INTRODUCTION
Dinoflagellates are a dominant group of phytoplankton that fulfill various ecological roles in marine ecosystems and are major contributors to harmful algal blooms (HABs) (Anderson et al. 2012, Jeong et al. 2021, Sakamoto et al. 2021). Dinoflagellates exhibit various nutritional strategies, including phototrophy, heterotrophy, and mixotrophy (Jacobson and Anderson 1996, Mitra et al. 2016, Jeong et al. 2021). Mixotrophic organisms exhibit heterotrophic uptake through mechanisms such as absorbotrophy, which involves the assimilation of low-molecular-weight molecules; biotrophy, encompassing a spectrum of interactions from parasitic to mutualistic that do not result in host mortality; and phagotrophy, characterized by the ingestion of organic matter through the predation of other living entities (Beisner et al. 2019). Many mixotrophic dinoflagellates enhance their ecological advantage by combining photosynthesis with phagotrophy, a process known as phago-mixotrophy (Li et al. 1999, Jeong et al. 2005a, 2005b, Mitra et al. 2016). This trophic mode enables them to thrive in environments with fluctuating light or nutrient levels, outcompete strict phototrophs and heterotrophs (Hansen 2011, Jeong et al. 2015) and sustain high growth rates even in light- or nutrient-limited environments (Li et al. 1999, Jeong et al. 2005a, 2010).
The ecological impacts of mixotrophy in dinoflagellates extend beyond their ability to adapt to a wide range of environmental conditions (Blossom et al. 2012, Stoecker et al. 2017, Lim et al. 2019b, Ok et al. 2019). For instance, under nutrient-limited conditions such as nitrogen or phosphorus depletion, phago-mixotrophic dinoflagellates can outcompete strict phototrophs, allowing them to sustain growth and even dominate the planktonic community (Jeong et al. 2010). Such traits enable mixotrophic dinoflagellates to thrive in stratified, low-light, or organically enriched environments, thereby expanding their ecological niches (Thingstad et al. 1996, Stoecker et al. 2017). Furthermore, mixotrophy has been associated with allelopathic interactions and toxin production in certain species such as Karlodinium veneficum, which may provide additional advantages during HABs formation (Anderson et al. 2012). Through the production of secondary metabolites, such as allelochemicals and reactive oxygen species, they inhibit the growth of competing organisms and cause cellular damage in fish and shellfish (Blossom et al. 2012, Cho et al. 2022). Several mixotrophic species secrete mucilage-like compounds that exacerbate anoxic conditions, leading to mass mortality of marine life (Anderson et al. 2012). Certain species of the genus Gonyaulax produce potent toxins, such as yessotoxin, which can accumulate in filter-feeding organisms, such as bivalves (Paz et al. 2004, Álvarez et al. 2016, Ferreiro et al. 2016). This accumulation can lead to serious risks to human health, including gastrointestinal upset and long-term damage to organs, such as the liver and pancreas (Paz et al. 2004, Ferreiro et al. 2016). Numerous reports have documented the formation of red tides by some Gonyaulax species worldwide, including South Africa, the USA, and Japan, highlighting their importance in marine ecosystems and fisheries (Grindley and Taylor 1962, Kamykowski 1980, Lam and Yip 1990, Lin et al. 1993, Koizumi et al. 1996, 2001, Gárate-Lizárraga et al. 2001, Cho 2011, Dias et al. 2023). These studies suggest that some Gonyaulax species can proliferate in certain environments and cause ecological disruption, emphasizing the need for continued monitoring and management.
Recent studies have explored the mechanisms by which mixotrophic dinoflagellates form and maintain HABs (Kim et al. 2008, Place et al. 2012, Jeong et al. 2015, 2021, Johnson 2015, Stoecker et al. 2017, Ok et al. 2023, You et al. 2023). One of the key factors contributing to their ecological success is their photophysiological trait. The maximum photochemical quantum yield (Fv/Fm), one of the photosynthetic efficiency parameters, is used to assess how mixotrophic dinoflagellates response to biotic and environmental factors, acquire energy, and regulate their growth (Kolber and Falkowski 1993, Li et al. 1999, Park et al. 2025). Constitutive mixotrophy allows dinoflagellates to sustain photosynthesis without relying on external prey (Mitra et al. 2016), thereby optimizing energy acquisition under nutrient-limited conditions and further reinforcing their ecological dominance. Simultaneous photosynthesis and predation enable them to sustain rapid growth rates across diverse environmental conditions, allowing them to thrive and dominate red tide events (Raven 1997, Jeong et al. 2005a, 2021). Balancing photosynthetic activity and heterotrophic feeding is crucial under fluctuating light conditions (Li et al. 1999). Mixotrophic dinoflagellates optimize their photosynthetic performance and utilize prey-derived nutrients to enhance biomass production (Li et al. 1999, Anderson et al. 2012, Stoecker et al. 2017). By linking photophysiological parameters to growth dynamics, researchers could quantify the ecological advantages of mixotrophy and better understand its role in HAB formation and persistence. However, studies on the mixotrophic abilities and photophysiological characteristics of Gonyaulax species are limited; here we test the hypothesis that under low light (20 μmol photons m−2 s−1), prey availability (Synechococcus N78-1) increases both the maximum PSII efficiency (Fv/Fm) and the specific growth rate (μ), with Light × Prey as the primary endpoint and species-specific responses as secondary endpoints.
