numerically abundant in the zooplankton communities of the Arkona Sea (AS), Bornholm Basin (BB) and Gotland Basin, despite the low salinity both in the surface and deeper water layers (Hernroth and Ackefors, 1979; Viitasalo, 1992; Schulz et al., 2012). Because these species live at the lower range of their tolerance limits, hydrological factors mainly determine the distribution and population size of the species. Salinity is, thus, a fundamental predictor of the abundance of the marine species and their contribution to zooplankton communities in the Baltic Sea (Viitasalo et al., 1995; Möllmann et al., 2000; Vuorinen et al., 2003; Mäkinen et al., 2017). In contrast to many brackish water species, the physiological responses and salinity tolerance of these marine species are, however, only rudimentarily understood. This gap in knowledge limits the capability to predict future changes in the abundance and distribution of marine species and in the diversity of Baltic zooplankton. In the Baltic Sea, long-term studies have regularly assessed the variation in the abundance of the genus Acartia spp. However, grouping of the species at a genus level largely restricts insights into the factors controlling the stock dynamics, because species of potential contrasting physiology, A. tonsa, A. bifilosa and A. longiremis, are integrated. Acartia tonsa is a thermophilic, brackish water species with a very broad salinity tolerance (Gaudy et al., 2000; Calliari et al., 2006; Holste and Peck, 2006; Svetlichny and Hubareva, 2014). The species occurs in the inner coastal waters and bights of the southern Baltic and is observed only occasionally in offshore water (Arndt and Schneese, 1986; Díaz-Gil et al., 2014; Schulz et al. 2012). The brackish A. bifilosa is ubiquitous in the Baltic Sea and is found in coastal as well as open waters ranging from the Kiel Bight to the Bothnian Sea and the Gulf of Finland, while A. longiremis is of marine origin and occurs primarily in colder, open water of the southern Baltic (Hernroth and Ackefors, 1979; Viitasalo, 1992; Schulz et al., 2012). Both A. bifilosa and A. longiremis are abundant in the deep basins of the Baltic and at times can dominate the copepod community (Wasmund et al. 2016). While the species’ wide distribution in the Baltic Sea suggests a broad salinity tolerance, direct knowledge about their salinity preferences are restricted to a few studies of A. bifilosa in estuarine water of the North Atlantic (Lance, 1963; Chinnery and Williams, 2004). Our main objectives were to characterize the physiological tolerance of A. longiremis in relation to decreasing salinity and to identify the critical salinity level that might limit its distribution and population dynamics in the Baltic Sea. Investigating the salinity responses of Baltic marine copepods is particularly relevant for future projections of biodiversity associated with environmental changes (Vuorinen et al., 2015). Surface salinity in the Baltic Sea has decreased in recent decades, due to increased river run-off and infrequent inflows of saline water from the North Sea through the Danish Straits (Hänninen et al., 2000; Meier et al., 2006). Simulations suggest a further decline by 1.5–2 units in the Bornholm and Gotland basins until the end of the century (Meier et al., 2012; Vuorinen et al., 2015). In addition, knowledge on the physiological effects of salinity stress in Acartia spp is often limited due to the focus on single traits such as reproductive success (Castro-Longoria, 2003; Chinnery and Williams, 2004; Holste and Peck, 2006; Svetlichny et al., 2010; Peck et al., 2015) or survival of specific life stages (Lance, 1963, 1964b; Cervetto et al., 1999; Calliari et al., 2008; Hubareva et al., 2008). Respiratory responses in relation to the performance of vital rates, however, can indicate physiological adaptation and the ability of species to cope with salinity stress (Farmer and Reeve, 1978; Gaudy et al., 2000; Isla and Perisinotto, 2004; Calliari et al., 2006; Svetlichny et al., 2012, 2014). We, therefore, investigated the feeding, egg production, egg hatching success and respiration of A. longiremis, in order to evaluate how salinity affects the metabolic budget using two different approaches. In the first, we compared the performance of A. longiremis occurring naturally at two different salinities in the western Baltic Sea. Due to a preference for the cooler, subsurface water layers below the thermocline in summer, the species occurred at a higher salinity in the shallow AS (S > 14.5) than in the deeper BB (S < 8). We considered specimens to be acclimated to the local conditions and compared vital rates at in situ salinity and survival of females to an instantaneous change in salinity in order to evaluate whether the species occurrence in large parts of the Baltic at a salinity below 8 is already constrained in present day salinity conditions. In the second approach, we intended to characterize the species tolerance to reduced salinity by comparing vital rates and survival of females from the Bornholm Sea acclimated to a decreasing salinity ranging from 7 to 3. METHOD Source populations Experiments were conducted during the cruise of R/V Dana to the central Baltic Sea, in September 2015. Zooplankton samples were taken with a 100 μm WP-2 net equipped with a non-filtering cod end and a closing device, at two stations located in the AS and the BB (Fig. 1). Due to a large horizontal salinity gradient in the subsurface layer at these stations, the salinity at the origin of the two populations was different. Individuals from the shallow AS (max. depth 45 m, 54°58′ N, 14°03′ E) were caught in the saline deep-water layer at a depth of 40–30 m, characterized by vertical gradients in salinity of 14.5–16.4 and in temperature of 10–14°C, respectively. The majority of the adult population of A. longiremis (>90%, 6.2–11.1 × 103 Ind m−3) stayed in this saline layer during day and night (results not shown). In the BB, the species avoids the warm surface during summer and concentrates in the colder but less saline intermediate water (Schulz et al. 2012). Zooplankton was, therefore, collected at a central station (depth 91 m, 55°17′ N, 15°43′ E) in the intermediate water layer (50–35 m) characterized by vertical gradients in a salinity of 7.5–7.9 and a temperature of 7.3–10.5°C, respectively. Samples were transferred into 30-L containers filled with pre-collected water from the sampling depth at both stations (35 m in the AS, 40 m in the BB) and brought into a walk-in cooling chamber set to a temperature of 10°C. Additional water needed for the cultures and experiments was collected at the same depths (AS: 80 L, BB: 180 L, collected in two batches during the cruise), subsequently GF/F filtered and stored until use. Fig. 1. View largeDownload slide Location of the sampling stations in the western Baltic Sea. Fig. 1. View largeDownload slide Location of the sampling stations in the western Baltic Sea. Adult A. longiremis were isolated from the catch into batches of 100–300 females and 30–70 males in 10 L permanently aerated, GF/F filtered seawater for acclimation to different salinity conditions (Table I). For the experiments with the population from the AS, copepods were set-up at a salinity of 16.0. For the experiments in the BB, one batch was kept at the in situ salinity of 7.7, while four other batches were progressively diluted to yield populations acclimated to salinities of 6.0, 5.0, 4.0 and 3.0. Salinity was adjusted by additions of MilliQ water at a rate of 1 d−1. Cultures were fed ad-libitum (>400 μgCL−1) a mixture of the cryptophyte Rhodomonas salina and the heterotrophic dinoflagellate Oxyrrhis marina. The mixture was chosen to provide good feeding conditions for A. longiremis, which has shown a preference for heterotrophic food (Peters et al., 2013). The cryptophyte was kept in exponential growth in 2 L batch cultures using GF/F filtered seawater at the respective salinities (10 °C, 16:8 h light:dark cycle, 100 mmol photons m−2 s−1, B-medium; Hansen, 1989). O. marina was grown in GF/F filtered seawater under similar conditions, except darkness, and fed with R. salina. Carbon content of O. marina was estimated from geometric dimensions using conversion factors (Menden-Deuer and Lessard, 2000). For R. salina grown at a salinity of 7.0–16.0, a carbon content of 54.6 pg C cell−1 was used (Dutz, unpublished results). The batches of females and males were acclimated to the food and final salinity conditions for 2 days before the start of experiments. All salinity measurements were conducted with a Cond 3210 conductivity meter equipped with a TetraCon 325 probe (WTW, Weilheim) with an accuracy of 0.5% of the measured value ± 0.1 digits. Table I: Summary of the salinity conditions used to evaluate to effect of salinity on feeding, egg production, egg hatching success and instantaneous survival of females of A. longiremis. Variation was within the accuracy limits of the conductivity meter (±0.1 units). Origin of population Salinity at origin Acclimation salinity S for acclimated rate measurements S for instantaneous survival Arkona Sea 14.5–16.4 16.0 16.0 16.0, 12.0, 10.0, 8.0, 7.0, 6.0, 5.0 Bornholm Sea 7.7–7.9 7.7 7.7 7.7, 7.0, 6.0, 5.0, 4.0, 3.0 6.0 6.0 6.0, 5.0, 4.0, 3.0 5.0 5.0 5.0, 4.0, 3.0 4.0 4.0 4.0, 3.0 3.0 – 3.0 Origin of population Salinity at origin Acclimation salinity S for acclimated rate measurements S for instantaneous survival Arkona Sea 14.5–16.4 16.0 16.0 16.0, 12.0, 10.0, 8.0, 7.0, 6.0, 5.0 Bornholm Sea 7.7–7.9 7.7 7.7 7.7, 7.0, 6.0, 5.0, 4.0, 3.0 6.0 6.0 6.0, 5.0, 4.0, 3.0 5.0 5.0 5.0, 4.0, 3.0 4.0 4.0 4.0, 3.0 3.0 – 3.0 Table I: Summary of the salinity conditions used to evaluate to effect of salinity on feeding, egg production, egg hatching success and instantaneous survival of females of A. longiremis. Variation was within the accuracy limits of the conductivity meter (±0.1 units). Origin of population Salinity at origin Acclimation salinity S for acclimated rate measurements S for instantaneous survival Arkona Sea 14.5–16.4 16.0 16.0 16.0, 12.0, 10.0, 8.0, 7.0, 6.0, 5.0 Bornholm Sea 7.7–7.9 7.7 7.7 7.7, 7.0, 6.0, 5.0, 4.0, 3.0 6.0 6.0 6.0, 5.0, 4.0, 3.0 5.0 5.0 5.0, 4.0, 3.0 4.0 4.0 4.0, 3.0 3.0 – 3.0 Origin of population Salinity at origin Acclimation salinity S for acclimated rate measurements S for instantaneous survival Arkona Sea 14.5–16.4 16.0 16.0 16.0, 12.0, 10.0, 8.0, 7.0, 6.0, 5.0 Bornholm Sea 7.7–7.9 7.7 7.7 7.7, 7.0, 6.0, 5.0, 4.0, 3.0 6.0 6.0 6.0, 5.0, 4.0, 3.0 5.0 5.0 5.0, 4.0, 3.0 4.0 4.0 4.0, 3.0 3.0 – 3.0 Feeding, egg production and egg hatching success Adults were picked from the batches acclimated to the final salinities. For each salinity treatment, 15–20 females and 5–7 males were placed in each of the four replicate 610 ml bottles filled with the experimental food suspension. The food was kept suspended by rotation on a plankton wheel, at a speed of 1 rpm. Food consisted of mixtures of R. salina and O. marina provided both at 100 μg C L−1. Adults were acclimated to experimental conditions for 24 h, followed by the transfer of females into new, separate bottles with fresh food suspension for another 24 h, to determine the feeding rate. Two types of control bottles were set-up in triplicate. The first consisted of suspensions of R. salina alone, in order to control its growth rate; the second consisted of the same mixture of R. salina and O. marina as used in grazing experiments, but without copepods, in order to determine O. marina growth and its consumption of R. salina. Samples for initial and final cell concentrations were counted in quadruplicate using a Coulter Multisizer Model 3 (R. salina) or a microscope (O. marina), using the Utermöhl technique from 6 to 7 replicates of a 4-mL sample. Ingestion rates of O. marina and females of A. longiremis were estimated from changes in prey cell numbers in treatments compared to those in controls using the approach by Tang et al. (2001). Briefly, the ingestion rates of females feeding on R. salina and O. marina in mixtures were estimated from changes in cell concentration (R. salina = Rho, O. marina = Ox) in the experiments described by Rho(t)=Rho0∗exp[(μRho−gAc−Rho⁎Ac)t−gOx⁎V∫0tOx(t)dt] (1) Ox(t)=Ox0⁎exp[(μOx−gAc−Ox⁎Ac)t] (2) where Rho0 and Ox0 are the initial cell concentrations of R. salina and O. marina, V is the volume of the bottle and Ac is the number of females in the experiment. The specific growth rates μRho and μOx (time−1) were obtained from the first and second control experiments without A. longiremis, respectively. The feeding coefficient of O. marina feeding on R. salina (gOx, Oxyrrhis−1 time−1) was calculated as the difference between R. salina specific growth rates in the first and the second control. The feeding coefficient (gAc-Ox, female−1 time−1), clearance (mL, female−1 time−1) and ingestion rate (cells female−1 time−1) of A. longiremis feeding on O. marina were calculated according to Frost (1972). The feeding coefficient of A. longiremis on R. salina (gAc-Rho, female−1 time−1) was obtained by substituting equation (2) into the integral term in equation (1) in order to describe the change in concentration of O. marina due to growth and predation and its effect on R. salina cell number as in the following equation: Rho(t)=Rho0∗exp[(μRho−gAc−Rho⁎Ac)t−gOxOx0VgAc−Ox⁎Ac−μOx⁎[1−exp((μOx−gAc−Ox⁎Ac)t)]] (3) The feeding coefficient gAc-Rho