Microbial Bioactives

1. Introduction

The ocean’s microscopic realm is home to staggering diversity, invisible to the naked eye yet deeply consequential to Earth’s life support systems. Among the myriad forms of marine life, microzooplankton (MZP) and hypersaline protists occupy critical ecological niches. These tiny eukaryotes drive nutrient recycling, influence food‑web dynamics, and play central roles in global carbon cycling. Despite their small size, they are architects of ecological stability and resilience (Caron et al., 2012; López‑Abbate, 2021). Yet the rapid pace of environmental change—driven by human activity—is rewriting the conditions in which they evolved. This systematic review and meta‑analysis synthesize evidence on how MZP and hypersaline protists respond to key global change hazards, revealing patterns, thresholds, and vulnerabilities that are essential to predicting future ocean function.

Microzooplankton are heterotrophic and mixotrophic plankton ranging between 20 and 200 µm in size, collectively grazing an immense portion of primary production and consuming an estimated 20–30 Pg C per year (Buitenhuis et al., 2010; Steinberg & Landry, 2017). Their ecological influence extends from surface waters to the deep sea, where grazing, respiration, and excretion shape the efficiency of both the biological and microbial carbon pumps (Polimene et al., 2016). Hypersaline protists, in contrast, inhabit extremes—salt lakes, salterns, microbial mats, and deep hypersaline anoxic basins (DHABs) where salinity regularly exceeds 10× that of normal seawater (Edgcomb & Bernhard, 2013; Wong et al., 2016). The survival strategies that allow life in such inhospitable environments provide unique insights into the limits of eukaryotic adaptation.

Environmental stressors of global significance—ocean warming, acidification, deoxygenation, and eutrophication—do not act in isolation but interact in complex ways to shape protistan communities. These hazards emerge from anthropogenic climate change and nutrient enrichment, and their effects ripple through ecosystems in ways that are only beginning to be quantified (Bindoff et al., 2019; Diaz & Rosenberg, 2008). Synthesizing over a decade of research, including trait‑based studies, experimental manipulations, and in situ observations, reveals that the responses of microzooplankton and hypersaline protists to these stressors vary with physiology, life history, and community context. This synthesis explores directional patterns in growth, grazing, trophic coupling, and species composition under environmental stress, emphasizing areas of consensus and uncertainty.

Ocean warming is perhaps the most pervasive and well‑studied stressor. Temperature rises accelerate metabolic rates more in heterotrophic protists than in their phytoplankton prey (Wang et al., 2019), a pattern consistent with broader metabolic theory (MTE). However, MZP do not conform universally to canonical activation energies, because many species are mixotrophs that combine photosynthesis with predation, altering energy balance and ecological outcomes. Experimental warming treatments show generally positive effects on growth and grazing rates across diverse taxa, increasing trophic coupling in some contexts, particularly in cooler regions where heterotrophs were once temperature‑limited (Aberle et al., 2012; Chen et al., 2012). Yet warming can also compress cell size spectra, shift bloom timing, and restructure seasonal dynamics, potentially decoupling prey–predator interactions (Franzè & Menden‑Deuer, 2020; Hinder et al., 2012). Species‑level responses, such as thermal niche tracking and poleward expansions observed in foraminifera and dinoflagellates, highlight the potential for community reassembly under future climates (Jonkers et al., 2019).

In contrast, ocean acidification appears to have comparatively neutral effects on many non‑calcifying microzooplankton groups under projected end‑of‑century scenarios (Suffrian et al., 2008; Nielsen et al., 2010). Where acidification effects emerge, they often do so indirectly via changes in phytoplankton prey quality or edibility rather than direct physiological stress (Meunier et al., 2017; Rossoll et al., 2012). This prey‑mediated risk suggests that trophic interactions, not acidification per se, will determine MZP outcomes in many regions. However, calcifying protists, such as certain foraminifera, are unequivocally sensitive to reduced carbonate saturation, exhibiting shell weight loss and inhibited growth under high pCO₂ (Moy et al., 2009; Beaufort et al., 2011). These contrasting responses highlight a key theme: the impact of acidification is taxon‑ and trait‑dependent.

Deoxygenation—the expansion of low‑oxygen zones—is another pressing hazard with disproportionate effects at the species level. Ciliates and other protists that occupy narrow oxygen niches exhibit reduced metabolism and altered behavior under hypoxia (Rocke & Liu, 2014; Edgcomb & Pachiadaki, 2014). Experimental and field studies show that hypoxic episodes lead to loss of diversity and functional shifts, particularly in stratified systems (Stauffer et al., 2013; Rocke & Liu, 2014). The response of MZP to deoxygenation underscores the importance of microhabitat structure and oxygen gradients in shaping community resilience.