This study focused on four species of the genus Gonyaulax—Gonyaulax kunsanensis (LMBE_V571), G. whaseongensis (GSPSH160909), G. cochlea (CCMP1592), and G. geomunensis (GM25_k.91)—due to the limited understanding of their mixotrophic behavior and toxin production, in contrast to the well-documented G. polygramma and G. spinifera (Jeong et al. 2005b, Riccardi et al. 2009). By investigating their feeding mechanisms, growth rates, and photophysiological responses under varying light intensities and mixotrophic conditions, this study aimed to clarify how these organisms succeed in their ecological niches and contribute to red tide formation. The findings of this study provide valuable insights into mixotrophic dinoflagellate ecology, particularly their photo-physiological adaptation to changing light conditions and accelerating trophic transfer of carbon via prey ingestion. Understanding the link between photosynthetic efficiency and growth rate under mixotrophic conditions can inform the prediction of red tide dynamics, as well as the development of strategies for managing and mitigating the impact of HABs, which are becoming increasingly prevalent due to global environmental change.
MATERIALS AND METHODS
Preparation of experimental organisms
Eleven species of photosynthetic dinoflagellates, two species of raphidophytes, three species of cryptophytes, along with species of prasinophyte, prymnesiophyte, bacillariophyte, and a bacterium were used as potential prey for Gonyaulax spp. (Table 1). The mean equivalent spherical diameters of the phytoplankton were derived from prior studies (Jeong et al. 2015, 2016). All 20 prey species were cultured in fresh F/2 seawater medium (Sigma Aldrich, Merck, St. Louis, MO, USA) under controlled conditions at 20°C. Cultures were exposed to white LED light (20 μmol photons m−2 s−1) under a 14: 10 h light: dark cycle. G. kunsanensis (LMBE_V571), G. whaseongensis (GSPSH160909), G. cochlea (CCMP1592), and G. geomunensis (GM25_k.91) were also originally maintained in F/2 seawater medium at 20°C, under white LED light (50 μmol photons m−2 s−1) and a 14:10 h light:dark cycle. All cultures were transferred to fresh medium every 3–4 weeks. In all experiments, the cultures were acclimated to the target light intensity before the experiment, and we used cultures in the exponential growth phase.
Experimental setup
This study comprises four experimental setups: (1) prey interaction assays to observe behavioral responses of Gonyaulax species to a variety of potential prey; (2) phagotrophy confirmation tests using fluorescent microbeads to validate actual ingestion ability; (3) growth rate and photophysiological measurement under different light and feeding conditions to assess mixotrophic adaptability; and (4) light-shifting experiments to evaluate the compensatory role of mixotrophy during transitions from high to low light conditions.
Exploring the mixotrophic abilities of Gonyaulax species
Experiment 1 investigated the specific feeding of Gonyaulax spp. provided with unialgal diets containing diverse algal species (Table 1). To evaluate the interactions between Gonyaulax spp. and potential prey species, a specific cell concentration was used for each prey species (Table 1). We first added 5-mL of F/2 medium to 42-mL polycarbonate bottles. Cultures of each prey species were then added to achieve the required concentrations, before adding Gonyaulax spp. at 1,000–2,000 cells mL−1. The bottles were then filled with sterile filtered seawater. To explore the indirect effects of prey on Gonyaulax spp., co-incubation with prey filtrates was performed under the same experimental conditions. All co-cultures were placed on a rotating wheel at 0.9 rpm and incubated at 20°C under a 14:10 h light:dark cycle of white LED light (20 μmol photons m−2 s−1). After 2, 24, and 48 h of co-incubation, 5-mL aliquots were collected from each bottle, dispensed into confocal dishes, and 20 Gonyaulax cells were examined using a dissecting microscope (SZX9; Olympus, Tokyo, Japan) for 2 min per cell. Alexandrium catenella and Akashiwo sanguinea, which exhibited unique interactions with G. kunsanensis, were observed under a light microscope (Eclipse Ti2; Nikon, Tokyo, Japan) at 40× to 100× magnification. We recorded the presence of prey cells ingested by Gonyaulax, interior of the predator cells, number of contacts with potential prey, and behaviors of both the predator and prey. For smaller prey species, we investigated predator–prey interactions and prey ingestion using the light microscope (Eclipse Ti2; Nikon) at 1,500× magnification. We further investigated Gonyaulax spp. feeding activity showing strong interactions with certain prey species. To validate the feeding activity, we observed the uptake of bacterium-sized fluorescein 5(6)-isothiocyanate (FITC)-labeled beads (Sigma Aldrich) into the cytoplasm of Gonyaulax spp. using an inverted fluorescence microscope (Carl Zeiss MicroImaging, GmbH, Germany). Before co-incubation, the bacterium-sized microbeads were diluted in freshly filtered seawater and sonicated for 1 min to prevent particle agglomeration. The experimental conditions, including light intensity, temperature, and incubation settings, were identical to those used in Experiment 1. After 2, 24, and 48 h, 5-mL aliquots were taken from each bottle, placed into confocal dishes, and examined for feeding events or food vacuoles. For each sample, 20 cells were observed for 5 min. The behavior of Gonyaulax species toward potential prey and beads was defined as follows: intentional contact with prey for at least 2 s was classified as “Contact”; the presence of prey in the cytoplasm, “Feeding”; and capture and dragging of prey in addition to contact, “Attack.”