and the ingestion rate (IRAc-Rho, cells female−1 time−1) of females were calculated using the initial (Rho0) and final (RhoT) cell concentration of R. salina in the grazing experiment during the incubation time (T) according to equations (4) and (5). Carbon-specific ingestion rates (μg C female−1 time−1) were calculated using the cell-specific carbon content of the algae as described above: gAc−Rho=ln(Rho0)−ln(RhoT)+μRhoTAc⁎T−gOx⁎Ox⁎VAc⁎T⁎(gAc−Ox−μOx)⁎(1−exp[(μOx−gAc−Ox⁎Ac)⁎T]) (4) IRAc−Rho=V⁎gAc−Rho⁎(P0+PT)2 (5) After transfer to fresh food suspensions following the grazing experiments, females were incubated for an additional 24 h in order to determine the egg production rate. Males were reintroduced. The experiments were terminated by collecting females and eggs carefully onto 100 and 30 μm sieves, respectively. Eggs were rinsed into plastic petri dishes, counted and incubated until eggs started to hatch (~4 days). After an additional 24 h, eggs and nauplii were fixed with Lugol’s solution and counted to estimate egg hatching success. The females were collected and transferred to respiration chambers. Females not required for respiration measurements were fixed in formalin for later length measurement. Respiration Respiration was determined in quadruple groups of three to four females for each salinity treatment. Females were placed in respirometer glass chambers of 2 mL filled with pasteurized, oxygen-saturated GF/F-filtered seawater matching the salinity treatments. The chambers were sealed and immersed in a water bath with a constant temperature of 10 °C (±0.1). Oxygen consumption was measured through a thin capillary in the chamber lids with the micro-sensor (UniSense, Micro Respiration System). The micro-sensor (Clark-type) measured oxygen partial pressure every 2 s and data acquisition software provided oxygen values and an average rate of oxygen consumption. The animals were allowed to adapt for 5 min to the chambers and changes in the oxygen partial pressure were then followed for additional 10 min. Background respiration from the pasteurized filtered seawater was measured at least four times in each treatment in order to account for bacterial oxygen consumption. The measurements were repeated after the animals were starved for a period of 24 h in GF/F filtered seawater. At the end, females were fixed in formalin for later length measurements. Survival For the survival experiments, 30–40 females from each of the copepod batches were exposed to an instantaneous decrease in salinity. In the case of A. longiremis originating from the AS (S = 16), females were immediately transferred to salinities of 12, 10, 8, 7, 6 and 5. The batches of A. longiremis from the BB acclimated to a salinity of 7.7, 6, 5, 4 and 3 were immediately transferred to the salinities of 7, 6, 5, 4 and 3, when those were lower than the salinity that they were being kept. Females left in the original salinity served as a control. Animals were incubated in groups of six to seven in 20 mL dishes with six replicates in each treatment and fed a surplus of food of a mixture of Rhodomonas and Oxyrrhis (>400 μg C L−1). The survival of females was checked every 2 h during the first 12–16 h of the incubation, and thereafter every 6–8 h over a period of 5 days. Females were assigned to three categories: active, inactive and dead. Healthy and active females showed regular hop-and-sink behavior and responded immediately to fluid disturbance induced by pipette suction. Inactive females showed reduced pipette avoidance and activity. Food and water in all incubations was replaced daily. Weight-specific ingestion, egg production and respiration Weight-specific ingestion, egg production and respiration rates in terms of carbon were calculated using length measurements and length-body carbon conversion factors provided for salinities of 14 and 7 (Köster, 2003). Egg volume was determined from the measurement of the diameter of eggs collected from the acclimation batches and converted to carbon assuming 0.14 × 10−6 μg C μm−3 (Huntley and Lopez, 1992). Respiration rates were converted to carbon using a respiratory quotient of 0.87 used for feeding of A.tonsa on Rhodomonas (Kiørboe et al., 1985). Statistical analyses Feeding, egg production, respiration and egg hatching were tested for differences between salinity treatments by one-way analysis of variance (ANOVA) and a Tukey’s post hoc test for pairwise multiple comparison (P = 0.05). All data were normally distributed (Shapiro–Wilk test, P > 0.62). Mortality in survival experiments was compared between the salinity treatments by analysis of covariance (ANCOVA) on log transformed data. All analyses were conducted using the SigmaPlot 13.0 statistical package. RESULTS Feeding, egg production and egg hatching success Ingestion rates of A. longiremis differed significantly between the salinity treatments (one-way ANOVA, F = 26.9, P < 0.001) and decreased from 1.2 ± 0.16 μg C Ind−1 d−1 to 0.3 ± 0.06 μg C Ind−1 d−1 at the salinity of 16 and 4, respectively (Fig. 2a). Females at the salinity of 3 did not survive in sufficient numbers to conduct experiments. Feeding rates of 0.9 ± 0.23 μg C Ind−1 d−1 at the salinity of 7.7 were not significantly different from the maximal rate at the salinity of 16. However, the feeding rates at the salinity of 6 were significantly lower compared to higher salinities, but significantly higher than those at the salinity of 4 and 5. The diet of A. longiremis consisted of, on average, 64–75% of Rhodomonas salina at the salinity of 7 and 16. The contribution tended to decrease at lower salinities to <40% at the salinity of 5. However, the diet consisted of nearly exclusively R. salina at the salinity of 4. Weight-specific ingestion rates decreased from 0.49 ± 0.067 μg C μg C−1 d−1 at the salinity of 16 to 0.16 ± 0.029 μg C μg C−1 d−1 at the salinity of 4 (Table II). The decrease was significant (one-way ANOVA, F = 21.0, P < 0.001) and differences between treatments were similar to the per capita rates. Table II: Weight-specific ingestion, egg production, respiration and starvation respiration of female A. longiremis feeding on mixtures of R. salina and O. marina at a salinity of 4–16. Origin of population Acclimation salinity Ingestion (μg C μg C−1 d−1) Egg production (μg C μg C−1 d−1) Respiration (μg C μg C−1 d−1) Respiration (starv.) (μg C μg C−1 d−1) Arkona Sea 16.0 0.49 ± 0.067a 0.008 ± 0.0035a 0.48 ± 0.034a – Bornholm Sea 7.7 0.47 ± 0.116a 0.010 ± 0.0031a 0.39 ± 0.046b 0.28 ± 0.016b 6.0 0.32 ± 0.055b 0.007 ± 0.0020ab 0.48 ± 0.060a 0.31 ± 0.013ab 5.0 0.18 ± 0.037c 0.003 ± 0.0014b 0.49 ± 0.049a 0.35 ± 0.035a 4.0 0.16 ± 0.029c – 0.31 ± 0.014c 0.19 ± 0.035c 3.0 – – – – Origin of population Acclimation salinity Ingestion (μg C μg C−1 d−1) Egg production (μg C μg C−1 d−1) Respiration (μg C μg C−1 d−1) Respiration (starv.) (μg C μg C−1 d−1) Arkona Sea 16.0 0.49 ± 0.067a 0.008 ± 0.0035a 0.48 ± 0.034a – Bornholm Sea 7.7 0.47 ± 0.116a 0.010 ± 0.0031a 0.39 ± 0.046b 0.28 ± 0.016b 6.0 0.32 ± 0.055b 0.007 ± 0.0020ab 0.48 ± 0.060a 0.31 ± 0.013ab 5.0 0.18 ± 0.037c 0.003 ± 0.0014b 0.49 ± 0.049a 0.35 ± 0.035a 4.0 0.16 ± 0.029c – 0.31 ± 0.014c 0.19 ± 0.035c 3.0 – – – – Letters denote significant differences at P < 0.05. Table II: Weight-specific ingestion, egg production, respiration and starvation respiration of female A. longiremis feeding on mixtures of R. salina and O. marina at a salinity of 4–16. Origin of population Acclimation salinity Ingestion (μg C μg C−1 d−1) Egg production (μg C μg C−1 d−1) Respiration (μg C μg C−1 d−1) Respiration (starv.) (μg C μg C−1 d−1) Arkona Sea 16.0 0.49 ± 0.067a 0.008 ± 0.0035a 0.48 ± 0.034a – Bornholm Sea 7.7 0.47 ± 0.116a 0.010 ± 0.0031a 0.39 ± 0.046b 0.28 ± 0.016b 6.0 0.32 ± 0.055b 0.007 ± 0.0020ab 0.48 ± 0.060a 0.31 ± 0.013ab 5.0 0.18 ± 0.037c 0.003 ± 0.0014b 0.49 ± 0.049a 0.35 ± 0.035a 4.0 0.16 ± 0.029c – 0.31 ± 0.014c 0.19 ± 0.035c 3.0 – – – – Origin of population Acclimation salinity Ingestion (μg C μg C−1 d−1) Egg production (μg C μg C−1 d−1) Respiration (μg C μg C−1 d−1) Respiration (starv.) (μg C μg C−1 d−1) Arkona Sea 16.0 0.49 ± 0.067a 0.008 ± 0.0035a 0.48 ± 0.034a – Bornholm Sea 7.7 0.47 ± 0.116a 0.010 ± 0.0031a 0.39 ± 0.046b 0.28 ± 0.016b 6.0 0.32 ± 0.055b 0.007 ± 0.0020ab 0.48 ± 0.060a 0.31 ± 0.013ab 5.0 0.18 ± 0.037c 0.003 ± 0.0014b 0.49 ± 0.049a 0.35 ± 0.035a 4.0 0.16 ± 0.029c – 0.31 ± 0.014c 0.19 ± 0.035c 3.0 – – – – Letters denote significant differences at P < 0.05. Egg production of females was low at all salinities and ranged from 0.6 ± 0.24 eggs Ind−1 d−1 at the salinity of 16 to 0.2 ± 0.08 eggs Ind−1 d−1 at the salinity of 5 (Fig. 2b). No eggs were produced at the lowest salinity of 4. Although the egg production was significantly related to salinity (one-way ANOVA, F = 11.5, P < 0.001), the rates were not statistically different between the salinities of 16, 7 and 6 (Fig. 2b). Weight-specific egg production rates significantly decreased from 0.010 ± 0.0031 μg C μg C−1 d−1 to 0.003 ± 0.0014 μg C μg C−1 d−1 (one-way ANOVA, F = 11.9, P < 0.001, Table II). Again, they were similar at the salinity of 16, 7 and 6, while the specific egg production at the salinity of 5 was lower. The egg hatching success was very variable due to the low number of eggs in the replicates and not significantly different between treatments (Fig. 2c). The length of females (n = 30) in feeding and egg production experiments varied between 684 ± 20 μm and 689 ± 32 μm, and was not significantly different between treatments (data not shown). Egg size was averaged over all treatments due to low egg numbers and was 79.2 ± 2.34 μm. Fig. 2. View largeDownload slide Ingestion rates (μg C Ind.−1 d−1), egg production (eggs Ind.−1 d−1) and egg hatching success (%) of A. longiremis females feeding on mixtures of R. salina and O. marina. Different letters denote treatments that are significantly different from each other according to a Tukey HSD (P < 0.05). Fig. 2. View largeDownload slide Ingestion rates (μg C Ind.−1 d−1), egg production (eggs Ind.−1 d−1) and egg hatching success (%) of A. longiremis females feeding on mixtures of R. salina and O. marina. Different letters denote treatments that are significantly different from each other according to a Tukey HSD (P < 0.05). Respiration The treatment salinity had a significant effect on the respiration rate of non-starved females (one-way ANOVA, F = 21.6, P < 0.001, Fig. 3). Highest respiration rates were recorded at the salinity of 16 (0.10 ± 0.007 μl O2 Ind−1 h−1), while the respiration rate of 0.07 ± 0.008 μL O2 Ind−1 h−1 at the salinity of 7 was significantly lower. However, respiration increased again to 0.09 ± 0.009 μL O2 Ind−1 h−1 when salinity was lowered to S = 5, which was not different from the oxygen consumption at the salinity of 16. The lowest respiration was measured at the salinity of 4. When females were starved for 24 h, respiration rates remained high and amounted to, on average, 67% of the rates determined for non-starved females and were significantly higher at the salinity of 5 (0.06 ± 0.006 μL O2 Ind−1 h−1) than at the salinity of 7 or 4. Likewise, weight-specific respiration in terms of carbon was similar at the salinity of 5, 6 and 16 (0.48–0.49 μg C μg C−1 d−1) and significantly higher than the rates calculated for the salinity of 4 and 7 (one-way ANOVA, F = 27.3, P < 0.001, Table II). Weight-specific respiration rates of starving females were also significantly higher at the salinity of 5 than at the salinity of 4 and 7 (one-way ANOVA, F = 27.7, P < 0.001, Table II). Survival Females originating from the AS survived the immediate salinity reduction generally well (Fig. 4a), but the survival decreased gradually over the duration of the experiment. The average survival over 120 h following exposure to reduced salinities of 12–7 ranged from 86 ± 13% to 95 ± 8% and was similar to the control at the salinity of 16 (91 ± 11%). Survival decreased to 82 ± 15% and 70 ± 13% at a treatment salinity to 6 and 5, respectively. Incapacitation of the females was observed only initially (<12 h) and when the salinity was reduced to values lower than 10 (Fig. 4b). In this case, the proportion of inactive females was inversely related to salinity and increased from 15 ± 36 at the salinity of 8 to 95 ± 27% at the salinity of 5 within the first 2 h, but vanished rapidly afterwards. Cumulative mortality, in contrast, was low during the first 24 h in all treatments and did not exceed 10% (Fig. 4c). This indicated that most of the inactive females recovered from incapacitation. The number of dead females increased gradually in all treatments after 72 h. Mortality rates ranged from 0.02 ± 0.032 d−1 at the salinity of 16 to 0.05 ± 0.040 d-1 at the salinity of 5 (Table III) being significantly different at the salinity of 5 than at other salinities (ANCOVA, P < 0.05). Fig. 3. View largeDownload slide Respiration rates of (μl O2 Ind.−1 h−1) of A. longiremis females feeding on mixtures of R. salina and O. marina before and after starvation. Different letters denote treatments that are significantly different from each other according to Tukey HSD (P < 0.05). Fig. 3. View largeDownload slide Respiration rates of (μl O2 Ind.