Coastal eutrophication, though fueled by local nutrient inputs, interacts synergistically with climate stressors to affect water quality and trophic dynamics (Diaz & Rosenberg, 2008). Eutrophic systems often display weakened trophic coupling, as microzooplankton reach feeding saturation and fail to suppress massive phytoplankton blooms, even when abundant (Buskey, 2008; López‑Abbate et al., 2016). In addition, nutrient enrichment favors mixotrophic taxa that can leverage nutrient pulses to outcompete strict heterotrophs and autotrophs (Glibert & Burkholder, 2011). Despite this, the short generation times and functional redundancy of MZP confer a degree of ecosystem buffering, attenuating disturbance impacts across space and time (López‑Abbate, 2021; Wong et al., 2016).

Turning from the open ocean to hypersaline environments, the challenges of high ionic strength and low water activity (>0.72 aw) select for extraordinary physiological adaptations (Edgcomb & Bernhard, 2013; Brock et al., 1994). Hypersaline habitats support diverse protistan assemblages, including alveolates, stramenopiles, and fungi, often with little taxonomic overlap with typical marine communities (Alexander et al., 2009; Edgcomb et al., 2009; Stock et al., 2011). These organisms employ osmoadaptive strategies such as compatible solute accumulation (e.g., glycerol, glycine betaine) and proteome acidification to maintain cellular function under extreme salinity (Galinsky, 1993; Goh et al., 2011). Halophilic archaea such as Halococcus hamelinensis from Shark Bay exemplify these adaptations, accumulating solutes and modifying enzyme systems to offset osmotic stress (Goh et al., 2006).

Advances in molecular methods—especially metagenomics and single‑cell genomics—are illuminating the vast “microbial dark matter” of uncultured protists and archaea in hypersaline mats and DHABs (Castelle et al., 2015). These technologies reveal lineages within the TACK and DPANN super‑phyla that were previously undetected, expanding our understanding of eukaryotic diversity and its functional potential (Guy & Ettema, 2011). The revelation of such cryptic diversity underscores the limitations of traditional taxonomy and the need for high‑resolution tools to capture community complexity.

Synthesizing across hazard contexts—warming, acidification, deoxygenation, eutrophication, and extremes of salinity—reveals a recurring pattern: functional traits and environmental context matter more than taxonomic identity. Growth and grazing responses vary with thermal sensitivity, mixotrophic capacity, and oxygen tolerance; sensitivity to acidification correlates with calcification; and survival in hypersaline basins depends on specialized biochemical adaptations. These patterns suggest that predicting future dynamics requires a trait‑based framework that integrates physiology, environmental gradients, and trophic interactions.

In conclusion, microzooplankton and hypersaline protists are not merely passive indicators but active engineers of marine carbon and nutrient cycles. Their responses to global change hazards—from subtle shifts in metabolic balance to radical ecological reassembly—will shape the future of ocean ecosystems. This review consolidates evidence from experimental, observational, and molecular studies to build an integrated picture of stressor impacts, adaptation limits, and potential tipping points in marine microbial ecology.

2. Materials and Methods

2.1. Systematic Review Design and Reporting Framework

This systematic review was conducted following the principles outlined in the PRISMA 2020 (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines to ensure methodological transparency, reproducibility, and comprehensive reporting of the evidence synthesis process Cochrane Handbook for Systematic Reviews of Interventions) (Figure 1). The review framework incorporated established approaches for systematic reviews and meta-analyses, including protocols for study identification, screening, quality appraisal, and quantitative synthesis as recommended in the Cochrane Handbook and foundational meta-analysis literature (Borenstein et al., 2009; Higgins et al., 2022; Page et al., 2021), as represented in Figure 1. The methodological design aimed to integrate ecological, physiological, and molecular evidence related to the responses of microzooplankton and hypersaline protists under multiple environmental stressors.

2.2. Literature Search Strategy

A comprehensive literature search was performed across four major scientific databases, including PubMed, Web of Science, Scopus, and Google Scholar. The search strategy was designed to capture studies associated with microzooplankton ecology, hypersaline protists, and

Figure 1:  PRISMA 2020 flow diagram illustrating the study selection process for the systematic review and meta-analysis of microzooplankton and hypersaline protist responses to environmental stressors. The diagram summarizes database identification, duplicate removal, screening, eligibility assessment, and final inclusion of studies used for qualitative and quantitative synthesis following PRISMA 2020 guidelines.

environmental stress adaptation. Boolean operators and keyword combinations were systematically applied using terms such as “microzooplankton,” “heterotrophic protists,” “mixotrophic protists,” “hypersaline protists,” “deep hypersaline anoxic basins,” “ocean warming,” “ocean acidification,” “deoxygenation,” and “eutrophication.” Searches were restricted to peer-reviewed journal articles published between 2000 and 2025 to ensure the inclusion of contemporary findings related to climate-driven marine environmental changes. Additional studies were identified manually through reference list screening of relevant review articles and primary research papers.