Fv/Fm and growth rate (μ) under different light and prey availability conditions (Light × Prey factorial design)
We quantified the effect of light (20 vs. 100 μmol photons m−2 s−1) and prey availability (Synechococcus N78-1: high, low, non) on Fv/Fm and μ in Gonyaulax spp. in a 2 × 3 factorial design (Light × Prey; experimental unit = bottle, n = 3 biological replicates per treatment).
Prior to the experiments, Gonyaulax spp. were grown in monocultures for at least 10 d under white LED light (20 and 100 μmol photons m−2 s−1, 14: 10 h light:dark cycle) in F/2 seawater medium at 20°C. Cultures in the exponential growth phase were used in all experiments. Experimental flasks were prepared by adding 5 mL of F/2 medium followed by Gonyaulax spp. to a final concentration of 1,000 cells mL−1 and filled with filtered seawater to a total volume of 50 mL per flask. The prey concentrations were adjusted accordingly and the flasks were maintained under the designated light conditions while monitoring photophysiological performance and growth. The Fv/Fm ratio was measured to assess photosynthetic efficiency. Prior to measurement, all the samples underwent dark acclimation for at least 10 min. Then, 4-mL aliquots from each replicate were transferred to standard glass cuvettes and allowed to stabilize under modulated (non-actinic) light for a few minutes. After stabilization, fluorescence measurements were conducted using a PHYTO-PAM-II Phytoplankton Analyzer and Phyto-Win software v3.t42 (Heinz Walz GmbH, Effeltrich, Germany). Fv/Fm was calculated as (Fm − F0)/Fm, where F0 and Fm denote the minimal and maximal fluorescence of dark-adapted samples, respectively. This parameter provides key insights into the maximum photochemical efficiency of photosystem II under varying prey concentrations and light intensities. The PHYTO-PAM-II system was used to distinguish populations based on their major light-absorbing pigments. Synechococcus sp. was classified as a phycoerythrin (PE)-type alga because it contains PE as the main light-absorbing pigment, while Gonyaulax spp. were classified as brown-type algae based on their xanthophyll pigment content (Beutler et al. 2002).
The growth rates were determined by comparing the initial and final cell densities. After 2 d, 5-mL aliquots were collected from each flask, fixed with Lugol’s solution at a final concentration of 5%, transferred to a 1-mL Sedgwick-Rafter chamber, and a minimum of 300 cells were counted using a light microscope (BX41; Olympus). The specific growth rate was calculated as μ (d−1) = [ln(Nt) − ln(N0)]/t, where N0 and Nt are initial and final cell densities over time t.
Effect of the changing light intensity on growth rates and Fv/Fm in Gonyaulax kunsanensis
Previous studies demonstrated that G. kunsanensis is capable of prey ingestion and that its growth rate under low-light conditions is proportional to prey availability. Therefore, we investigated the relationship between changes in light intensity, prey availability, and physiological responses to determine how variations in light intensity and prey availability influenced the growth rate and Fv/Fm of G. kunsanensis. Specifically, we compared the growth rates and Fv/Fm values under phototrophic (without prey) and phago-mixotrophic (with prey, Synechococcus sp.) conditions when cultures were subjected to a transition from high- to low-light intensity. Cultures were initially maintained at 100 μmol photons m−2 s−1 under white LED light and a 14:10 h light:dark cycle at a constant temperature of 20°C. During the first 4 d, growth rates and Fv/Fm values were monitored under phototrophic conditions without altering prey availability. After day 4, light intensity was reduced from 100 to 20 μmol photons m−2 s−1, and measurements were taken every 2 d until the end of the experiment under prey-presence (Synechococcus: 2.0 × 106 mL−1) and prey-absent conditions (triplicate bottles per treatment). Cell density and Fv/Fm values were monitored at regular intervals to calculate specific growth rates (d−1) and assess photosynthetic performance across different treatments. All environmental conditions, including temperature, were maintained at constant levels throughout the experiment. To ensure reliable results, each treatment was performed in triplicate (n = 3) under varying light and feeding conditions.
