In estuaries, mixing of freshwater and seawater often creates the well-known salinity gradients, from pure freshwater near the head of the estuary to seawater near the mouth of the estuary. This salinity gradient exerts a non-negligible impact on the physiology of phytoplankton, such as decreasing photosynthesis and increasing respiration, and subsequently reducing growth (Rijstenbil et al., 1989a, b ; Lu and Zhang, 1999; D’ors et al., 2016), and even altering species communities (Licursi et al., 2006; Li et al., 2014; Wang et al., 2015b). Thus, the continuous salinity gradients in estuaries are often considered as one of the main drivers to shift the distributions of phytoplankton biomass and species compositions (Licursi et al., 2006; Muylaert et al., 2009). Usually, freshwater species of phytoplankton are replaced by marine ones when the salinity varies from 0.5 to 10 in estuaries (Huang et al., 2004; Domingues et al., 2010; Li et al., 2011a, 2014). According to the reports of Li et al. (2011b), succession of dominant species occurred from typical freshwater chlorophytes (e.g., Scenedesmus spp.) below salinity 5 to marine diatoms (e.g., Skeletonema costatum, Chaetoceros sp. and Coscinodiscus spp.) above salinity 10 in the Jiulong River Estuary. Similarly, the green algae and cryptophytes were also observed as dominant species in lower saline water in South African estuaries, dinoflagellates in marine-brackish water, whereas diatoms and cyanobacteria dominated in hypersaline conditions (Nche-Fambo et al., 2015). Such a species succession along the salinity gradient is often attributed to the fact that these phytoplankton species are stenohaline and suffer from the osmotic stress when they are exposed to changing salinity (Licursi et al., 2006; D'ors et al., 2016). In cyanobacteria Spirulina platensis, the salt stress has been detected to cause the decrease in Photosystem II (PSII) activity and increase in PSII susceptibility to photoinhibition (Lu and Zhang, 1999); while in diatoms, e.g., Skeletonema costatum and Ditylum brightwellii, this salt stress has been observed to induce the increase in cellular small amino acids pools and in turn to enhance cellular osmolity (Rijstenbil et al., 1989a, b). Bloom of phytoplankton has been reported to associate regularly with low salinity of the estuarine areas or coastal regions (Lim and Ogata, 2005). Such as the blooms of dinoflagelate Alexandrium minutum coincided with increasing rainfalls and freshwater runoffs in the Mediterranean Sea (Giacobbe et al., 1996). Similar findings were also reported about the blooms of diatom Pseudo-Nitzschia multiserie under lower salinity conditions (Doucette et al., 2008) and abundant diatoms Pseudo-Nitzschia spp. in coastal areas where salinity varied widely (Thessen et al., 2005). In estuaries, the tide-induced intrusion of open sea water usually extends to far upstream (Wang et al., 1986), which leads to an intensely turbulent diffusion at estuarine axis (Liu et al., 2008) and mixes the seawater-thrived phytoplankton up to freshwater (Li et al., 2011b). However, little has been documented about how these phytoplankton groups respond to such an acute salinity change, despite it is of great importance in understanding the causes for frequent phytoplankton blooms and species dynamics in estuaries.

The Jiulong River Estuary (Fig. 1) is strongly influenced by tidal changes. Therefore, drastic salinity changes prevail this estuary, with the salinity-decrease rate reached as high as 6.7 km^–1 at interface between seawater and freshwater (Liu et al., 2008). Additionally, rapid economic development along this estuary has accelerated its eutrophication process during the last decades (Yan et al., 2012; Wu et al., 2017), leading to the frequent occurrence of algal blooms (Cao et al., 2005; Tang et al., 2010; Chen et al., 2012); thus, more and more attentions have recently been paid to the ecological status of this estuary, including to characterize its hydrodynamics (Liu et al., 2008; Wang et al., 2013), investigate its chemical environments (Yan et al., 2012; Chen et al., 2013a, b; Cao et al., 2015; Wang et al., 2015a), depict its biological features (Liu et al., 2013; Zhang et al., 2013; Tian et al., 2014) and analyze the relationships between biotic and abiotic properties (Li et al., 2011a; Chen et al., 2012; Wang et al., 2015b). However, studies focused on the physiology of phytoplankton are scarce (Li et al., 2011b), which limits our full understanding of this estuarine ecosystem. In this paper, we therefore aimed to experimentally test the physiological responses of phytoplankton to the acute field salt stress and to particularly examine their acclimation processes in the Jiulong River Estuary. Experiments with natural phytoplankton assemblies were conducted to test the effects of acute salt stress on the photosynthetic effective quantum yield and carbon fixation.