−1 h−1) of A. longiremis females feeding on mixtures of R. salina and O. marina before and after starvation. Different letters denote treatments that are significantly different from each other according to Tukey HSD (P < 0.05). Table III: Mortality rates of A. longiremis females from the Arkona Basin (origin: S = 16) and the Bornholm Sea (S = 7.7) during 5 days exposure to a reduced salinity. Origin of population Acclimation salinity Treatment salinity Mortality rate d−1 (± STD) Arkona Sea 16.0 16.0 0.02 ± 0.032 16.0 12.0 0.02 ± 0.026 16.0 10.0 0.01 ± 0.021 16.0 8.0 0.01 ± 0.015 16.0 7.0 0.01 ± 0.015 16.0 6.0 0.03 ± 0.038 16.0 5.0 0.05 ± 0.040a Bornholm Sea 7.7 7.7 0 7.7 7.0 0.01 ± 0.013 7.7 6.0 0.01 ± 0.010 7.7 5.0 0.02 ± 0.021 7.7 4.0 0.05 ± 0.055a 7.7 3.0 0.44 ± 0.036b 6.0 6.0 0.01 ± 0.018 6.0 5.0 0.02 ± 0.025 6.0 4.0 0.03 ± 0.042 6.0 3.0 0.41 ± 0.070a 5.0 5.0 0 5.0 4.0 0.08 ± 0.042a 5.0 3.0 0.41 ± 0.082b 4.0 4.0 0.05 ± 0.049 4.0 3.0 0.38 ± 0.074a Origin of population Acclimation salinity Treatment salinity Mortality rate d−1 (± STD) Arkona Sea 16.0 16.0 0.02 ± 0.032 16.0 12.0 0.02 ± 0.026 16.0 10.0 0.01 ± 0.021 16.0 8.0 0.01 ± 0.015 16.0 7.0 0.01 ± 0.015 16.0 6.0 0.03 ± 0.038 16.0 5.0 0.05 ± 0.040a Bornholm Sea 7.7 7.7 0 7.7 7.0 0.01 ± 0.013 7.7 6.0 0.01 ± 0.010 7.7 5.0 0.02 ± 0.021 7.7 4.0 0.05 ± 0.055a 7.7 3.0 0.44 ± 0.036b 6.0 6.0 0.01 ± 0.018 6.0 5.0 0.02 ± 0.025 6.0 4.0 0.03 ± 0.042 6.0 3.0 0.41 ± 0.070a 5.0 5.0 0 5.0 4.0 0.08 ± 0.042a 5.0 3.0 0.41 ± 0.082b 4.0 4.0 0.05 ± 0.049 4.0 3.0 0.38 ± 0.074a Letters denote significant differences at P < 0.05. View Large Table III: Mortality rates of A. longiremis females from the Arkona Basin (origin: S = 16) and the Bornholm Sea (S = 7.7) during 5 days exposure to a reduced salinity. Origin of population Acclimation salinity Treatment salinity Mortality rate d−1 (± STD) Arkona Sea 16.0 16.0 0.02 ± 0.032 16.0 12.0 0.02 ± 0.026 16.0 10.0 0.01 ± 0.021 16.0 8.0 0.01 ± 0.015 16.0 7.0 0.01 ± 0.015 16.0 6.0 0.03 ± 0.038 16.0 5.0 0.05 ± 0.040a Bornholm Sea 7.7 7.7 0 7.7 7.0 0.01 ± 0.013 7.7 6.0 0.01 ± 0.010 7.7 5.0 0.02 ± 0.021 7.7 4.0 0.05 ± 0.055a 7.7 3.0 0.44 ± 0.036b 6.0 6.0 0.01 ± 0.018 6.0 5.0 0.02 ± 0.025 6.0 4.0 0.03 ± 0.042 6.0 3.0 0.41 ± 0.070a 5.0 5.0 0 5.0 4.0 0.08 ± 0.042a 5.0 3.0 0.41 ± 0.082b 4.0 4.0 0.05 ± 0.049 4.0 3.0 0.38 ± 0.074a Origin of population Acclimation salinity Treatment salinity Mortality rate d−1 (± STD) Arkona Sea 16.0 16.0 0.02 ± 0.032 16.0 12.0 0.02 ± 0.026 16.0 10.0 0.01 ± 0.021 16.0 8.0 0.01 ± 0.015 16.0 7.0 0.01 ± 0.015 16.0 6.0 0.03 ± 0.038 16.0 5.0 0.05 ± 0.040a Bornholm Sea 7.7 7.7 0 7.7 7.0 0.01 ± 0.013 7.7 6.0 0.01 ± 0.010 7.7 5.0 0.02 ± 0.021 7.7 4.0 0.05 ± 0.055a 7.7 3.0 0.44 ± 0.036b 6.0 6.0 0.01 ± 0.018 6.0 5.0 0.02 ± 0.025 6.0 4.0 0.03 ± 0.042 6.0 3.0 0.41 ± 0.070a 5.0 5.0 0 5.0 4.0 0.08 ± 0.042a 5.0 3.0 0.41 ± 0.082b 4.0 4.0 0.05 ± 0.049 4.0 3.0 0.38 ± 0.074a Letters denote significant differences at P < 0.05. View Large Females from BB acclimated to a salinity of 7.7–3 displayed only small differences in survival following an instantenous reduction in salinity (Fig. 5). Survival was generally high, and >89% when females acclimated to S = 7.7, 6, and 5 were exposed to a treatment salinity of 5 or higher. The mortality rates of females were not significantly different from controls in these experiments (Table III). In contrast, mortality in treatment salinities of 4 and 3 resulted in a generally increased mortality of females independent of the acclimation salinity. At a treatment salinity of 4, survival decreased to on average 50–64%. Mortality rates ranged from 0.05 ± 0.055 d−1 to 0.08 ± 0.042 d−1 were significantly enhanced compared to controls (survival at acclimation salinity) except in females that were acclimated to the salinity of 6 (Fig. 5, Table III, ANCOVA, P < 0.05). Exposure to the salinity of 3 resulted in high mortality and none of the females survived to the end of the experiment. Incapacitation of females occurred at the salinity of 3 and 4 (Fig. 5). In contrast to survival, the acclimation salinity apparently increased the resistance to a reduction in the salinity. The lower the acclimation salinity was, the longer females remained inactive and the longer inactive females survived. Fig. 4. View largeDownload slide Proportions of surviving (a), incapacitated (b) or dead (c) females of Acartia longiremis (%) acclimated to a salinity of 16 (origin: AS) and exposed to a reduced salinity for five consecutive days (120 h). Females incubated at the original salinity (S = 16) served as control. For clarity, standard deviations are omitted. Fig. 4. View largeDownload slide Proportions of surviving (a), incapacitated (b) or dead (c) females of Acartia longiremis (%) acclimated to a salinity of 16 (origin: AS) and exposed to a reduced salinity for five consecutive days (120 h). Females incubated at the original salinity (S = 16) served as control. For clarity, standard deviations are omitted. The mortality of females following the exposure to a decreased salinity for 120 h showed a clear population dependence (Fig. 6). At a common salinity of 5, 6 and 7, the mortality of females originating from the higher salinity in the AS displayed a significantly higher mortality at the salinity of 5 and 6 than those originating from the Bornholm Sea (ANCOVA, P < 0.05). Mortality rates were not different at the salinity of 7. Fig. 5. View largeDownload slide Proportions of surviving (a), incapacitated (b) or dead (c) females of A. longiremis (%) acclimated to a salinity of 3, 4, 5 and 6 (origin: Bornholm Sea) and exposed to a reduced salinity for five consecutive days (120 h). Females incubated at the original salinity (S = 7.7) served as control. For clarity, standard deviations are omitted. Fig. 5. View largeDownload slide Proportions of surviving (a), incapacitated (b) or dead (c) females of A. longiremis (%) acclimated to a salinity of 3, 4, 5 and 6 (origin: Bornholm Sea) and exposed to a reduced salinity for five consecutive days (120 h). Females incubated at the original salinity (S = 7.7) served as control. For clarity, standard deviations are omitted. Fig. 6. View largeDownload slide Mortality (% ±standard deviation) of females of A. longiremis after 120 h exposure to a reduced salinity. Females originated from the AS (acclimation S: 16) and from the Bornholm Sea (acclimation S: 7.7, 6, 5, 4). For clarity, overlying data have been slightly offset with regard to final salinity. Fig. 6. View largeDownload slide Mortality (% ±standard deviation) of females of A. longiremis after 120 h exposure to a reduced salinity. Females originated from the AS (acclimation S: 16) and from the Bornholm Sea (acclimation S: 7.7, 6, 5, 4). For clarity, overlying data have been slightly offset with regard to final salinity. DISCUSSION Salinity tolerance of A. longiremis in the Baltic The spatiotemporal variation in salinity is a major factor determining the composition and succession of zooplankton in coastal and estuarine ecosystems (Ambler et al., 1985; Sotaert and van Rijswijk, 1993; Kimmel and Roman, 2004; Islam et al., 2006). The tolerance of copepods to decreasing salinity is essentially species-specific and appears not to be directly related to the ability to regulate the osmotic composition of the hemolymph (Farmer and Reeve, 1978; Svetlichny et al., 2012). However, true estuarine species often possess mechanisms to hyperregulate the osmotic composition of the hemolymph and, therefore, can persist at very low salinity or invade freshwater environments (Bayly, 1969; Brand and Bayly, 1971; Lee et al., 2012). Many marine copepods are, in contrast, osmoconformers with a limited ability to adjust the ionic concentration of the hemolymph and to withstand low salinity (Lance, 1965; Bayly, 1969; Svetlichny and Hubareva, 2014). Among these, species of the genus Acartia display a large range in tolerances reflecting their preferred habitat. Studies of the reproduction and survival of the brackish water species A. tonsa have shown an exceptionally wide salinity tolerance ranging from 2 to more than 33 (Cervetto et al., 1999; Calliari et al., 2006, Holste and Peck, 2006; Svetlichny and Hubareva, 2014). Other species like A. discaudata, A. margalefi or A. bifilosa, however, are less tolerant and prefer a salinity higher than 10–15 (Lance, 1964b; Castro-Longoria, 2003; Chinnery and Williams, 2004). A rather narrow range of salinity tolerance has been observed in the euryhaline marine species A. clausi which did not tolerate a salinity lower than 15–20 (Lance, 1964b; Castro-Longoria, 2003; Chinnery and Williams, 2004; Calliari et al. 2006). In the present study, A. longiremis displayed a considerable tolerance of low salinity. Feeding rates and reproductive success were not different between females from the AS at a salinity of 16 and those from the BB at a salinity of 7.7. In addition, specimen from both populations displayed a high survival following immediate changes in salinity down to 6. The broad tolerance is typical of a brackish water species and agrees with the wide distribution of A. longiremis in the Kattegat and the central Baltic at a salinity higher than 6. However, this contrasts with the euryhaline marine characteristics of the species outside the Baltic Sea. Acartia longiremis is a boreal-arctic species of the shelf seas of the North Atlantic and North Pacific and records from estuaries or coastal areas are generally rare (Lee and McAlice, 1979; Manning and Bucklin, 2005; Debes and Eliasen, 2006). Whether this reflects a general broad physiological tolerance of the species or the adaptation of the Baltic population to a low salinity needs further investigation. Physiological responses to salinity The changes in the feeding, egg production and survival of Acartia longiremis in response to a reduced salinity were largely consistent with those of other copepod species (Lance, 1964b; Castro-Longoria, 2003; Chinnery and Williams, 2004; Calliari et al., 2006). The similar feeding and egg production rates of the two populations at their native salinity of 7.7 and 16 suggest that the present salinity conditions in the western Baltic Sea (S > 7) have little negative impact on the species. However, the rates decreased rapidly in response to a further reduction to a critical salinity of 4–6. The decline in feeding of A. longiremis agrees with a decrease in ingestion and fecal pellet production observed for A. bifilosa/A. discaudata, A. tonsa or A. clausi with a decreasing salinity (Lance, 1964a; Calliari et al., 2006). However, while feeding in these species declined gradually over a broad range in salinity of 8–20 units, feeding of A. longiremis was similar between 16 and 7.7 and declined upon approaching the species’ critical salinity. This discrepancy is likely explained by the origin of A. tonsa and A. clausi from a salinity >30 (Lance, 1964a; Calliari et al., 2006), while A. longiremis in the Baltic was acclimated to low salinity. Egg production of females was decoupled from feeding and was unaffected by the changing salinity over a range of 16–6, followed by an abrupt decline at the salinity of 5. This contrasts with the maximal egg production rates at an optimal salinity and a gradual decline at suboptimal salinity observed in several Acartia species (Castro-Longoria, 2003; Calliari et al., 2006). Although the maximal egg production rate of A. longiremis is limited to, on average, 7–10 eggs female d−1 (Gómez-Gutiérrez and Peterson, 1999; Hansen et al., 1999), the egg production rates in our experiments were low. Reproductive females together with an increasing number of females with undeveloped gonads were observed in polar populations of A. longiremis during the transition to overwintering in September/October (Norrbin, 1994, 2001). Similarly, a low spawning frequency associated with immature females preparing for overwintering could have caused the observed low egg production, although the feeding activity in our experiments indicated that females were physiologically active. Nevertheless, our results indicate that the salinity tolerance for reproduction was lower than for feeding or survival, and could become critical for the persistence of viable populations of A. longiremis in the Baltic Sea, at a salinity of 5–6. The lack of strong effects on egg hatching success is consistent with the response of salinity tolerant species such as A. tonsa (Castro-Longoria, 2003; Calliari et al., 2006). The survival of females emphasized the ability of Acartia longiremis to cope well with salinity stress at its physiological limit. This is consistent with the strong resistance to instantaneous salinity stress in tolerant species like A. tonsa, in contrast to less tolerant marine species (Lance, 1963, 1964b; Cervetto et al., 1999). That instantaneous changes in salinity >10 were becoming critical for the survival of A. longiremis agrees with observations in A. tonsa by Cervetto et al., (1999), but contrast with a considerably larger decrease in salinity of more than 27 units tolerated by A. tonsa in another study (Calliari et al., 2008). The relatively short incubation time in the latter study (12 h) could explain this discrepancy because mortality in response to instantaneous salinity stress continued for several days unless critical levels were exceeded (Lance, 1963; Cervetto, et al. 1999, present study). Acclimation to a changing salinity is known to increase the survival of copepods (Lance, 1963; Cervetto et al., 1999). In A. longiremis, however, the effect of acclimation was only moderate. The low mortality of A. longiremis following the large and instantaneous salinity