Statistical analysis
Data normality was tested using the Shapiro–Wilk test, and the homogeneity of variances was verified using Levene’s test. All data met the assumptions required for statistical analyses. For each species, a one-way analysis of variance (ANOVA) was used to evaluate the effect of prey concentration under the same light intensity and the effect of light intensity under the same prey concentration on the growth rate and Fv/Fm. In addition, two-way ANOVA (factors: Light, Prey) was conducted to assess the individual effects and their interaction on growth rate and Fv/Fm. Under the light-shifting conditions, the effect of prey presence/absence on growth rate and Fv/Fm was tested using a two-tailed t-test. For ANOVA, Tukey’s HSD was performed to identify pairwise differences. All statistical analyses were conducted using SPSS software version 27 (IBM SPSS, Armonk, NY, USA). Data are presented as mean ± standard error (SE), with statistical significance set at p < 0.05.
RESULTS
Interaction between Gonyaulax spp. and potential prey species
G. kunsanensis and G. geomunensis exhibited specific interactions with particular prey types, while only G. kunsanensis actually showed the phagotrophic ability (Table 1). G. kunsanensis and G. geomunensis displayed strong interactions with certain cryptophytes, including Teleaulax amphioxeia, Storeatula major, and Rhodomonas salina, and the prymnesiophyte Isochrysis galbana. We observed G. kunsanensis actively attacking prey using its entire body during frequent interactions, especially in the apical horn and sulcus regions (Fig. 1). It used its flagellum to recognize, capture, and move around its prey and exhibited frequent aggressive behavior (Fig. 1).
In the co-incubation tests, G. kunsanensis displayed negative interactions with A. catenella (GJ210606) and A. sanguinea (SA200604) (Fig. 2). In the co-culture with A. catenella, the cell density of G. kunsanensis sharply decreased and became nearly undetectable under microscopic observation after 2 d incubation (Fig. 2D). On the other hand, we observed large quantities of cell debris from A. sanguinea in the bottle, which was in co-culture with G. kunsanensis (Fig. 2E & F).
Microscopic observations using FITC-labeled fluorescent beads confirmed the ingestion and internalization of bacterium-sized particles by G. kunsanensis, providing clear evidence of completed phagotrophic feeding. Fluorescent beads were distinctly observed within the cytoplasm of G. kunsanensis cells, suggesting that G. kunsanensis is capable of actively engulfing particles of bacterial size (Fig. 3). This method bypassed chlorophyll interference, allowing clear visualization of internalized particles. Among the four Gonyaulax species, only G. kunsanensis exhibited phagotrophic ability, only for bacterium-sized beads. Based on the phagotrophy results, Synechococcus sp., a picocyanobacterium, was selected as the prey item for subsequent light-feeding experiments. Although G. geomunensis used its flagella to recognize and capture prey (T. amphioxeia, S. major, R. salina, and I. galbana) during frequent contact, no ingestion was observed.
Effects of light intensities and interspecies interactions on growth and photophysiology of Gonyaulax spp
We observed species-specific responses in terms of Fv/Fm and growth rate (μ) under different light intensities (20 and 100 μmol photons m−2 s−1) and Synechococcus sp. (N78-1) concentrations for G. kunsanensis (LMBE_V571), G. geomunensis (GM25_k91), and G. whaseongensis (GSPSH160909) (Fig. 4). The growth rates varied depending on light intensity (Fig. 4A & B). Under high-light conditions (100 μmol photons m−2 s−1), G. kunsanensis exhibited high growth rates regardless of prey availability (mean ± SE, 0.15 ± 0.02 d−1 with high prey, 0.17 ± 0.02 d−1 with low prey, 0.16 ± 0.02 d−1 with non-prey; ANOVA, F(2,6) = 0.27, p > 0.05) (Fig. 4A). Similarly, G. geomunensis maintained relatively high growth rates regardless prey availability (0.19 ± 0.01 d−1 with high prey, 0.18 ± 0.02 d−1 with low prey, 0.14 ± 0.01 d−1 with non-prey; ANOVA, F(2,6) = 4.33, p > 0.05). Conversely, G. whaseongensis exhibited relatively low growth rates under high-light conditions and the presence of Synechococcus sp. did not affect its growth (0.06 ± 0.01 d−1 with high prey, 0.11 ± 0.03 d−1 with low prey, 0.10 ± 0.02 d−1 with non-prey; ANOVA, F(2,6) = 1.97, p > 0.05).