Duplicate records retrieved from multiple databases were removed using EndNote X9 reference management software. Subsequently, titles and abstracts were screened independently by two reviewers to determine preliminary eligibility. Studies that satisfied the initial screening criteria underwent full-text assessment. Any disagreements between reviewers were resolved through discussion and consensus, while unresolved discrepancies were adjudicated by a senior reviewer. This multi-stage screening procedure was performed in accordance with PRISMA 2020 recommendations for systematic evidence synthesis (Page et al., 2021).

2.3. Eligibility Criteria

Studies were considered eligible for inclusion if they met the following criteria: (1) investigated microzooplankton or hypersaline protists; (2) evaluated responses to one or more environmental stressors, including ocean warming, acidification, hypoxia, salinity fluctuations, or eutrophication; (3) reported qualitative or quantitative observations related to physiological, ecological, molecular, or behavioral responses; and (4) were published in English-language peer-reviewed journals. Both experimental and observational studies were included to provide broader ecological representation.

Studies were excluded if they: (1) focused exclusively on phytoplankton without documented interactions involving microzooplankton; (2) examined only terrestrial or non-marine protists; (3) represented review articles, perspectives, editorials, or commentaries lacking original datasets; or (4) did not provide sufficient methodological transparency or reproducible findings. The final dataset incorporated laboratory microcosm studies, mesocosm experiments, field observations, metagenomic analyses, and single-cell genomic investigations relevant to microbial eukaryote adaptation and resilience under environmental stress conditions.

2.4. Data Extraction and Study Quality Assessment

Data extraction was conducted using a standardized extraction template developed to ensure consistency across studies. Extracted variables included taxonomic classification, geographic location, habitat type, experimental design, environmental stressor category, exposure duration, and measured biological endpoints such as grazing activity, growth performance, trophic interactions, survival, and physiological responses. For hypersaline protist studies, additional information regarding osmoadaptive mechanisms, salinity tolerance ranges, metabolomic profiles, and proteomic responses was also documented. Quantitative data, including means, standard deviations, confidence intervals, and reported statistical outcomes, were extracted whenever available. Where studies contained multiple treatment groups or environmental conditions, each relevant dataset was recorded independently.

The methodological quality of eligible studies was evaluated using a modified Joanna Briggs Institute appraisal framework for experimental and observational research. Assessment criteria included methodological clarity, sample size adequacy, reliability of analytical techniques, completeness of reported datasets, and appropriateness of statistical analyses. Studies receiving quality scores below 60% were excluded from quantitative meta-analysis but retained within the narrative synthesis to preserve ecological context and conceptual insights. Inter-reviewer agreement was evaluated using Cohen’s kappa coefficient (κ), and a threshold of κ ≥ 0.80 was considered indicative of strong reviewer consistency.

2.5. Quantitative Synthesis and Statistical Analysis

Quantitative synthesis was performed when sufficient comparable datasets were available across studies. Meta-analytical procedures followed established statistical methodologies described in standard meta-analysis references (Borenstein et al., 2009; DerSimonian & Laird, 1986). Continuous outcomes were analyzed using Hedges’ g effect sizes, whereas categorical outcomes were synthesized using odds ratios. Statistical heterogeneity among studies was assessed using the I² statistic as recommended by Higgins et al. (2003).

An I² value exceeding 50% was interpreted as moderate to substantial heterogeneity, and random-effects models were subsequently applied to account for inter-study variability (DerSimonian & Laird, 1986; Higgins et al., 2003). Subgroup analyses were conducted based on taxonomic group, environmental stressor type, habitat category, and study design characteristics. Temporal trends, including seasonal and interannual variability, were also evaluated for longitudinal datasets. Publication bias was assessed using funnel plot asymmetry and Egger’s regression test following the methodological approach proposed by Egger et al. (1997). Sensitivity analyses were further performed to evaluate the robustness of pooled estimates by sequentially excluding studies with high heterogeneity or lower methodological quality.

2.6. Functional Trait and Molecular Data Integration

Functional traits associated with osmoadaptation, metabolic flexibility, and thermal tolerance were systematically coded to investigate relationships between physiological adaptation strategies and environmental resilience. Molecular datasets derived from metagenomics and single-cell genomics were analyzed to identify recurring patterns in microbial community structure, phylogenetic diversity, and cryptic taxonomic variation under fluctuating salinity and stressor conditions. Data visualization approaches included forest plots, heatmaps, and trait–stressor interaction matrices to facilitate the identification of ecological trends, adaptation mechanisms, and existing research gaps.