changes emphasizes the species ability to adjust rapidly to large salinity changes and its apparently broad physiological plasticity in the control of osmotic stress, irrespective of acclimation. Future work should include estimates of offspring survival and development in order to identify potential bottlenecks in the species’ population dynamics. The sensitivity to decreasing salinity can vary among developmental stages, with nauplii or copepodites being less tolerant than adult stages (Lance, 1964b; Cervetto et al., 1999; Lee et al., 2007). Metabolic costs Despite the large tolerance in feeding, egg production and survival of Acartia longiremis, oxygen consumption rates under natural and reduced salinity conditions point to a high-metabolic demand and, therefore, potential osmotic stress. Changes in respiration rates when salinity departs from natural conditions have been used as indicators of saline stress and physiological adaptation of copepods with, however, variable and contradictory responses (Lance, 1965; Farmer and Reeve, 1978; Gaudy et al., 2000; Calliari et al., 2006). As pointed out by Calliari et al. (2006), respiration alone is insufficient to evaluate effects of salinity stress because the metabolic rate also correlates positively with feeding and production, due to specific dynamic action reflecting the increasing energetic requirements for biosynthesis and transport associated with enhanced food uptake (Kiørboe et al., 1985). They showed that respiration of the brackish copepod A. tonsa largely varied with feeding and egg production and found no evidence for elevated respiration and costs related to species’ salinity tolerance. In contrast, a decoupling of feeding and respiration indicated the disruption of the metabolic balance in the less tolerant species A. clausi (Calliari et al., 2006). In our experiments, respiration rates of A. longiremis were already high under natural conditions. Physiological data concerning this species are lacking, and a comparison to other species is impeded by the different thermal adaption of temperate and boreal copepods, and the divergent experimental conditions. Nevertheless, feeding rates and daily rations of A. longiremis of 0.9–1.2 μg C Ind−1 d−1 or 0.47–0.49 d−1, respectively, were well within the range of rates reported for Acartia species in other studies (Kiørboe et al., 1985; Calliari et al., 2006). The respiration rates of females, however, appear considerably elevated. Oxygen consumption rates of 1.2–1.7 nL O2 Ind−1 min−1 at 10 °C exceeded those of 0.2–1.0 and 0.6–1.6 nL O2 Ind−1 min−1 reported for Acartia species at 10 and 18–20 °C, respectively (Anraku, 1964; Calliari et al., 2006; Hubareva et al., 2008). The weight-specific respiration rates for fed copepods of 0.39–49 d−1 were also higher than the 0.14–0.21 d−1 reported for A. tonsa and A. clausi at a higher experimental temperature (Calliari et al. 2006). This points to an unusually high-metabolic rate in this species, which was reinforced by the high starvation metabolism of 0.28–0.35 d−1. In contrast, respiration rates in A. tonsa decreased rapidly to less than 50% of the original rates within 24 h of starvation (Kiørboe et al., 1985). The increase in the respiration rate of Acartia longiremis when salinity decreased is in line with studies that showed osmotic stress by increased oxygen consumption or detrimental effects on the metabolic budget of less tolerant copepods (Gaudy et al., 2000; Calliari et al., 2006). The simultaneous decrease in feeding rates indicated increasing metabolic expenses that were not correlated with the feeding activity and, thus, likely reflected increasing osmotic stress. The high survival over the broad range of instantaneous salinity changes together with the fast recovery of females suggests that efficient physiological mechanisms were active, counteracting rapid internal osmotic changes. These were apparently metabolically expensive. When salinity further decreased below five, mortality increased while the oxygen consumption rates declined. This probably indicated that the physiological mechanisms regulating the internal osmolality became inefficient and collapsed. At present, we can only speculate about the physiological mechanisms behind the wide salinity tolerance of A. longiremis and the associated metabolic costs. While osmoregulators like the estuarine copepod Eurytemora affinis have been found to tolerate low salinity and freshwater conditions associated with the hyperregulation of the hemolymph and the activity of ion regulation enzymes in specialized organs (Lee et al., 2012; Johnson et al., 2014), very little is known about the adaptations of marine copepods to osmotic stress except for A. tonsa or about the physiological differences between tolerant and less tolerant Acartia species. While the hemolymph of A. tonsa was found to be slightly hyperosmotic at low salinity, it is isosmotic to seawater over a large range in salinity, similar to other osmoconforming, euryhaline copepod genera like Temora or Centropages (Lance, 1965; Bayly, 1969; Svetlichny and Hubareva, 2014). Such osmoconformers need to regulate the osmotic and ionic level of their body cells, which is achieved in the majority of cases through organic osmolytes such as free amino acids, peptides or ninhydrine positive substances (Farmer and Reeve, 1978; Péquex, 1995). In A. tonsa, the lowering of the free amino acid pool was found to be energetically expensive due to protein catabolism and ammonia excretion, but was achieved rapidly and accounted for short-term metabolic costs only (Farmer and Reeve, 1978). This is consistent with an unaffected metabolism following acclimation in this species (Calliari et al., 2006). The permanently enhanced respiration rates in A. longiremis, thus, could have been caused by less efficient regulation of the free amino acid pool and additional energetic costs of maintaining a homeostatic intracellular ionic composition by active ion pumps when the osmotic difference between hemolymph and cells was getting larger. Metabolic costs may also result from the regulation of other osmolytes or the expression of stress proteins maintaining cellular metabolic functions (Gonzales and Bradley, 1994; Péquex, 1995). Such costs may also not necessarily relate to osmoregulation. Centropages hamatus, for instance, was found to maintain the Mg-ion concentration in the hemolymph below the external medium, while Na was kept above which was interpreted to reflect the facilitation of nervous activity and locomotory control (Bayly, 1969). The mechanisms for the osmotic regulation in marine species that are tolerant and less tolerant to reduced salinity needs, therefore, further study. CONCLUSIONS Acartia longiremis is a widely distributed, marine species in the shelf seas of the northern hemisphere and can at times dominate the zooplankton in the brackish western Baltic Sea. Our study suggests that this dominance of the species at the low salinity is related to a broad salinity tolerance that is rather exceptional for a marine copepod species. The high feeding rates and plasticity in survival in response to seawater dilution indicate that the species possesses efficient mechanisms allowing a rapid adjustment to a changing salinity. However, high-respiration rates under natural and diluted conditions suggest that mechanisms of osmotic and cellular volume control need to be permanently expressed and are, therefore, metabolically expensive. Whether this is a general characteristic of the species or reflects a physiological adaptation to the low salinity conditions in the central Baltic Sea remains to be investigated. Acartia longiremis has not been reported from estuarine environments outside the Baltic Sea. The relatively stable salinity conditions in the Baltic Sea, thus, might have provided an environment under which adaptation could have evolved and allowed the species to persist and at times to dominate the copepod community. However, A. longiremis might persist in the Baltic Sea close to its physiological limit, which is indicated by the match between the lower salinity threshold for successful reproduction and survival at a salinity of 5–6 and the species’ present distribution in the Baltic Sea in offshore waters above a salinity of 6–7 (Chojnacki, 1984; Ojaveer et al., 1998; Díaz-Gil et al., 2014). Unfavorable conditions such as suboptimal temperature will further increase the sensitivity of copepods to changing salinity (Rippingale and Hodgkin, 1977; Lee et al., 2013). Acartia longiremis is a spring species in the Baltic Sea that avoids the warm surface layer during summer by submergence into the cold intermediate water layer (Hernroth and Ackefors, 1979; Schulz et al. 2012), which indicates a sensitivity to higher temperature. However, while warm surface temperatures might cause the absence of the species in the shallow coastal areas, salinity likely determines its distribution in the deep, open basins, which makes A. longiremis vulnerable to small changes in future salinity. The projected decrease of the salinity of 1.5–2 units until the end of the century, therefore, might cause a contraction of the distributional range of A. longiremis to the westernmost areas of the Baltic Sea. If the other abundant copepods of marine origin in the Baltic, like Temora longicornis or Pseudocalanus spp. demonstrate a similar sensitivity to changing salinity, the projected decrease would strongly alter the zooplankton diversity with a general decline of marine, spring–summer copepods and a shift to summer–autumn, brackish water tolerant species like A. bifilosa. In consequence, the productivity might decrease and the temporal survival windows for fish larvae feeding on these copepods might be strongly altered (e.g. Voss et al. 2012). At present, long-term changes in stocks of Acartia spp. and the environmental factors driving these changes are often assessed on the genus level, integrating the brackish water species A. tonsa and A. bifilosa with a large range in salinity tolerance (S < 6) and the marine species A. longiremis with a narrower tolerance (S > 6). In addition to salinity, the seasonal and vertical distributions of the species indicate different temperature preferences that are also insufficiently known. The present assessment of drivers of population changes integrating time series of the different Acartia species in the Baltic might be, therefore, misleading. ACKNOWLEDGEMENTS We are grateful for the support the scientific party and the crew of RV Dana during our experiments. Thanks to Marja Koski for statistical advice and valuable comments on an earlier version of the manuscript. FUNDING This work was supported by the BIO-C3 project (Biodiversity changes—causes, consequences and management implications, www.bio-c3.eu), belonging to BONUS, the joint Baltic Sea research and development programme (Art185), funded jointly from the European Union’s Seventh Programme for research, technological development and demonstration and the Danish Agency for Science Technology and Innovation (Ministry of Science, Technology and Innovation)—Denmark. The BIO-C3 cruise on RV DANA in September 2015 was funded by the Danish Centre for Marine Research (grant 2015-04). REFERENCES Ambler , J. W. , Cloern , J. E. and Hutchinson , A. ( 1985 ) Seasonal cycles of zooplankton from San Francisco Bay . Hydrobiologia , 129 , 177 – 179 . Google Scholar CrossRef Search ADS Anraku , M. ( 1964 ) Influence of the Cape Cod Canal on the hydrography and on the copepods in Buzzards Bay and Cape Cod Bay, Massachusetts. II. Respiration and feeding . Limnol. Oceanogr. , 9 , 195 – 206 . 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Google Scholar CrossRef Search ADS Wasmund , N. , Dutz , J. , Pollehne , F. , Siegel , H. and Zettler , M. L. ( 2016 ) Biological assessment of the Baltic Sea 2015 . Mar. Sci. Rep. , 102 , doi:10.12754/msr-2016-0102 . Author notes Corresponding editor: Roger Harris © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Plankton Research Oxford University Press

Broad plasticity in the salinity tolerance of a marine copepod species, Acartia longiremis, in the Baltic Sea

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Abstract