Under low-light conditions (20 μmol photons m−2 s−1), we observed species-specific patterns (Fig. 4B). Synechococcus sp. concentration strongly affected the growth rate of G. kunsanensis (ANOVA, F(2,6) = 9.96, p < 0.05). Growth rates were higher in the high prey condition (0.16 ± 0.02 d−1) than in the non-prey condition (0.05 ± 0.02 d−1). G. geomunensis exhibited relatively low growth rates independent of Synechococcus sp. concentration (0.06 ± 0.00 d−1 with high prey, 0.06 ± 0.00 d−1 with low prey, 0.06 ± 0.00 d−1 with non-prey; ANOVA, F(2,6) = 0.03, p > 0.05). G. whaseongensis maintained high growth rates regardless of Synechococcus sp. concentration (0.16 ± 0.02 d−1 with high prey, 0.14 ± 0.03 d−1 with low prey, 0.15 ± 0.02 d−1 with non-prey; ANOVA, F(2,6) = 0.25, p > 0.05).
Under high light, the Fv/Fm of G. kunsanensis differed among prey treatments (ANOVA, F(2,6) = 13.80, p < 0.01). The Fv/Fm of G. geomunensis and G. whaseongensis showed significant differences depending on Synechococcus concentrations (0.19 ± 0.01 with high prey, 0.13 ± 0.01 with low prey, 0.12 ± 0.01 with non-prey; ANOVA, F(2,6) = 10.79, p < 0.05 for G. geomunensis; 0.27 ± 0.02 with high prey, 0.23 ± 0.01 with low prey, 0.22 ± 0.01 with non-prey; F(2,6) = 5.73, p < 0.05 for G. whaseongensis). Under low-light conditions (20 μmol photons m−2 s−1), two species exhibited high Fv/Fm values regardless Synechococcus concentrations (Fig. 4D). The Fv/Fm of G. kunsanensis and G. geomunensis did not differ significantly according to Synechococcus concentrations (0.52 ± 0.00 with high prey, 0.53 ± 0.01 with low prey, 0.52 ± 0.01 with non-prey; F(2,6) = 2.09, p > 0.05 for G. kunsanensis; 0.41 ± 0.02 with high prey, 0.40 ± 0.01 with low prey, 0.38 ± 0.00 with non-prey; F(2,6) = 1.45, p > 0.05 for G. geomunensis), whereas the Fv/Fm of G. whaseongensis showed a significant difference among prey concentrations (0.59 ± 0.01 with high prey, 0.54 ± 0.00 with low prey, 0.53 ± 0.02 with non-prey; F(2,6) = 9.75, p < 0.05).
Light intensity affected growth rates across all species (two-way ANOVA, F(1,12) = 15.67, p < 0.01, ηp2 = 0.57 for G. kunsanensis; F(1,12) = 198.02, p < 0.001, ηp2 = 0.94 for G. geomunensis; F(1,12) = 14.24, p < 0.01, ηp2 = 0.54 for G. whaseongensis). Prey concentration also significantly affected the growth rate of G. geomunensis, though the effect size was smaller compared to light intensity (F(2,12) = 4.31, p < 0.05, ηp2 = 0.42) (Supplementary Table S1). The interaction of light intensity and prey concentration greatly affected the growth rates of G. kunsanensis (F(2,12) = 5.32, p < 0.05, ηp2 = 0.47) and G. geomunensis (F(2,12) = 4.06, p < 0.05, ηp2 = 0.40), though less so than the main effect of light intensity.
Light intensity greatly affected Fv/Fm across all species (two-way ANOVA, F(1,12) = 1,426.39, p < 0.001, ηp2 = 0.99 for G. kunsanensis; F(1,12) = 657.93, p < 0.001, ηp2 = 0.98 for G. geomunensis; F(1,12) = 1,283.84, p < 0.001, ηp2 = 0.99 for G. whaseongensis). Prey concentration strongly affected the Fv/Fm of G. kunsanensis (F(2,12) = 9.60, p < 0.01, ηp2 = 0.62) and G. whaseongensis (F(2,12) = 14.98, p < 0.001, ηp2 = 0.71), though less so than light intensity (Supplementary Table S1). The light and prey interaction was significant only for G. kunsanensis (F(2,12) = 15.82, p < 0.001, ηp2 = 0.73).