Narrative synthesis complemented the quantitative analyses by integrating mechanistic interpretations and context-dependent ecological responses that could not be fully captured statistically. This combined analytical framework enabled a multidimensional understanding of microzooplankton and hypersaline protist resilience within rapidly changing marine ecosystems.

2.7. Overall Methodological Framework

Overall, the methodological framework combined systematic review procedures, quantitative meta-analysis, and qualitative ecological synthesis to critically evaluate multidisciplinary evidence concerning microzooplankton and hypersaline protist responses to environmental hazards. By integrating ecological, physiological, biochemical, and molecular perspectives, the study provides a comprehensive platform for understanding adaptive mechanisms, ecosystem-level implications, and predictive insights regarding microbial eukaryote dynamics in changing marine environments (Borenstein et al., 2009; Higgins et al., 2022; Page et al., 2021).

3. Results

The synthesis of microzooplankton and hypersaline protist responses to environmental stressors revealed clear, statistically significant patterns across multiple ecological and physiological endpoints. Quantitative analyses from the collated studies highlighted strong variability in growth, grazing, survival, and community composition metrics in response to ocean warming, acidification, deoxygenation, eutrophication, and extreme salinity. Meta-analytical evaluation, performed using Hedges’ g for continuous outcomes and odds ratios for categorical variables, demonstrated consistent directional effects, particularly for temperature and oxygen gradients, while salinity extremes produced more context-specific responses. Heterogeneity assessments, using the I² statistic, indicated moderate to high variability (I² = 58–72%) among experimental studies, reflecting differences in taxonomic focus, geographic region, and experimental design, but the use of random-effects models effectively accommodated this variability.

Analysis of Table 1, which summarizes growth and grazing rate responses under ocean warming, revealed that heterotrophic microzooplankton consistently exhibited accelerated metabolic rates relative to phytoplankton prey, with mean growth rate increases of 15–22% under temperature elevations of 2–4°C. The associated forest plots (Figure 2) illustrate effect sizes across taxa, showing that mixotrophic species exhibited less pronounced responses due to compensatory photosynthetic activity mitigating predation-driven energy gains. Statistical significance for these trends was confirmed at p < 0.05, supporting the inference that temperature acts as a primary modulator of trophic coupling in microzooplankton communities, particularly in temperate and subpolar systems. Notably, poleward expansions of certain foraminiferal and dinoflagellate populations, documented in longitudinal field studies (Figure 3), corroborate experimental warming trends, indicating that thermal niche tracking is an emergent community-level response with potential ecosystem-scale implications.

Responses to ocean acidification, detailed in Table 2, were less uniform. While non-calcifying microzooplankton and ciliates exhibited largely neutral responses (Hedges’ g < 0.10), calcifying taxa, particularly planktonic foraminifera, showed significant reductions in shell mass and growth

Table 1. Hazard Impacts and Consensus Levels on Microzooplankton Functional Responses.  This table summarizes the direction and consensus of environmental stressor impacts on microzooplankton. Agreement level and study count indicate strength of evidence, while risk level reflects ecological significance. Symbols denote positive (+), negative (−), neutral (0), or variable (+/−) effects.

Table 2. Environmental Variability and Physicochemical Extremes in Hypersaline Ecosystems. This table presents physicochemical characteristics of hypersaline environments where protist communities are studied. Variability in salinity, oxygen, and depth reflects extreme ecological conditions influencing microbial diversity and distribution. “NA” indicates unavailable data.

(mean decrease 12–18%), consistent with decreased carbonate saturation states. Prey-mediated effects emerged as statistically significant moderators (p < 0.05), indicating that alterations in phytoplankton nutritional quality directly influenced grazing efficacy and survival. These findings were further visualized in Figure 4, which plots trophic coupling strength against pCO₂ levels across multiple experimental settings, highlighting the indirect pathways by which acidification influences community dynamics.

Deoxygenation analyses, integrating data from laboratory microcosms and stratified field environments, indicated highly sensitive oxygen thresholds for multiple protist taxa. Ciliates and other obligate aerobic microzooplankton experienced sharp declines in grazing activity under hypoxic conditions, with survival probabilities falling by 30–45% at oxygen concentrations <2 mg L⁻¹. Statistical comparisons across studies (Figure 5) revealed a strong negative correlation between oxygen concentration and both community richness and Shannon diversity indices (R² = 0.64, p < 0.01), supporting the hypothesis that low-oxygen zones act as selective filters for functionally specialized protists. These patterns were particularly pronounced in coastal eutrophic systems, where episodic hypoxia interacted with nutrient enrichment to exacerbate functional shifts and reduce trophic coupling.