From the open sea water going upstream to the tidal limit water of the Jiulong River Estuary, salinity decreased by 1.24 km^–1 from 30.4 (S1) to 7.52 (S5) and the euphotic zone depth decreased from 3.68 to 0.18 m (Table 1). The concentrations of dissolved inorganic nitrogen (DIN) increased from 38.6 to 145 μmol/L and silicate increased (SiO₃^2–) from 58.2 to 241 μmol/L; while that of phosphate (PO₄^3–, 2.74–3.48 μmol/L) showed a limited change (Table 1). Field Chl a content increased from 1.92 to 6.01 μg/L during the sampling period (Table 1).

Photosynthetic performances of the high-saline (salinity 30) cell assemblies changed greatly when being transferred to low-saline field water, with different extent depending on the degree that salinity changed (Fig. 2). For example, effective PSII quantum yield (ΔF/F_m′) sharply decreased from 0.67 to 0.14 within 5 min when being transferred to salinity 7.5 water (S5), followed by a marked increase to 0.45 in the following 40 min and then a gradual increase to 0.61 within 320 min, to the similar as control (Fig. 2a). Oppositely, non-photochemical quenching (NPQ) showed a sudden increase from 0 to 0.85 when being transferred to salinity 7.5 water, and decreased nearly to 0 within 70 min and stayed steady till the end of the experiments (Fig. 2b). When the high-saline cell assemblies were transferred to series intermediate salinity field water (salinity 13, 17 and 25), similar response pattern was observed, that is, decreased ΔF/F_m′ at first, then recovery leading to increased ΔF/F_m′ and decreased NPQ (Fig. 2). A similar response pattern also occurred when transferred low-saline (salinity 7.5) cell assemblies to series high-saline field waters (salinity 13, 17, 25 and 30), i.e., the quantum yield sharply decreased at first 2 min, then gradually increased and almost completely recovered within the following 200 min (Fig. 3a); while NPQ markedly increased at first then decreased to nearly 0 in the following 50 min and kept steady till the end of the experiments (Fig. 3b). The durations of the decrease of ΔF/F_m′ lasted 20 s after the acute salinity changes of 7.5 to 30 (Fig. 3a inset).

In outdoor conditions, phytoplankton assemblies showed similar photochemical performances as that in indoor conditions (Fig. 4). As solar PAR irradiation increased from dawn to noon (Fig. 4a), the ΔF/F_m′ of high-saline (salinity 30) cell assemblies decreased (Fig. 4b), and it then gradually increased up to initials in dusk. When the high-saline cell assemblies were exposed to lower-saline field water, the ΔF/F_m′ displayed a sudden decrease in morning, followed by a gradual increase to noon and finally up to the similar values as control in dusk (Fig. 4b). Consistently, the environmental stress, defined as: (Y_{Sal 30} – Y_{Sal x})/Y_{Sal 30} × 100%, displayed an opposite trend as ΔF/F_m′ and decreased throughout the day (Fig. 4c), for e.g. the stress degree decreased from 80% to 21% from dawn to dusk when the high-saline (salinity 30) cell assemblies were transferred to low-saline (salinity 7.5) field water. Particularly, the lowest values of ΔF/F_m′ in most salinity treatments occurred before 10:00 am but not 12:00 am when solar irradiation was highest (Fig. 4b), indicating the interactive light shock effects from dawn to noon upon the photosynthetic capacity.

Photosynthetic carbon fixation rate of high-saline cell assemblies reached 2.54 μg C (μg Chl a)^–1 h^–1 in salinity 28 field water; it sharply decreased by 41% after 20 min exposure to salinity 7.5 field water, and by 8.9% and 5.9% to intermediate salinity 15 and salinity 20 field water, respectively (Fig. 5). However, the carbon fixation rate under the salt stressful condition (i.e., high-saline cell assemblies were exposed to low-saline field water) markedly increased with increasing incubated duration, and showed even no significant differences (p>0.05) among the control (salinity 28), salinity 15 and salinity 20 treatment conditions after 200 min (Fig. 5).

Freshwater runoffs and tidal-induced exchanges with open sea water often drastically change the salinity along the estuaries, osmotically stressing phytoplankton therein. In the Jiulong River Estuary however, phytoplankton species are often plentiful (Li et al., 2011b; Huang et al., 2012) and even bloom (Li et al., 2011a). Such a contradiction of salt stress and abundant phytoplankton in this estuary spikes our interest to study the physiological responses of phytoplankton to the acute salinity changes. We found that the seawater-thrived phytoplankton assemblies from this estuary could recover within several hours from the field osmotic stresses on the base of chlorophyll fluorescence and carbon fixation, implying their fast acclimations to the acute salinity changes and subsequent blooms.