Growth rates and Fv/Fm in phototrophic and mixotrophic cultures under shifting light levels
The transition from high-light (100 μmol photons m−2 s−1) to low-light conditions (20 μmol photons m−2 s−1) had distinct effects on the growth rates of mixotrophic and phototrophic cultures, and prey addition significantly enhanced growth rates under reduced light availability on days 4–6 and days 6–8 (two-tailed t-test, p < 0.05 and p < 0.01, respectively) (Fig. 5A). Under high-light conditions, G. kunsanensis exhibited stable growth rates, showing no significant differences in the presence (Syn:100) or absence of prey (Non:100) (two-tailed t-test, p > 0.05) (Fig. 5A). When the cultures experienced low-light conditions (20 μmol photons m−2 s−1) by day 4, the growth rate of G. kunsanensis in the presence of prey (Syn:20) was 0.08 ± 0.01 d−1 on days 4–6 and increased to 0.14 ± 0.01 d−1 on days 6–8. Conversely, in the absence of prey (Non:20), the growth rate remained constant, measured at 0.05 ± 0.01 on days 4–6 and 0.06 ± 0.01 d−1 on days 6–8. On days 6–8, the growth rate in the presence of prey (Syn:20, 0.14 d−1) was approximately 2.3-fold higher (0.14 vs. 0.06 d−1) than that in the absence of prey (Non:20, 0.06 d−1) (two-tailed t-test, p < 0.01) (Fig. 5A).
The transition from high to low light also affected the Fv/Fm of G. kunsanensis (Fig. 5B). During the initial high-light conditions (days 0–4), Fv/Fm values remained stable, ranging between 0.32 and 0.39. After the shift to low-light conditions on day 4, Fv/Fm values gradually increased in both treatment groups. In the presence of prey (Syn:20), Fv/Fm increased from 0.48 ± 0.01 on day 4 to 0.57 ± 0.02 on day 6, and 0.60 ± 0.02 on day 8. In the absence of prey (Non:20), the values increased as the incubation time went by, reaching 0.61 ± 0.02 on day 6 and 0.67 ± 0.01 on day 8. Under low light, no prey effect on Fv/Fm was detected at day 6 (two-tailed t-test, p > 0.05); at day 8, Fv/Fm was higher without prey than with prey (0.67 ± 0.01 vs. 0.60 ± 0.02) (two-tailed t-test, p < 0.05). Under high-light condition, on day 6, Syn:100 and Non:100 treatments showed a significant difference (0.39 ± 0.03 and 0.30 ± 0.02, respectively) (two-tailed t-test, p < 0.05), which persisted through day 8 (0.40 ± 0.01 for Syn:100; 0.28 ± 0.02 for Non:100) (two-tailed t-test, p < 0.05).
DISCUSSION
This study demonstrated the distinct prey responses and feeding behaviors of Gonyaulax spp., revealing potential species-specific interactions. G. kunsanensis and G. geomunensis exhibited strong interactions with specific cryptophytes, prymnesiophyte, and bacteria, suggesting that these prey types may act as a potential prey. However, Gonyaulax is known to be an engulfing feeder, thus its feeding behavior is influenced by prey size limitations (Jeong et al. 2005a, 2005b). Frequent contact and pre-consumptive exploratory behavior were observed in Gonyaulax spp., indicating that they might recognize their prey. These behaviors were mediated by structural components such as the flagellum, apical horn, and sulcus, which appear to facilitate physical interactions with prey (Jeong et al. 2005b, Nielsen and Kiørboe 2015) and may play crucial roles in initial prey detection/interaction, capture, and processing. While direct ingestion of live prey in G. geomunensis was not observed, the coordinated movements and persistent prey-contact behaviors indicate a potential for phagocytosis-like feeding, warranting further investigation on more diverse prey items.
Feeding is not determined solely by the structural characteristics of the cell, but also by the ability to recognize prey. Verity (1991) reported that various planktonic protozoans can detect chemical cues released from prey, in addition to relying on physical properties such as size or shape and modify their motility accordingly. This enables selective feeding rather than random encounters. Karlodinium armiger, for example, exhibits a distinct pre-capture behavior characterized by increased swimming speed and rotation near prey cells, especially when the prey is damaged and leaking chemical compounds (Berge et al. 2008). This chemotactic behavior increases the encounter rate and enhances feeding efficiency. In this study, Gonyaulax cochlea and G. whaseongensis did not exhibit any chemotactic or feeding responses under experimental conditions. This suggests that these species may lack the chemosensory systems or intracellular signaling pathways necessary for prey recognition and ingestion. Therefore, further investigation into the role of these structures in prey acquisition is required to characterize the physio-ecological strategies of Gonyaulax spp.
Microscopic observations using fluorescent beads suggested that G. kunsanensis possesses phagotrophic capabilities, providing direct evidence of its mixotrophic nutritional strategy. Along with G. kunsanensis, only four Gonyaulax species exhibit phago-mixotrophy (Jacobson and Anderson 1996, Jeong et al. 2005a, 2005b), and the phago-mixotrophic behavior of only six phototrophic species has been investigated among more than 80 in the genus (algaebase.org, retrieved from Mar 24, 2025). For example, G. polygramma showed phagotrophic capabilities for diverse prey species with cell sizes <20 μm (Jeong et al. 2005b). G. spinifera can feed on the cyanobacterium Synechococcus (Jeong et al. 2005a), while food vacuoles containing ciliate remains were found inside G. diegensis (Jacobson and Anderson 1996). The results suggest that G. kunsanensis selectively feeds on the small prey particles, indicating that prey size limitations may play a key role in shaping its trophic strategy. These patterns of prey preference and selective ingestion suggest that Gonyaulax species may occupy distinct ecological niches, with phago-mixotrophic species like G. kunsanensis being better adapted to environments with fluctuating light or nutrient conditions. Their ability to exploit specific prey types may provide a competitive advantage, particularly during bloom events where prey composition and size structure vary dynamically.