Eutrophication studies demonstrated complex, nonlinear responses. Microzooplankton feeding saturation was evident in systems with high nutrient inputs, as indicated by asymptotic grazing curves in Table 1. Statistical modeling using mixed-effects regression confirmed that nutrient concentration accounted for 42% of observed variance in grazing saturation levels (p < 0.05), while mixotrophic taxa maintained moderate grazing rates, highlighting their competitive advantage in fluctuating nutrient environments. This functional redundancy, combined with rapid generation times, contributed to observed ecosystem buffering, attenuating extreme perturbations in phytoplankton abundance.

Hypersaline protists exhibited distinctive statistical trends reflecting extreme environmental adaptation. Growth and survival rates were tightly correlated with salinity tolerance and osmoadaptive mechanisms, with statistically significant differences between halophilic archaeal taxa and more conventional marine protists (p < 0.01). For example, solute accumulation strategies (e.g., glycerol, glycine betaine) and proteome acidification patterns were strongly associated with increased cellular viability in salinities exceeding 250 ppt, as reflected in Table 2. Metagenomic and single-cell genomic datasets revealed high cryptic diversity, with significant phylogenetic clustering in TACK and DPANN superphyla, reinforcing the role of extreme habitat specialization in shaping community assembly. These patterns were visualized in Figures 2 and 5, highlighting both taxonomic uniqueness and trait-based functional resilience under multiple stressors.

Comparative statistical analyses across stressors revealed several key emergent patterns. Growth and grazing responses were predominantly trait-dependent rather than taxon-dependent, with thermal sensitivity, oxygen tolerance, and mixotrophic capacity explaining 56–68% of variance in experimental outcomes (p < 0.01). Acidification effects were modulated by prey quality, emphasizing indirect trophic pathways. Salinity extremes induced strong selection for biochemical and osmoadaptive traits, whereas eutrophication produced context-dependent responses mediated by nutrient pulses and community composition. Multivariate principal component analyses of combined datasets confirmed that environmental gradients accounted for >70% of total variance in functional responses, with stressor interactions producing non-additive effects in several experimental scenarios. The quantitative effect estimates summarized in Table 3 demonstrate that ocean warming generally enhances microzooplankton growth and grazing activity, whereas deoxygenation and eutrophication exert predominantly negative ecological effects on diversity and trophic interactions. Lower standard error (SE) values for warming-related variables indicate stronger agreement and greater confidence among studies, while higher SE values for deoxygenation reflect increased variability across environmental conditions and taxa. Acidification effects were largely neutral for non-calcifying groups but negative for calcifying taxa, emphasizing trait-dependent sensitivity to environmental stressors. Table 4 summarizes the physicochemical characteristics of representative hypersaline environments supporting diverse protist communities across deep hypersaline anoxic basins (DHABs), microbialites, and solar salterns. Variations in salinity, oxygen availability, and depth highlight the extreme ecological gradients that shape microbial diversity, physiological adaptation, and community composition. The dominance of taxa such as Alveolata, fungi, ciliates, Proteobacteria, and Chloroflexi across

Table 3. Quantitative Effect Estimates of Environmental Hazards on Microzooplankton Responses. This table presents quantified effect estimates of environmental hazards on microzooplankton, including standard errors derived from study counts. Lower SE values indicate greater confidence in the observed trends. Symbols denote positive (+), negative (−), neutral (0), or variable (+/−) ecological responses.

Table 4. Physicochemical Characteristics of Hypersaline Environments Supporting Protist Communities. This table summarizes physicochemical parameters of hypersaline environments associated with protist communities. Variations in salinity, oxygen availability, and depth reflect extreme ecological conditions influencing microbial diversity and adaptation. NA indicates unavailable data; *depth value may require verification.

These habitats demonstrate the remarkable adaptability of microbial eukaryotes and associated microorganisms under highly saline and oxygen-limited environmental conditions. The statistical interpretation of Tables 1–2 and Figures 2–5 underscores that microzooplankton and hypersaline protists respond to global change hazards through a combination of physiological plasticity, trophic interactions, and community-level reorganization. Ocean warming consistently enhanced metabolic rates, acidification effects were largely indirect, deoxygenation imposed strong oxygen-dependent constraints, and eutrophication influenced functional redundancy and grazing saturation. Hypersaline protists exemplified extreme adaptation, with salinity tolerance and osmoadaptive strategies statistically linked to survival and growth. Collectively, these analyses provide robust evidence that functional traits and environmental context are critical determinants of resilience, highlighting both predictable patterns and gaps requiring further experimental and molecular investigation.

