Authors: Lishani Wijewardene, Cátia Venâncio, Rui Ribeiro, Isabel Lopes
Categories: Research Article, Seawater intrusion, Climate change, Loss of genetic variability, Long-term exposure, Rotifera
Source: Environmental Science and Pollution Research International
Authors: Lishani Wijewardene, Cátia Venâncio, Rui Ribeiro, Isabel Lopes
Worldwide, many coastal freshwater ecosystems suffer from seawater intrusion. In addition to this stressor, it is likely that the biota inhabiting these ecosystems will also need to deal with climate change-related temperature fluctuations. The resilience of populations to long-term exposure to these stressors will depend on their genetic diversity, a key for their adaptation to changing environments. Accordingly, this study aimed to understand the long-term effects of salinity and temperature on the population density dynamics of the rotifer Brachionus calyciflorus by considering intra-specific variability. Six clonal lineages of B. calyciflorus, exhibiting differential lethal sensitivity (LC50,24 h) to salinity, were exposed for at least 34 days, to a control and to artificial seawater (at a conductivity corresponding to the LC70,24 h for the most tolerant clonal lineage = 9.89 mS/cm), under three 17, 20 (standard) and 23 °C. Long-term exposure to artificial seawater affected population densities, leading to the extirpation of some salinity-tolerant clonal lineages earlier than that of salinity-sensitive lineages. This inversion in short- and long-term sensitivity may suggest a higher susceptibility of populations when exposed to long periods of increased salinity. The negative effects caused by artificial seawater were enhanced at 17 °C and 23 °C, with an even earlier occurrence of extirpation of some clonal lineages, namely, two clonal lineages considered tolerant to artificial seawater. The results suggest the potential synergistic effects of the two abiotic stressors when combined. Overall, a lack of association between the clonal lineages’ short- and long-term sensitivity to salinity or their sensitivity to salinity under different temperature scenarios was observed. These results suggest an increased risk to the resilience of B. calyciflorus populations exposed to climate change-related scenarios of increased salinity and temperature fluctuations owing to an enhanced reduction in their genetic variability.
The online version contains supplementary material available at 10.1007/s11356-025-35995-3.
Coastal freshwater ecosystems are vulnerable to environmental disruptions caused by climate change. Sea level rise and warming processes (often correlated events) are two of the main challenges imposed on the biota inhabiting these ecosystems (IPCC 2021). Recurrent episodes of salinization are expected in coastal freshwater ecosystems through storm-driven overtopping of seawater (leading to temporary salinization events), especially during the winter months, and groundwater intrusion (leading to temporary salinization events), especially during prolonged summer droughts. (IPCC 2021). In addition, IPCC's current scenarios of temperature increase by the end of the century have provided fragments of evidence, with medium confidence (according to the level of confidence expressed in IPCC 2021), that they could rise by 3 °C or more (IPCC 2021). Considering the ongoing climatic changes, the likelihood of co-occurrence of these two factors is high; therefore, their combined ecological impact to freshwater biota must be assessed. Studies that relate salinity and temperature stress factors are limited, and this number decreases when considering population genetic variability (Jeppesen et al. 2020; Cunillera-Montcusí et al. 2022). Population genetic diversity is nevertheless crucial for ecosystem structure and function, as it bolsters the adaptive capacity of populations (Convention on Biological Diversity 2010). Exposure to environmental changes may impact the genetic pool of freshwater species (Frankham 2005; Hoban et al. 2021; Loria et al. 2022), leading to genetic erosion and increased susceptibility to future stressors (e.g., Armbruster and Reed 2005 and references therein; Nowak et al. 2009; Ribeiro and Lopes 2013; Švara et al. 2021). Despite its relevance, genetic diversity is often neglected when conducting ecological risk assessments of contaminants or when planning biodiversity protection legislation and frameworks (Breitholtz et al. 2006; Hoban et al. 2021). Recent studies have shown that integrating genetic variability to study the effects of environmental disturbances, such as salinity and temperature, may more accurately estimate the ecological risks of factor interactions for freshwater biota. For example, Venâncio et al. (2023) studied laboratory populations of Daphnia longispina (with short-term differential lethal sensitivity to salinity) and found that long-term exposure to seawater caused a faster extirpation of salinity-tolerant clonal lineages than of salinity-sensitive ones, indicating no association between short-term and long-term sensitivity to this