Furthermore, the ecological advantages of mixotrophy under nutrient-limited or fluctuating light conditions might be associated with flexible nutrient allocation strategies. Mixotrophic protists can utilize prey-derived nutrients, such as nitrogen and phosphorus, in a manner analogous to inorganic nutrient uptake, thereby influencing their intracellular C: N:P stoichiometry (Mitra et al. 2014). This capacity allows them to selectively acquire limiting elements and maintain growth when external nutrient supply is scarce. Similarly, several studies have demonstrated that phagotrophy under low-light or nutrient-deficient conditions enhances nutrient acquisition and supports growth by providing essential nutrients (González-Olalla et al. 2021, Mena et al. 2025). Notably, Mena et al. (2025) reported that the harmful dinoflagellates Alexandrium minutum, Heterocapsa triquetra, and Prorocentrum micans exhibited reduced C: N ratios under low-light conditions, primarily due to increased nitrogen assimilation relative to carbon fixation. Together, these findings highlight that stoichiometric plasticity via phagotrophy is a key mechanism in sustaining cellular function and growth under environmental stress. In our study, the observed growth enhancement of G. kunsanensis under prey-rich, low-light conditions likely reflects a comparable metabolic compensation strategy, emphasizing the importance of nutrient flexibility in dynamic marine environments. Many studies have shown that mixotrophy can greatly increase the growth rate of dinoflagellates and affect algal bloom formation (Li et al. 1999, Jeong et al. 2015, Mitra et al. 2016, Lim et al. 2019a, Mena et al. 2025). Therefore, further research is needed on the phago-mixotrophic abilities, prey species preferences, and physiological characteristics of other Gonyaulax species, especially red tide-associated and toxin-producing species, such as G. fragilis and G. taylorii (Pompei et al. 2003, Álvarez et al. 2016).
Co-incubation experiments revealed complex interactions between G. kunsanensis and other species. When co-cultured with A. catenella, the cell numbers of G. kunsanensis decreased sharply and nearly disappeared over the experimental period. This is possibly due to lysis caused by the allelochemical produced by A. catenella, as many Alexandrium species exert allelopathic effects on microalgae (Tillmann et al. 2008, Tillmann and Hansen 2009, Ma et al. 2011, Blossom et al. 2012, Lim et al. 2015, 2019a). Conversely, large quantities of mucus produced by A. sanguinea were observed in co-culture, accompanied by a reduction in A. sanguinea cell numbers compared to the control (only A. sanguinea). This suggests that G. kunsanensis may release substances that induce lysis of A. sanguinea and inhibit its growth, similar to the allelopathic effects of other dinoflagellates (Lim et al. 2019a, Yan et al. 2019). These results indicated that G. kunsanensis can be affected by allelopathic chemicals from certain species and may exert lytic effects on other species. This dual role highlights the complex interactions of G. kunsanensis within marine microbial communities and suggests its potential to influence the survival and distribution of certain species.
Gonyaulax species exhibited distinct growth responses to variations in light intensity and trophic mode (phototrophic vs. phago-mixotrophic), suggesting that each species employs a different strategy for energy acquisition and utilization. G. kunsanensis demonstrated a strong dependence on prey ingestion to enhance growth under low-light conditions, whereas G. geomunensis and G. whaseongensis exhibited different physiological adaptative strategies, showing distinct responses to changes in light intensity.
The growth rates of G. geomunensis in high-light environments remained relatively high, but were significantly lower in low-light environments, indicating a limitation in optimizing energy acquisition under low-light conditions. The Fv/Fm of G. geomunensis was generally low in high-light environments but increased in the presence of Synechococcus sp. These results indicate that total photosynthetic production can be maintained in environments with sufficient photon flux even if photosystem II efficiency is low (Oh et al. 2012). The increase in Fv/Fm values with Synechococcus sp. supplementation suggests that compounds derived from the prey may contribute to photosynthetic system stability. Synechococcus produces various metabolites such as vitamin B12 that can promote the growth of neighboring microorganisms (Bonnet et al. 2010, Fiore et al. 2015, Tandon et al. 2017, Lee et al. 2021). Moreover, these vitamins may also contribute to the photosynthetic ability of microalgae (Tandon et al. 2017). Thus, the metabolites produced by Synechococcus may be involved in maintaining the photosynthetic efficiency of G. geomunensis. In low-light environments, the Fv/Fm values remained relatively constant, indicating that G. geomunensis was able to sustain its basic photosynthetic capacity even under low-light conditions. Conversely, G. whaseongensis maintained a comparatively high growth rate in low light compared with that under high-light conditions, suggesting that it is better adapted to low-light environments. The Fv/Fm of G. whaseongensis showed a distinct change with Synechococcus concentration in both high- and low-light environments, suggesting that Synechococcus-derived compounds may contribute to the photosynthetic capacity of G. whaseongensis as in G. geomunensis. In other words, although G. whaseongensis is primarily dependent on phototrophy for growth, it may benefit from biochemical interactions with the external environment to maintain its physiological stability.