Abstract We assessed feeding, reproduction, survival and respiration in the boreal–polar copepod Acartia longiremis over the salinity range of 3–16 in the Baltic Sea. Feeding and egg production were not affected at a salinity of 7–16, representing the range in which the species naturally occurs, but decreased significantly at a lower salinity. Survival experiments showed a broad physiological plasticity with no increase in mortality upon immediate exposure to salinities of 16–7. Acclimation of females to low salinity extended the survival range to a salinity of 5. While the response in vital rates was characteristic of a tolerant, brackish water species, unusually high respiration rates at a salinity of 7–16 indicated that the species experienced osmotic stress, and that the mechanism maintaining physiological integrity was energetically expensive. Divergent responses of an increase in respiration rate and a decrease in feeding rate at a salinity below 7 indicated a disruption of the energetic balance under which the osmotic stress could not be counteracted. Our results show that A. longiremis persists close to its physiological limit in the Baltic Sea, which makes the species vulnerable to small changes in future salinity. INTRODUCTION Physical oceanographic factors have a central role in determining the distribution and abundance of pelagic organisms in aquatic environments. In estuarine and coastal ecosystems, salinity gradients, together with the seasonal variation in temperature, food availability and predators, strongly influence the composition and spatial succession of zooplankton communities (Collins and Williams, 1982; Ambler et al., 1985; Kimmel and Roman, 2004; Kayfetz and Kimmerer, 2017). Physiological studies on copepods have demonstrated large differences in the ability of different species to adjust the osmolyte content of the hemolymph (Lance, 1965; Brand and Bayly,1971; Lee et al., 2012; Svetlichny and Hubareva, 2014) and to maintain rates of feeding (Lance,1964b; Calliari et al., 2006), reproductive success (Chinnery and Williams, 2004; Calliari et al., 2006; Beyrend-Dur et al., 2009; Hemraj et al., 2018) or survival and development at changing salinities (Lee et al., 2007; Devreker et al. 2007). The distribution of copepods in their original habitat often reflects these species-specific tolerance levels, showing a spatial succession from stenohaline marine to true estuarine species along salinity gradients (Collins and Williams, 1982; Sotaert and van Rijswijk, 1993; Islam et al., 2006). A large proportion of euryhaline marine species characterizes the zooplankton of the brackish Baltic Sea. Temora longicornis, Centropages hamatus, Acartia longiremis and Pseudocalanus acuspes, among others, are often numerically abundant in the zooplankton communities of the Arkona Sea (AS), Bornholm Basin (BB) and Gotland Basin, despite the low salinity both in the surface and deeper water layers (Hernroth and Ackefors, 1979; Viitasalo, 1992; Schulz et al., 2012). Because these species live at the lower range of their tolerance limits, hydrological factors mainly determine the distribution and population size of the species. Salinity is, thus, a fundamental predictor of the abundance of the marine species and their contribution to zooplankton communities in the Baltic Sea (Viitasalo et al., 1995; Möllmann et al., 2000; Vuorinen et al., 2003; Mäkinen et al., 2017). In contrast to many brackish water species, the physiological responses and salinity tolerance of these marine species are, however, only rudimentarily understood. This gap in knowledge limits the capability to predict future changes in the abundance and distribution of marine species and in the diversity of Baltic zooplankton. In the Baltic Sea, long-term studies have regularly assessed the variation in the abundance of the genus Acartia spp. However, grouping of the species at a genus level largely restricts insights into the factors controlling the stock dynamics, because species of potential contrasting physiology, A. tonsa, A. bifilosa and A. longiremis, are integrated. Acartia tonsa is a thermophilic, brackish water species with a very broad salinity tolerance (Gaudy et al., 2000; Calliari et al., 2006; Holste and Peck, 2006; Svetlichny and Hubareva, 2014). The species occurs in the inner coastal waters and bights of the southern Baltic and is observed only occasionally in offshore water (Arndt and Schneese, 1986; Díaz-Gil et al., 2014; Schulz et al. 2012). The brackish A. bifilosa is ubiquitous in the Baltic Sea and is found in coastal as well as open waters ranging from the Kiel Bight to the Bothnian Sea and the Gulf of Finland, while A. longiremis is of marine origin and occurs primarily in colder, open water of the southern Baltic (Hernroth and Ackefors, 1979; Viitasalo, 1992; Schulz et al., 2012). Both A. bifilosa and A. longiremis are abundant in the deep basins of the Baltic and at times can dominate the copepod community (Wasmund et al. 2016). While the species’ wide distribution in the Baltic Sea suggests a broad salinity tolerance, direct knowledge about their salinity preferences are restricted to a few studies of A. bifilosa in estuarine water of the North Atlantic (Lance, 1963; Chinnery and Williams, 2004). Our main objectives were to characterize the physiological tolerance of A. longiremis in relation to decreasing salinity and to identify the critical salinity level that might limit its distribution and population dynamics in the Baltic Sea. Investigating the salinity responses of Baltic marine copepods is particularly relevant for future projections of biodiversity associated with environmental changes (Vuorinen et al., 2015). Surface salinity in the Baltic Sea has decreased in recent decades, due to increased river run-off and infrequent inflows of saline water from the North Sea through the Danish Straits (Hänninen et al., 2000; Meier et al., 2006). Simulations suggest a further decline by 1.5–2 units in the Bornholm and Gotland basins until the end of the century (Meier et al., 2012; Vuorinen et al., 2015). In addition, knowledge on the physiological effects of salinity stress in Acartia spp is often limited due to the focus on single traits such as reproductive success (Castro-Longoria, 2003; Chinnery and Williams, 2004; Holste and Peck, 2006; Svetlichny et al., 2010; Peck et al., 2015) or survival of specific life stages (Lance, 1963, 1964b; Cervetto et al., 1999; Calliari et al., 2008; Hubareva et al., 2008). Respiratory responses in relation to the performance of vital rates, however, can indicate physiological adaptation and the ability of species to cope with salinity stress (Farmer and Reeve, 1978; Gaudy et al., 2000; Isla and Perisinotto, 2004; Calliari et al., 2006; Svetlichny et al., 2012, 2014). We, therefore, investigated the feeding, egg production, egg hatching success and respiration of A. longiremis, in order to evaluate how salinity affects the metabolic budget using two different approaches. In the first, we compared the performance of A. longiremis occurring naturally at two different salinities in the western Baltic Sea. Due to a preference for the cooler, subsurface water layers below the thermocline in summer, the species occurred at a higher salinity in the shallow AS (S > 14.5) than in the deeper BB (S < 8). We considered specimens to be acclimated to the local conditions and compared vital rates at in situ salinity and survival of females to an instantaneous change in salinity in order to evaluate whether the species occurrence in large parts of the Baltic at a salinity below 8 is already constrained in present day salinity conditions. In the second approach, we intended to characterize the species tolerance to reduced salinity by comparing vital rates and survival of females from the Bornholm Sea acclimated to a decreasing salinity ranging from 7 to 3. METHOD Source populations Experiments were conducted during the cruise of R/V Dana to the central Baltic Sea, in September 2015. Zooplankton samples were taken with a 100 μm WP-2 net equipped with a non-filtering cod end and a closing device, at two stations located in the AS and the BB (Fig. 1). Due to a large horizontal salinity gradient in the subsurface layer at these stations, the salinity at the origin of the two populations was different. Individuals from the shallow AS (max. depth 45 m, 54°58′ N, 14°03′ E) were caught in the saline deep-water layer at a depth of 40–30 m, characterized by vertical gradients in salinity of 14.5–16.4 and in temperature of 10–14°C, respectively. The majority of the adult population of A. longiremis (>90%, 6.2–11.1 × 103 Ind m−3) stayed in this saline layer during day and night (results not shown). In the BB, the species avoids the warm surface during summer and concentrates in the colder but less saline intermediate water (Schulz et al. 2012). Zooplankton was, therefore, collected at a central station (depth 91 m, 55°17′ N, 15°43′ E) in the intermediate water layer (50–35 m) characterized by vertical gradients in a salinity of 7.5–7.9 and a temperature of 7.3–10.5°C, respectively. Samples were transferred into 30-L containers filled with pre-collected water from the sampling depth at both stations (35 m in the AS, 40 m in the BB) and brought into a walk-in cooling chamber set to a temperature of 10°C. Additional water needed for the cultures and experiments was collected at the same depths (AS: 80 L, BB: 180 L, collected in two batches during the cruise), subsequently GF/F filtered and stored until use. Fig. 1. View largeDownload slide Location of the sampling stations in the western Baltic Sea. Fig. 1. View largeDownload slide Location of the sampling stations in the western Baltic Sea. Adult A. longiremis were isolated from the catch into batches of 100–300 females and 30–70 males in 10 L permanently aerated, GF/F filtered seawater for acclimation to different salinity conditions (Table I). For the experiments with the population from the AS, copepods were set-up at a salinity of 16.0. For the experiments in the BB, one batch was kept at the in situ salinity of 7.7, while four other batches were progressively diluted to yield populations acclimated to salinities of 6.0, 5.0, 4.0 and 3.0. Salinity was adjusted by additions of MilliQ water at a rate of 1 d−1. Cultures were fed ad-libitum (>400 μgCL−1) a mixture of the cryptophyte Rhodomonas salina and the heterotrophic dinoflagellate Oxyrrhis marina. The mixture was chosen to provide good feeding conditions for A. longiremis, which has shown a preference for heterotrophic food (Peters et al., 2013). The cryptophyte was kept in exponential growth in 2 L batch cultures using GF/F filtered seawater at the respective salinities (10 °C, 16:8 h light:dark cycle, 100 mmol photons m−2 s−1, B-medium; Hansen, 1989). O. marina was grown in GF/F filtered seawater under similar conditions, except darkness, and fed with R. salina. Carbon content of O. marina was estimated from geometric dimensions using conversion factors (Menden-Deuer and Lessard, 2000). For R. salina grown at a salinity of 7.0–16.0, a carbon content of 54.6 pg C cell−1 was used (Dutz, unpublished results). The batches of females and males were acclimated to the food and final salinity conditions for 2 days before the start of experiments. All salinity measurements were conducted with a Cond 3210 conductivity meter equipped with a TetraCon 325 probe (WTW, Weilheim) with an accuracy of 0.5% of the measured value ± 0.1 digits. Table I: Summary of the salinity conditions used to evaluate to effect of salinity on feeding, egg production, egg hatching success and instantaneous survival of females of A. longiremis. Variation was within the accuracy limits of the conductivity meter (±0.1 units). Origin of population Salinity at origin Acclimation salinity S for acclimated rate measurements S for instantaneous survival Arkona Sea 14.5–16.4 16.0 16.0 16.0, 12.0, 10.0, 8.0, 7.0, 6.0, 5.0 Bornholm Sea 7.7–7.9 7.7 7.7 7.7, 7.0, 6.0, 5.0, 4.0, 3.0 6.0 6.0 6.0, 5.0, 4.0, 3.0 5.0 5.0 5.0, 4.0, 3.0 4.0 4.0 4.0, 3.0 3.0 – 3.0 Origin of population Salinity at origin Acclimation salinity S for acclimated rate measurements S for instantaneous survival Arkona Sea 14.5–16.4 16.0 16.0 16.0, 12.0, 10.0, 8.0, 7.0, 6.0, 5.0 Bornholm Sea 7.7–7.9 7.7 7.7 7.7, 7.0, 6.0, 5.0, 4.0, 3.0 6.0 6.0 6.0, 5.0, 4.0, 3.0 5.0 5.0 5.0, 4.0, 3.0 4.0 4.0 4.0, 3.0 3.0 – 3.0 Table I: Summary of the salinity conditions used to evaluate to effect of salinity on feeding, egg production, egg hatching success and instantaneous survival of females of A. longiremis. Variation was within the accuracy limits of the conductivity meter (±0.1 units). Origin of population Salinity at origin Acclimation salinity S for acclimated rate measurements S for instantaneous survival Arkona Sea 14.5–16.4 16.0 16.0 16.0, 12.0, 10.0, 8.0, 7.0, 6.0, 5.0 Bornholm Sea 7.7–7.9 7.7 7.7 7.7, 7.0, 6.0, 5.0, 4.0, 3.0 6.0 6.0 6.0, 5.0, 4.0, 3.0 5.0 5.0 5.0, 4.0, 3.0 4.0 4.0 4.0, 3.0 3.0 – 3.0 Origin of population Salinity at origin Acclimation salinity S for acclimated rate measurements S for instantaneous survival Arkona Sea 14.5–16.4 16.0 16.0 16.0, 12.0, 10.0, 8.0, 7.0, 6.0, 5.0 Bornholm Sea 7.7–7.9 7.7 7.7 7.7, 7.0, 6.0, 5.0, 4.0, 3.0 6.0 6.0 6.0, 5.0, 4.0, 3.0 5.0 5.0 5.0, 4.0, 3.0 4.0 4.0 4.0, 3.0 3.0 – 3.0 Feeding, egg production and egg hatching success Adults were picked from the batches acclimated to the final salinities. For each salinity treatment, 15–20 females and 5–7 males were placed in each of the four replicate 610 ml bottles filled with the experimental food suspension. The food was kept suspended by rotation on a plankton wheel, at a speed of 1 rpm. Food consisted of mixtures of R. salina and O. marina provided both at 100 μg C L−1. Adults were acclimated to experimental conditions for 24 h, followed by the transfer of females into new, separate bottles with fresh food suspension for another 24 h, to determine the feeding rate. Two types of control bottles were set-up in triplicate. The first consisted of suspensions of R. salina alone, in order to control its growth rate; the second consisted of the same mixture of R. salina and O. marina as used in grazing experiments, but without copepods, in order to determine O. marina growth and its consumption of R. salina. Samples for initial and final cell concentrations were counted in quadruplicate using a Coulter Multisizer Model 3 (R. salina) or a microscope (O. marina), using the Utermöhl technique from 6 to 7 replicates of a 4-mL sample. Ingestion rates of O. marina and females of