Suppression of cyst germination success in Gymnodinium catenatum and Ostreopsis cf. ovata by macroalgal extracts from the southern Mediterranean Sea

Dinoflagellates are one of the most important microorganisms in the aquatic ecosystems, playing a prominent role as primary producers in the trophic chain, especially in the marine environment (Bravo and Figueroa, 2014; Rukminasari and Tahir, 2021). Nonetheless, many dinoflagellate species form harmful algal blooms (HABs) and produce toxins that endanger aquaculture, fisheries, and public health (Mohamed, 2018; Mohamed & Al-Shehri, 2012). It has been established that over 200 of the more than 2000 species of dinoflagellates can form cysts as a part of their life cycle (Likumahua et al., 2021; Li et al., 2020). These cysts sink into the sea bottom, sustain unfavorable conditions and remain viable in sediments for up to 100 years (Cuellar-Martinez et al., 2023; Figueroa et al., 2007; Miyazono et al., 2012; Vahtera et al., 2014). When environmental conditions are restored, the cysts germinate to form vegetative cells that re-enter the water column, thereby triggering the formation of HABs (Butman et al., 2014).

Cyst germination is regulated by internal factors, including the obligatory dormancy (i.e., maturation period for cysts to germinate), which occurs immediately after cyst formation and can last from 12 hr to 12 months, depending on the species (Su et al., 2016). On the contrary, cyst germination can also be governed by external factors such as temperature, light, oxygen, and salinity (Cuellar-Martinez et al., 2023; Kremp & Anderson, 2000; Zheng et al., 2024). When these external factors are satisfied, temperature is the key determinant of cyst germination (Fischer & Brosnahan, 2022; Vahtera et al., 2014). Hence, dinoflagellate cyst germination is predicted to be enhanced by warming and climate change (Brosnahan et al., 2020).

Therefore, the inhibition of cyst germination in the natural environment would restrict the development of extensive coastal blooms. Most studies conducted on the mitigation of HABs by ecofriendly methods such as macroalgae or their extracts investigated the effects of macroalgal materials only on vegetative cells (Accoroni et al., 2015; Ben Gharbia et al., 2017; Benitt et al., 2022; Wang et al., 2007). Little is known about the inhibitory effects of macroalgae on the cyst germination of dinoflagellate species. The only one study that has addressed the inhibition of dinoflagellate cyst germination by the brown alga (Turbinaria ornate) is Mohamed et al. (2022)).

Our previous study recorded intense bloom of G. catenatum and O. cf. ovata in Tajoura coastal waters in September 2023, with a high prevalence of these species’ cysts in the surface sediments (Mohamed et al., 2025). Remarkably, an assemblage of seven macroalgae (Jania rubens, Laurencia obtusa, Cystoseira compressa, Cystoseira crinita, Cystoseira barbata, Ulva lactuca, and Ulva linza) was found on the Libyan Mediterranean coast, approximately 1 km away from the bloom area (Elfituri et al., 2022); nevertheless, these macroalgae were not found in this bloom area. Therefore, we hypothesized that these macroalgae might have had a role in the impairment of cyst germination and development of this bloom in the non-impacted area, but their absence permitted the bloom formation in the impacted area.

Therefore, this study was conducted to examine the effect of these macroalgae on the germination success of G. catenatum and O. cf. ovata cysts found in surface sediments of the Libyan Mediterranean Sea. This study also examined the use of these macroalgae as an ecofriendly bioagent of controlling HABs formation.

The results of experiments investigating the inhibitory effects of Libyan macroalgae on the germination success (i.e., germling viability) of cysts of two harmful dinoflagellates species (G. catenatum and O.cf. ovata) forming blooms in the Libyan Mediterranean coast are presented in Figs. 1 and 2.

Figure 1

Effect of aqueous macroalgal extracts on the success of cyst germination in harmful microalgae: (A) Ostreopsis cf. ovata and (B) Gymnodinium catenatum. Data points are means ± SD (n = 3). Different uppercase letters indicate statistically significant differences (p < 0.05) in the cyst germination among different macroalgae for each extract concentration, whereas different lowercase letters indicate statistically significant differences (p < 0.05) in the cyst germination between different extract concentrations for each macroalgal species (i.e., horizontal track). SD, standard deviation.

The cysts of G. catenatum and O. cf. ovata in the control and treatment groups (i.e., with macroalgal extracts) incubated in sterilized seawater at 25°C, passed the dormancy period and underwent excystment as early as the 2nd day (63%) and as late as the 20th day (98%). However, the percentage of successful cyst germination (i.e., empty cysts forming live motile cells) varied significantly between the control and treatment groups (p < 0.0001). The highest germination success in the control group (96%) reached on day 20 and remain constant until the end of the germination course (30 days). On the contrary, the highest germination success of cysts incubated with macroalgal extracts was recorded after 20 days and continued for up to 30 days, depending on the macroalgae species and the type of extract (i.e., aquatic/ethanolic extract). Therefore, the cyst germination experiment was terminated on day 30. The germination of G. catenatum and O. cf. ovata cysts in a short period in natural seawater at optimal temperatures of 23–25°C has also been reported in previous studies, which showed 1 day for Gymnodinium cysts (Liu et al., 2020) and 2–5 days for Ostreopsis cysts (Accoroni et al., 2014; Bravo et al., 2012).