G. kunsanensis exhibited a strong reliance on mixotrophy in low-light environments, and its growth rate significantly increased as Synechococcus sp. concentration increased. This indicates that mixotrophy serves as a crucial compensatory mechanism under light-limited conditions, enabling G. kunsanensis to acquire energy from auxiliary sources, consistent with the findings of previous studies on other mixotrophic dinoflagellates (Lim et al. 2019b, Ok et al. 2019). Despite this reliance on prey, no significant difference in Fv/Fm was observed in the presence or absence of prey, suggesting that prey ingestion primarily supports growth by supplementing energy and nutrients rather than directly enhancing photosynthetic performance. These findings align with the energy acquisition strategies described by Stoecker et al. (2017), emphasizing the role of mixotrophy in enhancing metabolic flexibility, allowing planktonic protists to optimize energy utilization in dynamic environments. The supplementation of nutrients in mixotrophy in turn promotes survival and growth in challenging environments (Li et al. 1999, Mitra et al. 2016, Stoecker et al. 2017, Lim et al. 2019b, Ok et al. 2019, Jeong et al. 2021, Costa et al. 2022, You et al. 2023).
The results from the shifting light intensity experiment showed that G. kunsanensis rapidly adapts to changes in light intensity by adjusting its photosynthetic mechanisms and mixotrophic strategies. Under high-light conditions, G. kunsanensis maintained a stable growth rate with or without prey, suggesting that photosynthesis alone was sufficient to provide energy. However, after switching to the low-light condition, the growth rate dramatically decreased under phototrophic conditions, whereas Fv/Fm increased to enhance photosynthetic performance (Fig. 5). The mixotrophic growth rate gradually increased over time under the low-light condition, confirming that G. kunsanensis can compensate for low growth rates by utilizing mixotrophy and that feeding plays a more important role than photosynthetic efficiency under low-light conditions. Future studies should quantitatively analyze the carbon and nutrient metabolic pathways between phototrophy and mixotrophy in G. kunsanensis and elucidate the interaction between photosynthetic regulation and mixotrophic strategies in low-light environments at the molecular level.
In conclusion, among the examined species, only G. kunsanensis exhibited a clear mixotrophic response, maintaining phototrophic growth under high light and increasing μ in the presence of prey under low light. Our findings indicate that Gonyaulax species use different ecological strategies in response to changing light environments and that adaptation to low light varies among species. G. kunsanensis maintained stable growth through photosynthesis in light-enriched environments and increased its growth rate through mixotrophy in light-limited environments. Conversely, G. whaseongensis could maintain continuous growth through photosynthesis alone in low-light environments; G. geomunensis showed high growth rates in high-light environments but low growth rates in low-light environments. G. kunsanensis maintained its growth rate through mixotrophy in low-light environments, indicating that mixotrophy is an important compensatory mechanism for energy and nutrient acquisition in light-limited environments. This suggests that the ability to flexibly switch between phototrophy and mixotrophy in response to environmental changes contributes to the ecological adaptability of G. kunsanensis.
In marine environment, especially in coastal waters where light decreases with depth due to turbidity, phago-mixotrophy can enhance nutrient acquisition and sustain growth dominance (Thingstad et al. 1996, Blossom et al. 2012, Stoecker et al. 2017, Lim et al. 2019b, Ok et al. 2019). The ability of G. kunsanensis to maintain growth under low-light conditions through prey ingestion suggests it can exploit heterotrophic pathways where strict phototrophs or heterotrophs may face limitations. This trophic flexibility may facilitate population growth and prolonged bloom events under suboptimal conditions when suitable prey is available, contributing to red tide initiation and maintenance. To elucidate phago-mixotrophy’s role in population dynamics and ecological interactions in coastal waters, future research should explore how light and prey availability influence Gonyaulax spp. population behavior, and how their mixotrophic strategies impact microbial community structure and nutrient cycling. As HAB formation is linked to environmental and nutrient fluctuations (Maso and Garcés 2006), these insights are essential for predicting blooms under changing climate conditions.
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