A. longiremis were estimated from changes in prey cell numbers in treatments compared to those in controls using the approach by Tang et al. (2001). Briefly, the ingestion rates of females feeding on R. salina and O. marina in mixtures were estimated from changes in cell concentration (R. salina = Rho, O. marina = Ox) in the experiments described by Rho(t)=Rho0∗exp[(μRho−gAc−Rho⁎Ac)t−gOx⁎V∫0tOx(t)dt] (1) Ox(t)=Ox0⁎exp[(μOx−gAc−Ox⁎Ac)t] (2) where Rho0 and Ox0 are the initial cell concentrations of R. salina and O. marina, V is the volume of the bottle and Ac is the number of females in the experiment. The specific growth rates μRho and μOx (time−1) were obtained from the first and second control experiments without A. longiremis, respectively. The feeding coefficient of O. marina feeding on R. salina (gOx, Oxyrrhis−1 time−1) was calculated as the difference between R. salina specific growth rates in the first and the second control. The feeding coefficient (gAc-Ox, female−1 time−1), clearance (mL, female−1 time−1) and ingestion rate (cells female−1 time−1) of A. longiremis feeding on O. marina were calculated according to Frost (1972). The feeding coefficient of A. longiremis on R. salina (gAc-Rho, female−1 time−1) was obtained by substituting equation (2) into the integral term in equation (1) in order to describe the change in concentration of O. marina due to growth and predation and its effect on R. salina cell number as in the following equation: Rho(t)=Rho0∗exp[(μRho−gAc−Rho⁎Ac)t−gOxOx0VgAc−Ox⁎Ac−μOx⁎[1−exp((μOx−gAc−Ox⁎Ac)t)]] (3) The feeding coefficient gAc-Rho and the ingestion rate (IRAc-Rho, cells female−1 time−1) of females were calculated using the initial (Rho0) and final (RhoT) cell concentration of R. salina in the grazing experiment during the incubation time (T) according to equations (4) and (5). Carbon-specific ingestion rates (μg C female−1 time−1) were calculated using the cell-specific carbon content of the algae as described above: gAc−Rho=ln(Rho0)−ln(RhoT)+μRhoTAc⁎T−gOx⁎Ox⁎VAc⁎T⁎(gAc−Ox−μOx)⁎(1−exp[(μOx−gAc−Ox⁎Ac)⁎T]) (4) IRAc−Rho=V⁎gAc−Rho⁎(P0+PT)2 (5) After transfer to fresh food suspensions following the grazing experiments, females were incubated for an additional 24 h in order to determine the egg production rate. Males were reintroduced. The experiments were terminated by collecting females and eggs carefully onto 100 and 30 μm sieves, respectively. Eggs were rinsed into plastic petri dishes, counted and incubated until eggs started to hatch (~4 days). After an additional 24 h, eggs and nauplii were fixed with Lugol’s solution and counted to estimate egg hatching success. The females were collected and transferred to respiration chambers. Females not required for respiration measurements were fixed in formalin for later length measurement. Respiration Respiration was determined in quadruple groups of three to four females for each salinity treatment. Females were placed in respirometer glass chambers of 2 mL filled with pasteurized, oxygen-saturated GF/F-filtered seawater matching the salinity treatments. The chambers were sealed and immersed in a water bath with a constant temperature of 10 °C (±0.1). Oxygen consumption was measured through a thin capillary in the chamber lids with the micro-sensor (UniSense, Micro Respiration System). The micro-sensor (Clark-type) measured oxygen partial pressure every 2 s and data acquisition software provided oxygen values and an average rate of oxygen consumption. The animals were allowed to adapt for 5 min to the chambers and changes in the oxygen partial pressure were then followed for additional 10 min. Background respiration from the pasteurized filtered seawater was measured at least four times in each treatment in order to account for bacterial oxygen consumption. The measurements were repeated after the animals were starved for a period of 24 h in GF/F filtered seawater. At the end, females were fixed in formalin for later length measurements. Survival For the survival experiments, 30–40 females from each of the copepod batches were exposed to an instantaneous decrease in salinity. In the case of A. longiremis originating from the AS (S = 16), females were immediately transferred to salinities of 12, 10, 8, 7, 6 and 5. The batches of A. longiremis from the BB acclimated to a salinity of 7.7, 6, 5, 4 and 3 were immediately transferred to the salinities of 7, 6, 5, 4 and 3, when those were lower than the salinity that they were being kept. Females left in the original salinity served as a control. Animals were incubated in groups of six to seven in 20 mL dishes with six replicates in each treatment and fed a surplus of food of a mixture of Rhodomonas and Oxyrrhis (>400 μg C L−1). The survival of females was checked every 2 h during the first 12–16 h of the incubation, and thereafter every 6–8 h over a period of 5 days. Females were assigned to three categories: active, inactive and dead. Healthy and active females showed regular hop-and-sink behavior and responded immediately to fluid disturbance induced by pipette suction. Inactive females showed reduced pipette avoidance and activity. Food and water in all incubations was replaced daily. Weight-specific ingestion, egg production and respiration Weight-specific ingestion, egg production and respiration rates in terms of carbon were calculated using length measurements and length-body carbon conversion factors provided for salinities of 14 and 7 (Köster, 2003). Egg volume was determined from the measurement of the diameter of eggs collected from the acclimation batches and converted to carbon assuming 0.14 × 10−6 μg C μm−3 (Huntley and Lopez, 1992). Respiration rates were converted to carbon using a respiratory quotient of 0.87 used for feeding of A.tonsa on Rhodomonas (Kiørboe et al., 1985). Statistical analyses Feeding, egg production, respiration and egg hatching were tested for differences between salinity treatments by one-way analysis of variance (ANOVA) and a Tukey’s post hoc test for pairwise multiple comparison (P = 0.05). All data were normally distributed (Shapiro–Wilk test, P > 0.62). Mortality in survival experiments was compared between the salinity treatments by analysis of covariance (ANCOVA) on log transformed data. All analyses were conducted using the SigmaPlot 13.0 statistical package. RESULTS Feeding, egg production and egg hatching success Ingestion rates of A. longiremis differed significantly between the salinity treatments (one-way ANOVA, F = 26.9, P < 0.001) and decreased from 1.2 ± 0.16 μg C Ind−1 d−1 to 0.3 ± 0.06 μg C Ind−1 d−1 at the salinity of 16 and 4, respectively (Fig. 2a). Females at the salinity of 3 did not survive in sufficient numbers to conduct experiments. Feeding rates of 0.9 ± 0.23 μg C Ind−1 d−1 at the salinity of 7.7 were not significantly different from the maximal rate at the salinity of 16. However, the feeding rates at the salinity of 6 were significantly lower compared to higher salinities, but significantly higher than those at the salinity of 4 and 5. The diet of A. longiremis consisted of, on average, 64–75% of Rhodomonas salina at the salinity of 7 and 16. The contribution tended to decrease at lower salinities to <40% at the salinity of 5. However, the diet consisted of nearly exclusively R. salina at the salinity of 4. Weight-specific ingestion rates decreased from 0.49 ± 0.067 μg C μg C−1 d−1 at the salinity of 16 to 0.16 ± 0.029 μg C μg C−1 d−1 at the salinity of 4 (Table II). The decrease was significant (one-way ANOVA, F = 21.0, P < 0.001) and differences between treatments were similar to the per capita rates. Table II: Weight-specific ingestion, egg production, respiration and starvation respiration of female A. longiremis feeding on mixtures of R. salina and O. marina at a salinity of 4–16. Origin of population Acclimation salinity Ingestion (μg C μg C−1 d−1) Egg production (μg C μg C−1 d−1) Respiration (μg C μg C−1 d−1) Respiration (starv.) (μg C μg C−1 d−1) Arkona Sea 16.0 0.49 ± 0.067a 0.008 ± 0.0035a 0.48 ± 0.034a – Bornholm Sea 7.7 0.47 ± 0.116a 0.010 ± 0.0031a 0.39 ± 0.046b 0.28 ± 0.016b 6.0 0.32 ± 0.055b 0.007 ± 0.0020ab 0.48 ± 0.060a 0.31 ± 0.013ab 5.0 0.18 ± 0.037c 0.003 ± 0.0014b 0.49 ± 0.049a 0.35 ± 0.035a 4.0 0.16 ± 0.029c – 0.31 ± 0.014c 0.19 ± 0.035c 3.0 – – – – Origin of population Acclimation salinity Ingestion (μg C μg C−1 d−1) Egg production (μg C μg C−1 d−1) Respiration (μg C μg C−1 d−1) Respiration (starv.) (μg C μg C−1 d−1) Arkona Sea 16.0 0.49 ± 0.067a 0.008 ± 0.0035a 0.48 ± 0.034a – Bornholm Sea 7.7 0.47 ± 0.116a 0.010 ± 0.0031a 0.39 ± 0.046b 0.28 ± 0.016b 6.0 0.32 ± 0.055b 0.007 ± 0.0020ab 0.48 ± 0.060a 0.31 ± 0.013ab 5.0 0.18 ± 0.037c 0.003 ± 0.0014b 0.49 ± 0.049a 0.35 ± 0.035a 4.0 0.16 ± 0.029c – 0.31 ± 0.014c 0.19 ± 0.035c 3.0 – – – – Letters denote significant differences at P < 0.05. Table II: Weight-specific ingestion, egg production, respiration and starvation respiration of female A. longiremis feeding on mixtures of R. salina and O. marina at a salinity of 4–16. Origin of population Acclimation salinity Ingestion (μg C μg C−1 d−1) Egg production (μg C μg C−1 d−1) Respiration (μg C μg C−1 d−1) Respiration (starv.) (μg C μg C−1 d−1) Arkona Sea 16.0 0.49 ± 0.067a 0.008 ± 0.0035a 0.48 ± 0.034a – Bornholm Sea 7.7 0.47 ± 0.116a 0.010 ± 0.0031a 0.39 ± 0.046b 0.28 ± 0.016b 6.0 0.32 ± 0.055b 0.007 ± 0.0020ab 0.48 ± 0.060a 0.31 ± 0.013ab 5.0 0.18 ± 0.037c 0.003 ± 0.0014b 0.49 ± 0.049a 0.35 ± 0.035a 4.0 0.16 ± 0.029c – 0.31 ± 0.014c 0.19 ± 0.035c 3.0 – – – – Origin of population Acclimation salinity Ingestion (μg C μg C−1 d−1) Egg production (μg C μg C−1 d−1) Respiration (μg C μg C−1 d−1) Respiration (starv.) (μg C μg C−1 d−1) Arkona Sea 16.0 0.49 ± 0.067a 0.008 ± 0.0035a 0.48 ± 0.034a – Bornholm Sea 7.7 0.47 ± 0.116a 0.010 ± 0.0031a 0.39 ± 0.046b 0.28 ± 0.016b 6.0 0.32 ± 0.055b 0.007 ± 0.0020ab 0.48 ± 0.060a 0.31 ± 0.013ab 5.0 0.18 ± 0.037c 0.003 ± 0.0014b 0.49 ± 0.049a 0.35 ± 0.035a 4.0 0.16 ± 0.029c – 0.31 ± 0.014c 0.19 ± 0.035c 3.0 – – – – Letters denote significant differences at P < 0.05. Egg production of females was low at all salinities and ranged from 0.6 ± 0.24 eggs Ind−1 d−1 at the salinity of 16 to 0.2 ± 0.08 eggs Ind−1 d−1 at the salinity of 5 (Fig. 2b). No eggs were produced at the lowest salinity of 4. Although the egg production was significantly related to salinity (one-way ANOVA, F = 11.5, P < 0.001), the rates were not statistically different between the salinities of 16, 7 and 6 (Fig. 2b). Weight-specific egg production rates significantly decreased from 0.010 ± 0.0031 μg C μg C−1 d−1 to 0.003 ± 0.0014 μg C μg C−1 d−1 (one-way ANOVA, F = 11.9, P < 0.001, Table II). Again, they were similar at the salinity of 16, 7 and 6, while the specific egg production at the salinity of 5 was lower. The egg hatching success was very variable due to the low number of eggs in the replicates and not significantly different between treatments (Fig. 2c). The length of females (n = 30) in feeding and egg production experiments varied between 684 ± 20 μm and 689 ± 32 μm, and was not significantly different between treatments (data not shown). Egg size was averaged over all treatments due to low egg numbers and was 79.2 ± 2.34 μm. Fig. 2. View largeDownload slide Ingestion rates (μg C Ind.−1 d−1), egg production (eggs Ind.−1 d−1) and egg hatching success (%) of A. longiremis females feeding on mixtures of R. salina and O. marina. Different letters denote treatments that are significantly different from each other according to a Tukey HSD (P < 0.05). Fig. 2. View largeDownload slide Ingestion rates (μg C Ind.−1 d−1), egg production (eggs Ind.−1 d−1) and egg hatching success (%) of A. longiremis females feeding on mixtures of R. salina and O. marina. Different letters denote treatments that are significantly different from each other according to a Tukey HSD (P < 0.05). Respiration The treatment salinity had a significant effect on the respiration rate of non-starved females (one-way ANOVA, F = 21.6, P < 0.001, Fig. 3). Highest respiration rates were recorded at the salinity of 16 (0.10 ± 0.007 μl O2 Ind−1 h−1), while the respiration rate of 0.07 ± 0.008 μL O2 Ind−1 h−1 at the salinity of 7 was significantly lower. However, respiration increased again to 0.09 ± 0.009 μL O2 Ind−1 h−1 when salinity was lowered to S = 5, which was not different from the oxygen consumption at the salinity of 16. The lowest respiration was measured at the salinity of 4. When females were starved for 24 h, respiration rates remained high and amounted to, on average, 67% of the rates determined for non-starved females and were significantly higher at the salinity of 5 (0.06 ± 0.006 μL O2 Ind−1 h−1) than at the salinity of 7 or 4. Likewise, weight-specific respiration in terms of carbon was similar at the salinity of 5, 6 and 16 (0.48–0.49 μg C μg C−1 d−1) and significantly higher than the rates calculated for the salinity of 4 and 7 (one-way ANOVA, F = 27.3, P < 0.001, Table II). Weight-specific respiration rates of starving females were also significantly higher at the salinity of 5 than at the salinity of 4 and 7 (one-way ANOVA, F = 27.7, P < 0.001, Table II). Survival Females originating from the AS survived the immediate salinity reduction generally well (Fig. 4a), but the survival decreased gradually over the duration of the experiment. The average survival over 120 h following exposure to reduced salinities of 12–7 ranged from 86 ± 13% to 95 ± 8% and was similar to the control at the salinity of 16 (91 ± 11%). Survival decreased to 82 ± 15% and 70 ± 13% at a treatment salinity to 6 and 5, respectively. Incapacitation of the females was observed only initially (<12 h) and when the salinity was reduced to values lower than 10 (Fig. 4b). In this case, the proportion of inactive females was inversely related to salinity and increased from 15 ± 36 at the salinity of 8 to 95 ± 27% at the salinity of 5 within the first 2 h, but vanished rapidly afterwards. Cumulative mortality, in contrast, was low during the first 24 h in all treatments and did not exceed 10% (Fig. 4c). This indicated that most of the inactive females recovered from incapacitation. The number of dead females increased gradually in all treatments after 72 h. Mortality rates ranged from 0.02 ± 0.032 d−1 at the salinity of 16 to 0.05 ± 0.040 d-1 at the salinity of 5 (Table III) being significantly different at the salinity of 5 than at other salinities (ANCOVA, P < 0.05). Fig. 3. View largeDownload slide Respiration rates of (μl O2 Ind.