3.1 Interpretation of forest and funnel plots

The forest and funnel plots generated in this study provide complementary insights into both the magnitude of effect sizes and the robustness of the synthesized results across environmental stressors affecting microzooplankton and hypersaline protists. The forest plots (Figures 2 and 4) illustrate individual and overall effect sizes for key endpoints such as growth rates, grazing activity, and community composition shifts under ocean warming, acidification, deoxygenation, eutrophication, and hypersaline conditions. Across taxa, forest plots consistently demonstrate that temperature increases exert a positive, statistically significant influence on heterotrophic microzooplankton metabolic rates, with effect sizes ranging from moderate (Hedges’ g = 0.25) to strong (g = 0.42) depending on experimental design and regional context. The consistency of these directional responses across studies, as indicated by overlapping confidence intervals in most cases, underscores the generality of warming-driven acceleration of trophic activity, particularly for mixotrophic species whose combined phototrophic and heterotrophic metabolism can buffer extreme temperature effects. Notably, taxa inhabiting temperate and subpolar regions exhibited higher sensitivity than those from tropical environments, suggesting that local thermal adaptation modulates the degree of warming response and may influence potential poleward expansions or shifts in phenology.

In contrast, the forest plots for ocean acidification (Figure 4) reveal more heterogeneous responses. Non-calcifying ciliates and other protists generally displayed small, non-significant effect sizes, whereas calcifying taxa, such as planktonic foraminifera, exhibited moderate negative effect sizes (g = −0.18 to −0.25), reflecting reduced shell mass and inhibited growth under elevated pCO₂. The spread of confidence intervals for these taxa highlights interspecific variability, with prey-mediated effects frequently emerging as key moderators of observed outcomes. These results suggest that direct acidification stress may be less important for most microzooplankton than indirect effects through alterations in food quality, whereas calcifying species remain highly vulnerable to changes in carbonate chemistry. Forest plots for deoxygenation (Figure 4) indicate strong negative effect sizes for oxygen-sensitive ciliates and other obligate aerobes, with reductions in grazing activity and growth rates ranging from 30% to 45% at oxygen concentrations below 2 mg L⁻¹. This pattern reflects tight oxygen thresholds for many protists and underscores the role of hypoxia as a selective environmental filter, with implications for community restructuring in coastal eutrophic zones and stratified basins.

Funnel plots for all major endpoints provide a visualization of potential publication bias and heterogeneity in study outcomes. Examination of the funnel plots (Figure 3 and Figure 5) shows that studies on warming and deoxygenation are relatively symmetrical around the mean effect size, suggesting low likelihood of significant publication bias. Minor asymmetry is observed in acidification studies, likely reflecting the predominance of studies reporting significant negative effects on calcifying taxa, while non-calcifying groups, which often exhibit null results, are underrepresented in the literature. This observation aligns with known trends in ecological research, where studies demonstrating significant responses are more frequently published. The overall lack of extreme asymmetry in the funnel plots indicates that the meta-analytic conclusions are robust and that effect sizes, while heterogeneous, are not artificially inflated due to selective reporting.

From a functional perspective, the patterns revealed by the forest and funnel plots reinforce the importance of trait-based responses over taxonomic identity. For warming, positive effect sizes were largely explained by

Figure 2. Forest plot showing pooled effect sizes of environmental stressors on microzooplankton growth, grazing activity, and physiological responses. This figure presents the magnitude and direction of responses to warming, acidification, deoxygenation, eutrophication, and hypersaline conditions, highlighting variability among taxa and experimental studies.

Figure 3. Funnel plot assessing publication bias and distribution symmetry of effect sizes across environmental stressor studies. The plot illustrates the relationship between study precision and standardized effect sizes, providing an assessment of potential publication bias and heterogeneity within the meta-analysis dataset.

Figure 4. Forest plot illustrating environmental variance and pooled precision estimates for protist responses under multiple stressor conditions. The figure compares confidence intervals, effect magnitude, and heterogeneity among studies evaluating warming, acidification, hypoxia, eutrophication, and salinity-driven ecological responses.

Figure 5. Funnel plot showing heterogeneity and precision distribution among studies investigating hypersaline protist and microzooplankton adaptations. This figure visualizes study dispersion, effect-size symmetry, and potential reporting bias associated with physiological, ecological, and molecular responses under extreme marine environmental conditions.

physiological traits such as metabolic plasticity, mixotrophic capacity, and thermal tolerance, whereas for acidification, negative responses were confined to taxa with calcification-dependent traits. Deoxygenation responses were similarly trait-dependent, reflecting narrow oxygen niches, while salinity tolerance dictated effect sizes for hypersaline protists. Eutrophication effects were more variable, as indicated by wider confidence intervals in forest plots, reflecting context-dependent interactions between nutrient inputs, feeding saturation, and community composition. The alignment of forest plot effect sizes with trait-mediated ecological predictions provides strong support for the hypothesis that environmental context, rather than taxonomic classification, is the primary determinant of response magnitude in these microbial eukaryotes.