Apart from light limitation (Cloern, 1999), the salinity changes are usually over the burden of phytoplankton cells in estuaries, thus causing the great increase in respiration and decrease in photosynthesis and growth (Rijstenbil et al., 1989a, b; Lu and Zhang, 1999; D'ors et al., 2016), and even making the cells to die (Licursi et al., 2006). In this view of the point, estuarine environments are rough and not suitable for phytoplankton to grow well (Cloern, 1999) although the nutrients contents therein e.g. nitrogen (N) and phosphorus (P) are usually high due to the land-derived runoffs and mixing-caused relaxations from sediments (Li et al., 2011b, 2014; Yan et al., 2012). On the other hand, the excessive N loads often create a surplus supply of estuarine N, leading P to be relatively scarce and again negatively limiting the growth of phytoplankton, e.g., in the Jiulong River Estuary (Mo et al., 2013) or Pearl (Zhujiang) River Estuary (Yin et al., 2001; Yi et al., 2014). Moreover, osmotic stress induced by salinity changes has been shown to suppress the synthesis of e.g. D1 protein at both the transcriptional and translational levels (Allakhverdiev et al., 2002), which may again cause the decrease in photosynthetic activity of photosystem II (Figs 2-4) and carbon fixation (Fig. 5). In many estuaries however, high nutrients contents usually favor the growth of phytoplankton, which could overrun the adverse effects of salt stress or light limitation and make them to grow well and even bloom (Huang et al., 2004; Li et al., 2011a, 2014; Gao et al., 2018). According to Brand (1984), most phytoplankton species from the estuaries are more euryhaline than those from coastal or oceanic waters, which may make them to better tolerate the osmotic stress and thus lead them to be more abundant. What’s more, it is common that primary inhibition of photosynthetic activity by salt stress is followed by partial or full restoration, but the time required for recovery is species-specific and depends on the osmotic stress degree (Radchenko and Il’iash, 2006). For example, the osmotic stress was testified to stimulate the production of intracellular ROS, but it does not induce cellular toxicity before ROS formation exceeding antioxidant defense capability or disrupting redox signaling, and affecting cell functionality (Häubner et al., 2014). Our results showed that phytoplankton assemblies from the Jiulong River Estuary could rapidly (i.e., within several hours) recover from the acute osmotic stresses, under either indoor (Figs 2 and 3) or outdoor conditions (Fig. 4), which not only contributes to the explanations about the better adaptation of estuarine phytoplankton to the acute salinity changes but also implies a potential reason why phytoplankton often bloom in this estuary although the environments therein are rough for their growing if considering the drastic salinity changes.

Differential nutrients levels among the five sampling sites (Table 1) might temper our findings, although the acute osmotic stress was not covered by the nutrient changes (Figs 2-4). Moreover, the dominant species are Nitzschia longissima and Thalassiosira spp. after the 5-day-indoor cultivation of natural cell assemblies in f/2-enriched field water, and no significant difference was observed between indoor and outdoor species compositions (data not shown). It indicates the elimination of the effects of environmental changes from outdoor- to indoor- condition, as well as the elimination of the effects of zooplankton prey by pre-filtrating the sample through a 180 μm-pore mesh before collecting phytoplankton assemblies. Thus, our cell assemblies could still mimic the field condition, despite we indeed observed the species changes after inoculating the high-saline cell assemblies into f/2-enriched low-saline field water for another three weeks—just left the unknown pennates as dominating species (data not shown). It is consistent with the results of Hernando et al. (2015) who reported the big centric diatoms are replaced by small pennate ones after 8 d exposure to low salinity in a coastal area of Antarctica. In this study, we used solely larger cells (>20 µm) that took up ~40% of total field cell assemblies (Huang et al., 2012). For small cells however, they have high surface to volume ratios and are more vulnerable to environmental changes than large ones, no exception to the salt stress (Raven, 1998; Finkel et al., 2010). So, rapid recovery of phytoplankton from the acute osmotic stress would be tempered if considering the whole cell assemblies.

Phytoplankton assemblies from the Jiulong River Estuary proved strongly capable to recover from osmotic stress (Figs 2-5), which, together with a wide salinity range (Table 1), points to the mechanisms regulating intracellular salt pressure. Up to now, it still remains unclear that the mechanisms of the acute salt stress effects on photosynthetic performances of phytoplankton assemblies. In particular the metabolic processes in a certain algal species under the changed salinity are not known, although the release of low molecular weight compounds of e.g. amino acids and glucoses to environments has been proposed to be one of the main mechanisms of response to osmotic stress in diatoms (Rijstenbil et al., 1989a, b). Some authors considered that the osmotic stress mainly contribute adverse effects to the growth of phytoplankton (Lu and Zhang, 1999; Licursi et al., 2006; D'ors et al., 2016); while others suggested that abundant nutrients interact with high salt tolerance to make them to bloom frequently in estuaries although the salinity varies drastically (Lim and Ogata, 2005; Doucette et al., 2008). However, our results indicate that estuarine phytoplankton could rapidly recover from the osmotic stress; it to some extent reconcile the phenomenon that phytoplankton are abundant in estuaries in spite of the rough salinity environments.

We are grateful to the experimental helps of Ying Zheng, Wei Li and Guiyuan Yang and nutrient analysis of Yongming Huang.