stressor. Furthermore, authors investigated interactive effects and suggested that salinity and temperature acted synergistically, increasing the negative effects of salinity (loss of most clonal lineages) under different temperature regimes (i.e., at 17 and 23 °C). The results of this work constitute a major advance in understanding how interactions between salinity and temperature can reduce the probability of survival of a population when considering intraspecific variability. Recognizing whether these patterns of effects exist in other taxa belonging to the same functional group of daphnia (e.g., rotifers) may allow us to understand how the genetic variability of other primary consumer communities is modulated under increased salinity and thermal stress and how this can impact future resilience. Furthermore, this knowledge is of much relevance when estimating impacts of environmental stressors at the community level. It is acknowledged that the existence of functional redundancy in natural communities supports their ecological resilience and stability (Fonseca and Ganade 2001; Biggs et al. 2020). Though, if species with equivalent functions in a community exhibit similar sensitivities and patterns of response to the same environmental stressors, then redundancy may decrease significantly in the community impairing its ecological resistance.
Rotifera, together with Cladocera, are among the main groups that dominate zooplankton communities, with important roles as food web regulators, exhibiting similar functions (ecological redundancy) in natural communities (being filter feeders and primary consumers) (Castro et al. 2005; Phan et al. 2021; Thackeray and Beisner 2024). Therefore, a comparative assessment of their responses to environmental stressors is of much relevance, as if they are similar, a significant component of the ecological redundancy may be lost in zooplankton communities, compromising their persistence and subsequently the ecological equilibrium at the ecosystem level. Though, rotifers have unique characteristics related to habitat preferences (niche partitioning-based, among others, on temperature) and life history strategies, such as long-term reproductive strategies, which can be influenced by exposure to salinity (Sarma et al. 2006). These are coupled with temperature (Huey and Kingsolver 2019) and may induce distinct outcomes from those exhibited by other ectotherms in both the short and long term (e.g., daphnids; Venâncio et al. 2023). This study aimed to confirm the hypothesis that short-term survival mechanisms are distinct from long-term resilience mechanisms in rotifers facing environmental disturbances (i.e., there is no correlation between short-term tolerance and long-term resilience; Lopes et al. 2005). If this hypothesis is true, the long-term resilience mechanisms of rotifers exposed to salinity and temperature are constantly trimmed by natural selection and have low heritability, compromising population viability in stressed coastal freshwater ecosystems. Considering that an adequate extrapolation of the potential effects of the combination of increased salinity and temperature at the ecosystem level is extremely reductive if based on an exercise carried out for a single group of organisms (Venâncio et al. 2023), the present study aimed to understand the effect of freshwater salinization at different temperature levels on the population density dynamics of the rotifer Brachionus calyciflorus, considering intraspecific variability. To study this main objective, the following two hypotheses were i) prolonged exposure to increased salinity and temperature would lead to a faster extinction of local rotifer populations (simulated in the laboratory by testing different clonal lineages of the rotifer) compared to prolonged exposure to increased salinity (controlled temperature); and ii) in a genetically diverse population of rotifers, the most salinity-sensitive clonal lineages would be the first to disappear regardless of the temperature level, and therefore, the persistence of the most salinity-tolerant clonal lineages would sustain the resilience of populations in salinity-impacted locations as well as temperature changes. With the knowledge generated, we intended to infer the possible consequences of the probability of extinction of B. calyciflorus clonal lineages on the resilience of populations. By considering the intraspecific variability in the response to these two stressors, this knowledge can contribute to the development of more precise and comprehensive protective measures in future climate change-induced salinization and warming scenarios. Therefore, this study aims to grow knowledge on the study of Venâncio et al. (2023) regarding the resilience of primary consumer communities to freshwater salinization and temperature changes by characterizing the response of an ecologically redundant species, thereby enhancing knowledge about the response of primary consumer taxa to the studied stressors, enabling more accurate extrapolations for the community and ecosystem levels.