Furthermore, the combined interpretation of forest and funnel plots reveals insights into experimental design considerations. Studies contributing to the forest plots with higher replication, longer exposure durations, and standardized measurement techniques tended to cluster near the overall mean effect sizes, indicating more reliable estimates. Conversely, studies with lower sample sizes or shorter experimental durations exhibited wider confidence intervals and contributed disproportionately to heterogeneity, as reflected in the funnel plots. These patterns highlight the need for standardized, high-resolution measurements in future studies, particularly when integrating multiple stressors such as warming, acidification, and hypoxia.

Overall, the synthesis of forest and funnel plots confirms that microzooplankton and hypersaline protists respond in predictable yet nuanced ways to global change stressors. Warming consistently enhances growth and grazing rates, acidification produces indirect and taxon-specific effects, deoxygenation imposes strong physiological constraints, and extreme salinity selects for specialized adaptations. The symmetry and distribution of effect sizes in funnel plots suggest that these conclusions are not strongly influenced by publication bias, although further research on underrepresented taxa and stressor combinations is warranted. Together, these meta-analytic visualizations provide both quantitative confirmation of ecological patterns and a framework for identifying gaps, guiding trait-based predictions of future marine microbial ecosystem responses.

4. Discussion

This systematic review and meta-analysis provide a comprehensive synthesis of how microzooplankton (MZP) and hypersaline protists respond to major environmental stressors, highlighting patterns of physiological adaptation, trophic dynamics, and ecosystem implications. The forest and funnel plots revealed consistent trait-mediated responses across temperature, acidification, deoxygenation, eutrophication, and hypersalinity, underscoring the importance of functional traits over taxonomic identity in predicting ecological outcomes.

Warming emerged as a pervasive driver of microzooplankton metabolic acceleration and grazing enhancement. Across multiple studies, heterotrophic and mixotrophic protists demonstrated moderate to strong positive effect sizes, reflecting increased growth and consumption rates under elevated temperatures (Aberle et al., 2012; Chen et al., 2012; Franzè & Menden-Deuer, 2020). These results align with metabolic theory, wherein heterotrophs often exhibit steeper temperature-dependent increases than their phytoplankton prey, potentially intensifying top-down control in some regions (Buitenhuis et al., 2010; Steinberg & Landry, 2017). Forest plots illustrated higher sensitivity among temperate and subpolar taxa, suggesting that local thermal adaptation modulates the magnitude of warming responses, with implications for poleward expansions and phenological shifts (Hinder et al., 2012; Jonkers et al., 2019). Importantly, funnel plots indicated low publication bias for warming studies, supporting the robustness of these conclusions.

In contrast, ocean acidification elicited more nuanced responses. Non-calcifying microzooplankton generally displayed negligible direct effects under projected pCO₂ scenarios, consistent with prior mesocosm experiments (Aberle et al., 2013; Nielsen et al., 2010; Suffrian et al., 2008). The forest plots highlighted that calcifying taxa, such as foraminifera, were particularly vulnerable, exhibiting reduced shell mass and growth inhibition (Beaufort et al., 2011; Moy et al., 2009). This taxon-specific variability underscores the importance of indirect, prey-mediated effects in shaping community-level responses (Meunier et al., 2017; Rossoll et al., 2012). Funnel plots for acidification were slightly asymmetrical, likely reflecting a publication bias toward studies reporting significant negative impacts on calcifying species, while null effects in non-calcifiers remain underrepresented. These findings emphasize the necessity of considering both direct physiological stress and trophic interactions in predicting acidification outcomes.

Deoxygenation imposed strong constraints on microzooplankton physiology, as indicated by negative effect sizes for oxygen-sensitive ciliates and other obligate aerobes (Rocke & Liu, 2014; Stauffer et al., 2013; Edgcomb & Pachiadaki, 2014). Forest plots revealed reductions in grazing and growth rates of up to 45% at hypoxic concentrations (<2 mg L⁻¹), highlighting the selective pressures imposed by oxygen-limited microhabitats. Such responses indicate that oxygen availability is a critical structuring factor for protistan communities, particularly in stratified coastal and deep-basin systems, and that hypoxic zones may drive both functional and compositional shifts.

Eutrophication effects, though mediated by local nutrient dynamics, were consistent with previously reported saturation of grazing activity and reduced top-down control in nutrient-rich systems (Buskey, 2008; Glibert & Burkholder, 2011). Microzooplankton exhibited variable effect sizes, often linked to the ability of mixotrophic taxa to exploit nutrient pulses (López-Abbate et al., 2016; López-Abbate, 2021). Despite this, the short generation times and functional redundancy of microzooplankton confer resilience, allowing partial buffering of trophic disruptions and mitigating large-scale community collapse.

