KEYWORDS
salinity stress, phytoplankton resilience, lipids, monogalactosyldiacylglycerol, fatty acids, estuaries
1INTRODUCTION
Estuaries are highly dynamic systems, extremely important in providing a wealth of scientific information as they are exposed to a variety of stressors of natural (variable salinity, light attenuation, notable changes in nutrient availability and temperature) and anthropogenic origin (pollutants, various commercial activities in coastal areas, changes in water circulation due to construction, the effects of global warming, which are more easily felt in coastal areas than in the open oceans) (Miller et al., 2009; Sheela and Dhinagaran, 2023), They are also characterized by high productivity (Corell, 1978) and are often exploited as mariculture sites (Yang et al., 2017). In such environments, plankton develop mechanisms to acclimate or adapt to rapid environmental changes through physiological and/or community responses. Much of the productivity and microbiological diversity of estuaries is related to phytoplankton and its composition. Studies of phytoplankton communities in estuaries have shown that phytoplankton communities along the salinity gradient consist of marine phytoplankton at higher salinities, freshwater species at low salinity and species that develop at intermediate salinity (Lancelot and Muylaert, 2011). The osmotic shock caused by the increase in salinity influences the decline in abundance of freshwater phytoplankton downstream (Burić et al., 2007). The phytoplankton community is dependent on the flushing rate in the estuary (Lionard et al., 2008). Their studies in the Scheldt Estuary (Belgium) have shown that diatoms dominate the community at low flushing rates, while chlorophytes from the Scheldt are more important at high flushing rates. Diatoms have also been reported to develop in the lower parts of the Krka River Estuary, while the upper estuary due to the constant salinity changes short-lived nanoflagellates tend to develop (Burić et al., 2007).
Autotrophic plankton is the most important lipid producer in the seas (Gašparović et al., 2014). Lipid concentrations in seawater are relatively low, although they are involved in many important biological processes (Arts et al., 2001). Their quantity and quality depend on environmental factors and the stage in the life cycle of the primary producers (Zhukova and Aizdaicher, 2001). One of the most important functions of lipids is the formation of membrane lipid bilayers for cells and organelles. The adaptability and flexibility of the membrane structure imposed by the nature of environment are only possible with a broad spectrum of lipid mixtures (Dowhan et al., 2008). Increased synthesis of glycolipids by phytoplankton has been observed under conditions of P scarcity, high temperatures and high light intensities (Gašparović et al. 2013; Novak et al., 2019).
The thylakoid membranes of chloroplasts consist mainly of glycolipids, monogalactosyldiacylglycerols (MGDG), digalactosyldiacylglycerols and sulfoquinovosyldiacylglycerols , with a small proportion of phosphatidyldiacylglycerols (Douce and Joyard, 1990). They are highly unsaturated (Selstam, 1998). These membranes are crucial for plant cell metabolism (Douce and Joyard, 1990), reflecting the importance of their adaptability to environmental changes. The effect of different stressors, such as changes in temperature and salinity, drought, and exposure to pollutants, on thylakoid membrane lipid remodelling has often been studied in higher plants (e.g. Ristic and Cass, 1991; Stefanov et al., 1995; Zheng et al., 2011; Omoto et al., 2016), but rarely in autotrophic plankton population. The literature indicates that different photosynthetic organisms use different strategies to cope with stress in terms of the quantity and quality of thylakoid membrane lipids.
Phytoplankton in estuaries are exposed to constant stress due to salinity changes. To maintain membrane homeostasis, the composition of membrane lipids is expected to change. The scientific question we tried to answer is: Does the change in salinity lead to membrane lipid remodeling in the estuarine phytoplankton to acclimate/adapt to such changes? Since photosynthesis is one of the most sensitive cellular processes, we hypothesized that regulating the state of the thylakoid membrane would contribute to the maintenance of photosynthesis under osmotic stress and thus to cells survival. To address this hypothesis, we analyzed the lipid profiles of particles from the subtropical, eutrophic Wenchang River Estuary and the temperate, mesotrophic Krka River Estuary. We used thin–layer chromatography–flame ionization detection (TLC/FID) to investigate the changes in the composition of lipid classes. To explore the stress responses, in which fatty acids play an important role, the redistribution of fatty acids within a single lipid class, was investigated, using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI/MS/MS). In addition, phytoplankton pigments were analyzed to gain insight into the phytoplankton community responses within the salinity gradients along the estuaries. This allowed us to propose a mechanism for cell stress acclimation through thylakoid membrane remodeling.
2MATERIALS AND METHODS
2.1Study sites and sample collection
Two estuaries, the Krka River Estuary and the Wenchang River Estuary, were selected because of their multiple differences, including temperature and riverine nutrient loading. We only sampled surface water to avoid other possible influencing factors on lipid synthesis, e.g. attenuated light at different depths.
The Krka River is a karst river that forms a 25 km long salt wedge estuary spreading from the Skradinski Buk waterfalls to the Šibenik Channel (Figure1a). Because of the physical barrier of the Skradinski Buk, riverine water, rich in phytoplankton developing at the Visovac lake flows into the estuary by the waterfall contributing to the estuarine phytoplankton community (Cetinić et al 2006, Šupraha et al 2016). The main sources of nutrients in this estuary are the Krka River and numerous submarine groundwater discharges connected to the karst aquifer. The Krka River is the most pristine and organic matter–poor river with a dissolved organic carbon (DOC) of only 0.5 mg L-1 (Louis et al., 2009; Hao et al, 2021), while the DOC concentration in the Krka River Estuary averages 1 mg L-1 (Lechtenfeld et al., 2013). The Wenchang and Wenijao rivers flow into the Wenchang River Estuary (Figure 1b). Input from the rivers, groundwater discharge and aquaculture wastewater are the main sources of nutrients entering the Wenchang River Estuary (Liu et al., 2011). The DOC content in the Wenchang River Estuary reaches values of up to 3.8 mg L-1 (Hao et al., 2021) and even up to 20 mg L-1 in ponds (Herbeck et al., 2013), which indicates eutrophic character.