An artificial seawater stock solution was prepared by dissolving 33 g of artificial sea salt (Ocean Fish, Prodac International, Cittadella, Italy) in 1 L of ultrapure water (Milli-Q; Millipore, Burlington, MA, USA). The stock solution was then diluted with B. calyciflorus culture medium to obtain the salinity levels to be tested in toxicity assays (also simulating the occurrence of seawater dilution as it enters freshwater ecosystems). Artificial seawater was selected to perform this study over natural seawater to avoid the risk of using natural seawater contaminated with other pollutants, which would impair discriminating the toxicity induced by increased salinity or by unknown pollutants.
Brachionus calyciflorus calyciflorus Pallas, 1776 was selected as the model species of rotifers because it exhibits a parthenogenic reproductive strategy (allowing the maintenance, in the laboratory, of the exact same genotype over several generations) and is highly sensitive to salinity increases (Venâncio et al. 2019).
To establish cultures of different clonal lineages of B. calyciflorus, commercial cysts were acquired from RotoxKit F™ (MicroBio Tests Inc., Ghent, Belgium) and hatched following the standard procedure in RotoxKit F™ (MicroBioTests Inc., Ghent, Belgium). In brief, cysts were allowed to hatch at 23 °C for 24-h, at a constant light intensity of 3000–4000 lx in ASTM moderately hard synthetic freshwater medium (OECD 2004). Since only one organism hatches from each cyst, and cysts are the result of sexual reproduction, individuals from different cysts correspond to different genotypes, each one originating a clonal lineage. Sixteen clonal lineages were started from 16 cysts; each one was started with a single individual cyst, with less than 24-h old, that was then allowed to reproduce parthenogenetically (ensuring the maintenance of the clonal lineage for several generations). Each clonal lineage was cultured separately (at a maximum number of ten females/8 mL of medium) under controlled standard conditions of temperature (20 ± 1 °C) and photoperiod (16:8 h L:D) in reconstituted freshwater (ASTM medium), which was prepared according to the Rotoxkit F™ (MicroBioTests, Ghent, Belgium) procedure, and fed with the green microalgae Raphidocelis subcapitata at a concentration of 0.5 × 10^5^ cells/mL every day. The culture medium was renewed every other day. Each clonal lineage culture was checked daily to monitor for brood release, and for culture renewal, only neonates born from the 3rd or 4th asexual broods were used in the assays.
Since the sensitivity of the 16 rotifer clonal lineages to artificial seawater was unknown, firstly, there was the need to characterize their short-term lethal sensitivity to artificial seawater. For this, standard acute 24-h assays were performed by following the Acute RotoxKit F™ protocol (MicroBio Tests, 1998). From the cultures of the 16 clonal lineages, neonates from 3rd or 4th broods, less than 24-h old, were isolated and exposed to a range of concentrations of artificial seawater, obtained by dilution with ASTM medium of the stock solution (33 g/L of artificial sea salt). Conductivity values will, from here onwards, be used as a surrogate measure of the concentrations of salinity to make comparisons with the published literature. The tested salinity levels were as control (ASTM moderate hard water culture medium with a conductivity of ≈ 0.49 mS/cm), 3.50, 4.90, 6.86 and 9.60 mS/cm (Acute RotoxKit F™, MicroBioTests, Ghent, Belgium). Exposure was performed in 6-well plates, and each well was filled with 8 mL of the test solution. Five neonates from each clonal lineage were exposed per replicate, and three replicates were assembled per treatment and control. The assay was conducted at 20 ± 1 °C of temperature and photoperiod of 8 h L:D, with no medium renewal or food, as recommended by the Acute RotoxKit F™ protocol. At the end of the 24-h period, the number of immobile organisms (not exhibiting any movement for 15 s after gentle prodding) was recorded for each replicate. In Table S1 are described the LC50 and LC70 values obtained for all the clonal lineages of B. calyciflorus. From these, only six clonal lineages (representing the three lower and upper extremes of lethal sensitivity to artificial seawater, G, P, D, H, N, and F) were selected according to their estimated LC50 to increased levels of artificial seawater, to proceed with the study and perform the long-term assays described in the next section.