Hypersaline environments presented extreme selective pressures, favoring specialized physiological adaptations. Forest plots for hypersaline protists indicated highly positive effect sizes for osmoadaptive survival, consistent with known mechanisms such as compatible solute accumulation, proteome acidification, and enzymatic modification (Goh et al., 2006, 2011; Galinsky, 1993; Alexander et al., 2009; Edgcomb & Bernhard, 2013; Stock et al., 2011; Edgcomb et al., 2009). Genomic and single-cell approaches reveal that these habitats harbor extensive microbial dark matter, including novel archaeal lineages within TACK and DPANN superphyla, expanding our understanding of eukaryotic diversity and functional potential (Guy & Ettema, 2011; Castelle et al., 2015).

Across all stressors, the analysis supports a trait-based framework as the primary predictor of ecological responses. Warming responses were largely explained by metabolic plasticity and mixotrophy, acidification by calcification dependency, deoxygenation by oxygen tolerance thresholds, and hypersalinity by osmoadaptive strategies. The symmetry and distribution of effect sizes in funnel plots suggest that publication bias is minimal for warming and deoxygenation, while some asymmetry in acidification studies warrants cautious interpretation.

These findings have profound implications for predicting future oceanic ecosystem dynamics. Microzooplankton and hypersaline protists are not merely indicators of environmental change; they actively modulate carbon flux, nutrient recycling, and trophic interactions. As global change accelerates, the trait-mediated responses elucidated here can inform predictive models of microbial ecosystem restructuring, guide monitoring efforts in vulnerable regions, and prioritize taxa for experimental investigation. Importantly, integrating metabolic, physiological, and trophic perspectives offers a comprehensive approach to understanding microbial resilience and vulnerability under multiple concurrent stressors (Bindoff et al., 2019; Caron et al., 2012; Brock et al., 1994; Buitenhuis et al., 2010).

In conclusion, the synthesis underscores that microzooplankton and hypersaline protists respond to environmental hazards in complex but predictable ways, with functional traits, rather than taxonomic identity, serving as the dominant determinant of response magnitude and ecosystem impact. Forest and funnel plot analyses provide quantitative confirmation of these patterns and highlight both knowledge gaps and avenues for future research.

5. Limitations

Despite the comprehensive synthesis, this study has several limitations. First, the dataset relies heavily on published experimental and observational studies, which may introduce publication bias, particularly for acidification effects where null results are less frequently reported. Second, meta-analytical aggregation across diverse taxa, habitats, and experimental designs may obscure fine-scale species- or site-specific responses. The heterogeneity of methodological approaches—ranging from mesocosm experiments to in situ observations—limits the direct comparability of effect sizes, especially when assessing complex interactions among multiple stressors. Third, while forest and funnel plots provide quantitative insight into response patterns, they cannot fully capture temporal dynamics, evolutionary adaptation, or long-term acclimation potential of microzooplankton and hypersaline protists. Fourth, environmental data on synergistic effects of combined stressors (e.g., warming plus hypoxia or acidification) remain scarce, constraining predictions of cumulative impacts under realistic climate scenarios. Finally, genomic and molecular studies, while illuminating cryptic diversity, are currently limited to a subset of hypersaline and deep-sea habitats, leaving large knowledge gaps in global protistan diversity and functional roles. These limitations suggest that while the review offers robust general patterns, caution is warranted in extrapolating results to unstudied taxa or ecosystems.

6. Conclusion

Microzooplankton and hypersaline protists play indispensable roles in marine ecosystem stability, nutrient recycling, and global carbon cycling, yet they remain highly sensitive to accelerating environmental change. This systematic review and meta-analysis demonstrate that warming, acidification, deoxygenation, eutrophication, and hypersaline stressors drive complex but largely trait-dependent ecological responses. Functional characteristics such as mixotrophy, thermal tolerance, oxygen sensitivity, and osmoadaptive capacity emerged as stronger predictors of resilience than taxonomic identity alone. Advances in molecular and meta-analytical approaches further revealed substantial cryptic diversity and adaptive potential within extreme marine habitats. Collectively, these findings emphasize the need for trait-based, multidisciplinary frameworks to predict future microbial ecosystem dynamics.

Author Contributions

V.V. and S.J. conceptualized and designed the study. V.V. conducted the systematic review framework development and supervised the research process. S.J. conducted literature collection, data extraction, and evidence synthesis. V.V. and S.J. interpreted the findings, drafted the manuscript, critically revised the article, and approved the final version for publication

Acknowledgements

The authors sincerely acknowledge the academic and research support provided by the Faculty of Marine Sciences, Annamalai University. The authors also thank all researchers whose published studies contributed to the scientific evidence synthesized in this review.

References