−1 h−1) of A. longiremis females feeding on mixtures of R. salina and O. marina before and after starvation. Different letters denote treatments that are significantly different from each other according to Tukey HSD (P < 0.05). Fig. 3. View largeDownload slide Respiration rates of (μl O2 Ind.−1 h−1) of A. longiremis females feeding on mixtures of R. salina and O. marina before and after starvation. Different letters denote treatments that are significantly different from each other according to Tukey HSD (P < 0.05). Table III: Mortality rates of A. longiremis females from the Arkona Basin (origin: S = 16) and the Bornholm Sea (S = 7.7) during 5 days exposure to a reduced salinity. Origin of population Acclimation salinity Treatment salinity Mortality rate d−1 (± STD) Arkona Sea 16.0 16.0 0.02 ± 0.032 16.0 12.0 0.02 ± 0.026 16.0 10.0 0.01 ± 0.021 16.0 8.0 0.01 ± 0.015 16.0 7.0 0.01 ± 0.015 16.0 6.0 0.03 ± 0.038 16.0 5.0 0.05 ± 0.040a Bornholm Sea 7.7 7.7 0 7.7 7.0 0.01 ± 0.013 7.7 6.0 0.01 ± 0.010 7.7 5.0 0.02 ± 0.021 7.7 4.0 0.05 ± 0.055a 7.7 3.0 0.44 ± 0.036b 6.0 6.0 0.01 ± 0.018 6.0 5.0 0.02 ± 0.025 6.0 4.0 0.03 ± 0.042 6.0 3.0 0.41 ± 0.070a 5.0 5.0 0 5.0 4.0 0.08 ± 0.042a 5.0 3.0 0.41 ± 0.082b 4.0 4.0 0.05 ± 0.049 4.0 3.0 0.38 ± 0.074a Origin of population Acclimation salinity Treatment salinity Mortality rate d−1 (± STD) Arkona Sea 16.0 16.0 0.02 ± 0.032 16.0 12.0 0.02 ± 0.026 16.0 10.0 0.01 ± 0.021 16.0 8.0 0.01 ± 0.015 16.0 7.0 0.01 ± 0.015 16.0 6.0 0.03 ± 0.038 16.0 5.0 0.05 ± 0.040a Bornholm Sea 7.7 7.7 0 7.7 7.0 0.01 ± 0.013 7.7 6.0 0.01 ± 0.010 7.7 5.0 0.02 ± 0.021 7.7 4.0 0.05 ± 0.055a 7.7 3.0 0.44 ± 0.036b 6.0 6.0 0.01 ± 0.018 6.0 5.0 0.02 ± 0.025 6.0 4.0 0.03 ± 0.042 6.0 3.0 0.41 ± 0.070a 5.0 5.0 0 5.0 4.0 0.08 ± 0.042a 5.0 3.0 0.41 ± 0.082b 4.0 4.0 0.05 ± 0.049 4.0 3.0 0.38 ± 0.074a Letters denote significant differences at P < 0.05. View Large Table III: Mortality rates of A. longiremis females from the Arkona Basin (origin: S = 16) and the Bornholm Sea (S = 7.7) during 5 days exposure to a reduced salinity. Origin of population Acclimation salinity Treatment salinity Mortality rate d−1 (± STD) Arkona Sea 16.0 16.0 0.02 ± 0.032 16.0 12.0 0.02 ± 0.026 16.0 10.0 0.01 ± 0.021 16.0 8.0 0.01 ± 0.015 16.0 7.0 0.01 ± 0.015 16.0 6.0 0.03 ± 0.038 16.0 5.0 0.05 ± 0.040a Bornholm Sea 7.7 7.7 0 7.7 7.0 0.01 ± 0.013 7.7 6.0 0.01 ± 0.010 7.7 5.0 0.02 ± 0.021 7.7 4.0 0.05 ± 0.055a 7.7 3.0 0.44 ± 0.036b 6.0 6.0 0.01 ± 0.018 6.0 5.0 0.02 ± 0.025 6.0 4.0 0.03 ± 0.042 6.0 3.0 0.41 ± 0.070a 5.0 5.0 0 5.0 4.0 0.08 ± 0.042a 5.0 3.0 0.41 ± 0.082b 4.0 4.0 0.05 ± 0.049 4.0 3.0 0.38 ± 0.074a Origin of population Acclimation salinity Treatment salinity Mortality rate d−1 (± STD) Arkona Sea 16.0 16.0 0.02 ± 0.032 16.0 12.0 0.02 ± 0.026 16.0 10.0 0.01 ± 0.021 16.0 8.0 0.01 ± 0.015 16.0 7.0 0.01 ± 0.015 16.0 6.0 0.03 ± 0.038 16.0 5.0 0.05 ± 0.040a Bornholm Sea 7.7 7.7 0 7.7 7.0 0.01 ± 0.013 7.7 6.0 0.01 ± 0.010 7.7 5.0 0.02 ± 0.021 7.7 4.0 0.05 ± 0.055a 7.7 3.0 0.44 ± 0.036b 6.0 6.0 0.01 ± 0.018 6.0 5.0 0.02 ± 0.025 6.0 4.0 0.03 ± 0.042 6.0 3.0 0.41 ± 0.070a 5.0 5.0 0 5.0 4.0 0.08 ± 0.042a 5.0 3.0 0.41 ± 0.082b 4.0 4.0 0.05 ± 0.049 4.0 3.0 0.38 ± 0.074a Letters denote significant differences at P < 0.05. View Large Females from BB acclimated to a salinity of 7.7–3 displayed only small differences in survival following an instantenous reduction in salinity (Fig. 5). Survival was generally high, and >89% when females acclimated to S = 7.7, 6, and 5 were exposed to a treatment salinity of 5 or higher. The mortality rates of females were not significantly different from controls in these experiments (Table III). In contrast, mortality in treatment salinities of 4 and 3 resulted in a generally increased mortality of females independent of the acclimation salinity. At a treatment salinity of 4, survival decreased to on average 50–64%. Mortality rates ranged from 0.05 ± 0.055 d−1 to 0.08 ± 0.042 d−1 were significantly enhanced compared to controls (survival at acclimation salinity) except in females that were acclimated to the salinity of 6 (Fig. 5, Table III, ANCOVA, P < 0.05). Exposure to the salinity of 3 resulted in high mortality and none of the females survived to the end of the experiment. Incapacitation of females occurred at the salinity of 3 and 4 (Fig. 5). In contrast to survival, the acclimation salinity apparently increased the resistance to a reduction in the salinity. The lower the acclimation salinity was, the longer females remained inactive and the longer inactive females survived. Fig. 4. View largeDownload slide Proportions of surviving (a), incapacitated (b) or dead (c) females of Acartia longiremis (%) acclimated to a salinity of 16 (origin: AS) and exposed to a reduced salinity for five consecutive days (120 h). Females incubated at the original salinity (S = 16) served as control. For clarity, standard deviations are omitted. Fig. 4. View largeDownload slide Proportions of surviving (a), incapacitated (b) or dead (c) females of Acartia longiremis (%) acclimated to a salinity of 16 (origin: AS) and exposed to a reduced salinity for five consecutive days (120 h). Females incubated at the original salinity (S = 16) served as control. For clarity, standard deviations are omitted. The mortality of females following the exposure to a decreased salinity for 120 h showed a clear population dependence (Fig. 6). At a common salinity of 5, 6 and 7, the mortality of females originating from the higher salinity in the AS displayed a significantly higher mortality at the salinity of 5 and 6 than those originating from the Bornholm Sea (ANCOVA, P < 0.05). Mortality rates were not different at the salinity of 7. Fig. 5. View largeDownload slide Proportions of surviving (a), incapacitated (b) or dead (c) females of A. longiremis (%) acclimated to a salinity of 3, 4, 5 and 6 (origin: Bornholm Sea) and exposed to a reduced salinity for five consecutive days (120 h). Females incubated at the original salinity (S = 7.7) served as control. For clarity, standard deviations are omitted. Fig. 5. View largeDownload slide Proportions of surviving (a), incapacitated (b) or dead (c) females of A. longiremis (%) acclimated to a salinity of 3, 4, 5 and 6 (origin: Bornholm Sea) and exposed to a reduced salinity for five consecutive days (120 h). Females incubated at the original salinity (S = 7.7) served as control. For clarity, standard deviations are omitted. Fig. 6. View largeDownload slide Mortality (% ±standard deviation) of females of A. longiremis after 120 h exposure to a reduced salinity. Females originated from the AS (acclimation S: 16) and from the Bornholm Sea (acclimation S: 7.7, 6, 5, 4). For clarity, overlying data have been slightly offset with regard to final salinity. Fig. 6. View largeDownload slide Mortality (% ±standard deviation) of females of A. longiremis after 120 h exposure to a reduced salinity. Females originated from the AS (acclimation S: 16) and from the Bornholm Sea (acclimation S: 7.7, 6, 5, 4). For clarity, overlying data have been slightly offset with regard to final salinity. DISCUSSION Salinity tolerance of A. longiremis in the Baltic The spatiotemporal variation in salinity is a major factor determining the composition and succession of zooplankton in coastal and estuarine ecosystems (Ambler et al., 1985; Sotaert and van Rijswijk, 1993; Kimmel and Roman, 2004; Islam et al., 2006). The tolerance of copepods to decreasing salinity is essentially species-specific and appears not to be directly related to the ability to regulate the osmotic composition of the hemolymph (Farmer and Reeve, 1978; Svetlichny et al., 2012). However, true estuarine species often possess mechanisms to hyperregulate the osmotic composition of the hemolymph and, therefore, can persist at very low salinity or invade freshwater environments (Bayly, 1969; Brand and Bayly, 1971; Lee et al., 2012). Many marine copepods are, in contrast, osmoconformers with a limited ability to adjust the ionic concentration of the hemolymph and to withstand low salinity (Lance, 1965; Bayly, 1969; Svetlichny and Hubareva, 2014). Among these, species of the genus Acartia display a large range in tolerances reflecting their preferred habitat. Studies of the reproduction and survival of the brackish water species A. tonsa have shown an exceptionally wide salinity tolerance ranging from 2 to more than 33 (Cervetto et al., 1999; Calliari et al., 2006, Holste and Peck, 2006; Svetlichny and Hubareva, 2014). Other species like A. discaudata, A. margalefi or A. bifilosa, however, are less tolerant and prefer a salinity higher than 10–15 (Lance, 1964b; Castro-Longoria, 2003; Chinnery and Williams, 2004). A rather narrow range of salinity tolerance has been observed in the euryhaline marine species A. clausi which did not tolerate a salinity lower than 15–20 (Lance, 1964b; Castro-Longoria, 2003; Chinnery and Williams, 2004; Calliari et al. 2006). In the present study, A. longiremis displayed a considerable tolerance of low salinity. Feeding rates and reproductive success were not different between females from the AS at a salinity of 16 and those from the BB at a salinity of 7.7. In addition, specimen from both populations displayed a high survival following immediate changes in salinity down to 6. The broad tolerance is typical of a brackish water species and agrees with the wide distribution of A. longiremis in the Kattegat and the central Baltic at a salinity higher than 6. However, this contrasts with the euryhaline marine characteristics of the species outside the Baltic Sea. Acartia longiremis is a boreal-arctic species of the shelf seas of the North Atlantic and North Pacific and records from estuaries or coastal areas are generally rare (Lee and McAlice, 1979; Manning and Bucklin, 2005; Debes and Eliasen, 2006). Whether this reflects a general broad physiological tolerance of the species or the adaptation of the Baltic population to a low salinity needs further investigation. Physiological responses to salinity The changes in the feeding, egg production and survival of Acartia longiremis in response to a reduced salinity were largely consistent with those of other copepod species (Lance, 1964b; Castro-Longoria, 2003; Chinnery and Williams, 2004; Calliari et al., 2006). The similar feeding and egg production rates of the two populations at their native salinity of 7.7 and 16 suggest that the present salinity conditions in the western Baltic Sea (S > 7) have little negative impact on the species. However, the rates decreased rapidly in response to a further reduction to a critical salinity of 4–6. The decline in feeding of A. longiremis agrees with a decrease in ingestion and fecal pellet production observed for A. bifilosa/A. discaudata, A. tonsa or A. clausi with a decreasing salinity (Lance, 1964a; Calliari et al., 2006). However, while feeding in these species declined gradually over a broad range in salinity of 8–20 units, feeding of A. longiremis was similar between 16 and 7.7 and declined upon approaching the species’ critical salinity. This discrepancy is likely explained by the origin of A. tonsa and A. clausi from a salinity >30 (Lance, 1964a; Calliari et al., 2006), while A. longiremis in the Baltic was acclimated to low salinity. Egg production of females was decoupled from feeding and was unaffected by the changing salinity over a range of 16–6, followed by an abrupt decline at the salinity of 5. This contrasts with the maximal egg production rates at an optimal salinity and a gradual decline at suboptimal salinity observed in several Acartia species (Castro-Longoria, 2003; Calliari et al., 2006). Although the maximal egg production rate of A. longiremis is limited to, on average, 7–10 eggs female d−1 (Gómez-Gutiérrez and Peterson, 1999; Hansen et al., 1999), the egg production rates in our experiments were low. Reproductive females together with an increasing number of females with undeveloped gonads were observed in polar populations of A. longiremis during the transition to overwintering in September/October (Norrbin, 1994, 2001). Similarly, a low spawning frequency associated with immature females preparing for overwintering could have caused the observed low egg production, although the feeding activity in our experiments indicated that females were physiologically active. Nevertheless, our results