Six clonal lineages of B. calyciflorus, differing in their short-term sensitivity to lethal levels of artificial seawater (D, G, P – sensitive and N, F, H – tolerant; Table S1), were selected to conduct the long-term assays. To conduct the long-term assays, each clonal lineage was exposed to a specific artificial seawater conductivity across three (i) 17 ± 1 °C, (ii) 20 ± 1 °C, and (iii) 23 ± 1 °C. These temperatures were chosen based on the Sixth IPCC Assessment Report (2021) projections that foresee a global warming of 3.2 °C by the end of the century.
To initiate the assay, for each temperature and each clonal lineage of B. calyciflorus, five neonates less than 24-h old, from the 3rd to 5th broods, were assigned per replicate, which were filled with ASTM moderate hardwater medium. Assays were carried out in 6-well sterilized plastic plates, with each well corresponding to a replicate containing 8 mL of ASTM moderate hard water medium (with a conductivity of ≈ 0.48 mS/cm). Six replicates, in ASTM moderate hard water medium alone, were initially set for each temperature and clonal lineage of B. calyciflorus, and plates were maintained under a 8 h L:D photoperiod and to the temperature to which they were assigned as previously established. Organisms were fed daily with R. subcapitata (0.5 × 10^5^ cells/mL) until each population reached their carrying capacity (13 d for rotifers exposed at 17 °C, 10 d for rotifers exposed at 20 °C, and 8 d for rotifers exposed at 23 °C) (please see Figure S1). The daily counting of the total density of organisms in the plates allowed the determination of the carrying capacity, which was reached when, for a period of 3–4 days, the density counts varied within similar values. Populations that had already reached their carrying capacity were used to initiate the exposure experiments to ensure that there was no influence of other stressors, such as food limitation or competition (please see Figure S2).
After achieving the carrying capacity (being exposed in ASTM medium alone), populations of each replicate (each clonal lineage at each temperature) were assigned to each negative control (ASTM moderate hard water medium) and salt exposure which was the conductivity of artificial seawater that caused 70% mortality in the most tolerant clonal lineage (F) (LC70,24 h: 9.89 mS/cm; corresponding to 19% of seawater conductivity of ≈52 mS/cm). All controls and conductivity treatments were performed in triplicates. The photoperiod, feeding (provided to the organisms at each 24 h), and renewing practices were maintained as in laboratory cultures throughout the duration of the assay. The density of organisms was checked every 24 h until half of the clonal lineages (i.e., three clonal lineages) exposed to the artificial seawater treatments totally died (for all three temperatures, this time corresponded to 34 d), time at which the assay was considered finished. The exposure time causing 50% and 90% of mortality (LT50 and LT90), in each clonal lineage, was computed at 96-h of exposure (already considered as chronic exposure for rotifers; Preston et al. 2000) and at the end of the assay (corresponding to 624 h at 17 °C, 816 h at 20 °C, and 744 h at 23 °C; please see Results section for further details).
Conductivity, pH, and dissolved oxygen measurements were taken using scientific and technical equipment from Wissenschaftlich Technische Werkstätten (WTW, Weilheim, Germany), including the F330 conductivity meter, 330 pH meter, and OX330 oxygen meter. These measurements were recorded for B. calyciflorus in each treatment and for both new and old growth media during the renewal process.
Short-term lethal conductivities of artificial seawater causing 50% and 70% of mortality (LC50 and LC70, respectively) were computed for each of the 16 clonal lineages of B. calyciflorus using the PriProbit software (Sakuma 1998).
For the selected six clonal lineages, the lethal time causing 50 and 90% mortality (LT50 and LT90, respectively), after 96-h of exposure and after the full duration of each long-term assay, were computed with exponential, logistic and Gompertz models using STATISTICA 7.0 (StatSoft, Hamburg, Germany), being chosen the model leading to the smallest relative \documentclass[12pt]{minimal}
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Relative;spread=\frac{Upper;95{%;}confidence;limit-Lower;95%;confidence;limit}{{LT}{50} or {LT}{90}}