indicate that the salinity tolerance for reproduction was lower than for feeding or survival, and could become critical for the persistence of viable populations of A. longiremis in the Baltic Sea, at a salinity of 5–6. The lack of strong effects on egg hatching success is consistent with the response of salinity tolerant species such as A. tonsa (Castro-Longoria, 2003; Calliari et al., 2006). The survival of females emphasized the ability of Acartia longiremis to cope well with salinity stress at its physiological limit. This is consistent with the strong resistance to instantaneous salinity stress in tolerant species like A. tonsa, in contrast to less tolerant marine species (Lance, 1963, 1964b; Cervetto et al., 1999). That instantaneous changes in salinity >10 were becoming critical for the survival of A. longiremis agrees with observations in A. tonsa by Cervetto et al., (1999), but contrast with a considerably larger decrease in salinity of more than 27 units tolerated by A. tonsa in another study (Calliari et al., 2008). The relatively short incubation time in the latter study (12 h) could explain this discrepancy because mortality in response to instantaneous salinity stress continued for several days unless critical levels were exceeded (Lance, 1963; Cervetto, et al. 1999, present study). Acclimation to a changing salinity is known to increase the survival of copepods (Lance, 1963; Cervetto et al., 1999). In A. longiremis, however, the effect of acclimation was only moderate. The low mortality of A. longiremis following the large and instantaneous salinity changes emphasizes the species ability to adjust rapidly to large salinity changes and its apparently broad physiological plasticity in the control of osmotic stress, irrespective of acclimation. Future work should include estimates of offspring survival and development in order to identify potential bottlenecks in the species’ population dynamics. The sensitivity to decreasing salinity can vary among developmental stages, with nauplii or copepodites being less tolerant than adult stages (Lance, 1964b; Cervetto et al., 1999; Lee et al., 2007). Metabolic costs Despite the large tolerance in feeding, egg production and survival of Acartia longiremis, oxygen consumption rates under natural and reduced salinity conditions point to a high-metabolic demand and, therefore, potential osmotic stress. Changes in respiration rates when salinity departs from natural conditions have been used as indicators of saline stress and physiological adaptation of copepods with, however, variable and contradictory responses (Lance, 1965; Farmer and Reeve, 1978; Gaudy et al., 2000; Calliari et al., 2006). As pointed out by Calliari et al. (2006), respiration alone is insufficient to evaluate effects of salinity stress because the metabolic rate also correlates positively with feeding and production, due to specific dynamic action reflecting the increasing energetic requirements for biosynthesis and transport associated with enhanced food uptake (Kiørboe et al., 1985). They showed that respiration of the brackish copepod A. tonsa largely varied with feeding and egg production and found no evidence for elevated respiration and costs related to species’ salinity tolerance. In contrast, a decoupling of feeding and respiration indicated the disruption of the metabolic balance in the less tolerant species A. clausi (Calliari et al., 2006). In our experiments, respiration rates of A. longiremis were already high under natural conditions. Physiological data concerning this species are lacking, and a comparison to other species is impeded by the different thermal adaption of temperate and boreal copepods, and the divergent experimental conditions. Nevertheless, feeding rates and daily rations of A. longiremis of 0.9–1.2 μg C Ind−1 d−1 or 0.47–0.49 d−1, respectively, were well within the range of rates reported for Acartia species in other studies (Kiørboe et al., 1985; Calliari et al., 2006). The respiration rates of females, however, appear considerably elevated. Oxygen consumption rates of 1.2–1.7 nL O2 Ind−1 min−1 at 10 °C exceeded those of 0.2–1.0 and 0.6–1.6 nL O2 Ind−1 min−1 reported for Acartia species at 10 and 18–20 °C, respectively (Anraku, 1964; Calliari et al., 2006; Hubareva et al., 2008). The weight-specific respiration rates for fed copepods of 0.39–49 d−1 were also higher than the 0.14–0.21 d−1 reported for A. tonsa and A. clausi at a higher experimental temperature (Calliari et al. 2006). This points to an unusually high-metabolic rate in this species, which was reinforced by the high starvation metabolism of 0.28–0.35 d−1. In contrast, respiration rates in A. tonsa decreased rapidly to less than 50% of the original rates within 24 h of starvation (Kiørboe et al., 1985). The increase in the respiration rate of Acartia longiremis when salinity decreased is in line with studies that showed osmotic stress by increased oxygen consumption or detrimental effects on the metabolic budget of less tolerant copepods (Gaudy et al., 2000; Calliari et al., 2006). The simultaneous decrease in feeding rates indicated increasing metabolic expenses that were not correlated with the feeding activity and, thus, likely reflected increasing osmotic stress. The high survival over the broad range of instantaneous salinity changes together with the fast recovery of females suggests that efficient physiological mechanisms were active, counteracting rapid internal osmotic changes. These were apparently metabolically expensive. When salinity further decreased below five, mortality increased while the oxygen consumption rates declined. This probably indicated that the physiological mechanisms regulating the internal osmolality became inefficient and collapsed. At present, we can only speculate about the physiological mechanisms behind the wide salinity tolerance of A. longiremis and the associated metabolic costs. While osmoregulators like the estuarine copepod Eurytemora affinis have been found to tolerate low salinity and freshwater conditions associated with the hyperregulation of the hemolymph and the activity of ion regulation enzymes in specialized organs (Lee et al., 2012; Johnson et al., 2014), very little is known about the adaptations of marine copepods to osmotic stress except for A. tonsa or about the physiological differences between tolerant and less tolerant Acartia species. While the hemolymph of A. tonsa was found to be slightly hyperosmotic at low salinity, it is isosmotic to seawater over a large range in salinity, similar to other osmoconforming, euryhaline copepod genera like Temora or Centropages (Lance, 1965; Bayly, 1969; Svetlichny and Hubareva, 2014). Such osmoconformers need to regulate the osmotic and ionic level of their body cells, which is achieved in the majority of cases through organic osmolytes such as free amino acids, peptides or ninhydrine positive substances (Farmer and Reeve, 1978; Péquex, 1995). In A. tonsa, the lowering of the free amino acid pool was found to be energetically expensive due to protein catabolism and ammonia excretion, but was achieved rapidly and accounted for short-term metabolic costs only (Farmer and Reeve, 1978). This is consistent with an unaffected metabolism following acclimation in this species (Calliari et al., 2006). The permanently enhanced respiration rates in A. longiremis, thus, could have been caused by less efficient regulation of the free amino acid pool and additional energetic costs of maintaining a homeostatic intracellular ionic composition by active ion pumps when the osmotic difference between hemolymph and cells was getting larger. Metabolic costs may also result from the regulation of other osmolytes or the expression of stress proteins maintaining cellular metabolic functions (Gonzales and Bradley, 1994; Péquex, 1995). Such costs may also not necessarily relate to osmoregulation. Centropages hamatus, for instance, was found to maintain the Mg-ion concentration in the hemolymph below the external medium, while Na was kept above which was interpreted to reflect the facilitation of nervous activity and locomotory control (Bayly, 1969). The mechanisms for the osmotic regulation in marine species that are tolerant and less tolerant to reduced salinity needs, therefore, further study. CONCLUSIONS Acartia longiremis is a widely distributed, marine species in the shelf seas of the northern hemisphere and can at times dominate the zooplankton in the brackish western Baltic Sea. Our study suggests that this dominance of the species at the low salinity is related to a broad salinity tolerance that is rather exceptional for a marine copepod species. The high feeding rates and plasticity in survival in response to seawater dilution indicate that the species possesses efficient mechanisms allowing a rapid adjustment to a changing salinity. However, high-respiration rates under natural and diluted conditions suggest that mechanisms of osmotic and cellular volume control need to be permanently expressed and are, therefore, metabolically expensive. Whether this is a general characteristic of the species or reflects a physiological adaptation to the low salinity conditions in the central Baltic Sea remains to be investigated. Acartia longiremis has not been reported from estuarine environments outside the Baltic Sea. The relatively stable salinity conditions in the Baltic Sea, thus, might have provided an environment under which adaptation could have evolved and allowed the species to persist and at times to dominate the copepod community. However, A. longiremis might persist in the Baltic Sea close to its physiological limit, which is indicated by the match between the lower salinity threshold for successful reproduction and survival at a salinity of 5–6 and the species’ present distribution in the Baltic Sea in offshore waters above a salinity of 6–7 (Chojnacki, 1984; Ojaveer et al., 1998; Díaz-Gil et al., 2014). Unfavorable conditions such as suboptimal temperature will further increase the sensitivity of copepods to changing salinity (Rippingale and Hodgkin, 1977; Lee et al., 2013). Acartia longiremis is a spring species in the Baltic Sea that avoids the warm surface layer during summer by submergence into the cold intermediate water layer (Hernroth and Ackefors, 1979; Schulz et al. 2012), which indicates a sensitivity to higher temperature. However, while warm surface temperatures might cause the absence of the species in the shallow coastal areas, salinity likely determines its distribution in the deep, open basins, which makes A. longiremis vulnerable to small changes in future salinity. The projected decrease of the salinity of 1.5–2 units until the end of the century, therefore, might cause a contraction of the distributional range of A. longiremis to the westernmost areas of the Baltic Sea. If the other abundant copepods of marine origin in the Baltic, like Temora longicornis or Pseudocalanus spp. demonstrate a similar sensitivity to changing salinity, the projected decrease would strongly alter the zooplankton diversity with a general decline of marine, spring–summer copepods and a shift to summer–autumn, brackish water tolerant species like A. bifilosa. In consequence, the productivity might decrease and the temporal survival windows for fish larvae feeding on these copepods might be strongly altered (e.g. Voss et al. 2012). At present, long-term changes in stocks of Acartia spp. and the environmental factors driving these changes are often assessed on the genus level, integrating the brackish water species A. tonsa and A. bifilosa with a large range in salinity tolerance (S < 6) and the marine species A. longiremis with a narrower tolerance (S > 6). In addition to salinity, the seasonal and vertical distributions of the species indicate different temperature preferences that are also insufficiently known. The present assessment of drivers of population changes integrating time series of the different Acartia species in the Baltic might be, therefore, misleading. ACKNOWLEDGEMENTS We are grateful for the support the scientific party and the crew of RV Dana during our experiments. Thanks to Marja Koski for statistical advice and valuable comments on an earlier version of the manuscript. FUNDING This work was supported by the BIO-C3 project (Biodiversity changes—causes, consequences and management implications, www.bio-c3.eu), belonging to BONUS, the joint Baltic Sea research and development programme (Art185), funded jointly from the European Union’s Seventh Programme for research, technological development and demonstration and the Danish Agency for Science Technology and Innovation (Ministry of Science, Technology and Innovation)—Denmark. The BIO-C3 cruise on RV DANA in September 2015 was funded by the Danish Centre for Marine Research (grant 2015-04). REFERENCES Ambler , J. W. , Cloern , J. E. and Hutchinson , A. ( 1985 ) Seasonal cycles of zooplankton from San Francisco Bay . Hydrobiologia , 129 , 177 – 179 . Google Scholar CrossRef Search ADS Anraku , M. ( 1964 ) Influence of the Cape Cod Canal on the hydrography and on the copepods in Buzzards Bay and Cape Cod Bay, Massachusetts. II. Respiration and feeding . Limnol. Oceanogr. , 9 , 195 – 206 . 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Journal of Plankton ResearchOxford University Press

Published: May 3, 2018

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