The addition of aqueous or ethanol extracts of seven seaweeds (J. rubens, L. obtusa, C. compressa, C. crinita, C. barbata, U. lactuca, and U. linza) to cultures of G. catenatum and O. cf. ovata cysts caused varying reduction in the percentage of successful germination (i.e., viable germlings) of these cysts. The percentage of successful germination of the two cyst species decreased significantly (p < 0.001) with the increase in the concentration of aqueous or ethanol macroalgal extract (Figs. 1 and 2). The aqueous extracts of four macroalgae (J. rubens, L. obtusa, U. lactuca, and U. linza) showed weak inhibitory effects on the germination success of both G. catenatum (% germination inhibition = 12%–26%) and O. cf. ovata cysts (5%–27%); while the aqueous extracts of the three species of Cystoseira (C. compressa, C. barbata, and C. crinita) showed moderate inhibition on germination success of O. cf. ovata cysts (% inhibition = 53%–69%; Fig. 1A) and G. catenatum cysts (37%–59%) (Fig. 1B).

Figure 2

Effect of methanol macroalgal extracts on the success of cyst germination in harmful microalgae: (A) Ostreopsis cf. ovata and (B) Gymnodinium catenatum. Data points are means ± SD (n = 3). Different uppercase letters indicate statistically significant differences (p < 0.05) in the cyst germination among different macroalgae for each extract concentration, whereas different lowercase letters indicate statistically significant differences (p < 0.05) in the cyst germination between different extract concentrations for each macroalgal species (i.e., horizontal track). SD, standard deviation.

On the other hand, methanol extracts of these macroalgae exerted greater inhibitory effects on the cyst germination success compared with aqueous extracts (p < 0.05), and these effects varied significantly with the increase of macroalgal extracts (p < 0.05). On the species level of Cystoseira, the methanol extracts at a concentration of 10 mg · mL⁻¹ of the three species of Cystoseira (C. compressa, C. barbata, and C. crinita) exhibited the strongest inhibitory effects on the germination success of G. catenatum cysts (% inhibition = 83%, 90%, and 95%, respectively; Fig. 2A) and O. cf. ovata cysts (88%, 95%, and 100%, respectively; Fig. 2B). Whereas the methanol extracts of the remaining macroalgal species (J. rubens, L. obtusa, U. lactuca, and U. linza) at the highest concentration (10 mg · mL⁻¹) showed moderate inhibitory effects on the germination success of G. catenatum (% inhibition = 69% and 74%, and O. cf. ovata cysts (74%–77%, respectively; Figs. 2A and 2B).

Based on the IC₅₀ values, the inhibitory effects of methanol extracts of all macroalgae tested were greater than aqueous extracts (p < 0.05) on the germination success of G. catenatum and O. cf. ovata cysts (Tables 1 and 2). The IC₅₀ values also varied significantly among macroalgae tested (p < 0.05), either for aqueous or methanolic extracts. For aqueous extracts, C. crinita had the lowest IC₅₀ values (i.e., highest inhibition degree) against G. catenatum and O. cf. ovata cysts (IC₅₀ = 3.9 and 9.9 mg · g⁻¹, respectively), followed by C. compressa (IC₅₀ = 4.4 and 11.7 mg · g⁻¹) and C. barbata (IC₅₀ = 6.6 and 14.8 mg · g⁻¹). By contrast, the aqueous extracts of other macroalgae exhibited lower inhibition levels (IC₅₀ = 58.6–590 mg ⋅ g⁻¹) toward the germination success of the two cyst types (Tables 1 and 2).

For the methanol macroalgal extracts, the three Cystoseira species also showed the strongest inhibitory effects against the germination success of the two cyst types, with IC₅₀ values (0.05–0.23 mg · g⁻¹) lower than those reported for aqueous extracts of these macroalgae (Tables 1 and 2). On the contrary, the methanolic extracts of other macroalgal species demonstrated a moderate level of inhibition against cyst germination success (Tables 1 and 2); the order of inhibition efficiency (i.e., low IC₅₀ value) was as follows: J. rubens (1.1–1.4 mg · g⁻¹) > U. linza = U. lactuca (1.3–1.4 mg · g⁻¹) > L. obtusa (1.6–2.4 mg ⋅ g⁻¹). The variation in the inhibition of cyst germination among the different macroalgae in this study may be due to the difference in the type and amount of bioactive compounds in the macroalgal extracts, as suggested by earlier research (Ben Gharbia et al., 2017).

The results of this study showed that the macroalgal extracts did not directly affect the germination of dinoflagellate cysts (i.e., excystment), but rather hindered the survival of the germling cells, preventing them from producing motile vegetative cells. Thus, these extracts would hinder cysts’ capacity to successfully repopulate new planktonic populations in the aquatic environment. The results corroborate the fact that the viability of germling cells after excystment represents a bottleneck in the bloom initiation (Genovesi et al., 2009; Mardones et al., 2016), and hence the inhibition of germling cell viability of dinoflagellate cysts retards the recurrence of relevant species in the water column. Previous studies have shown that dinoflagellate cysts can germinate (i.e., undergo excystment) but the germling cells would not be able to divide and would die soon under unfavorable abiotic conditions such as temperature, light, and oxygen-availability (Brosnahan et al., 2020; Genovesi et al., 2009; Vahtera et al., 2014). However, Mohamed et al. (2022) investigated the biological interaction between macroalgae and dinoflagellate cyst germination. They demonstrated the inhibitory effects of ethanol extracts of the brown macroalga, Turbinaria ornata, on the viability of germlings and formed excystment of some dinoflagellate cysts (e.g., A. catenella, C. polykrikos, S. trochoidea, P. minimum, and D. acuminata cysts) collected from the surface sediments in the Red Sea. Therefore, the present study is the second to explore the inhibition of germination success of dinoflagellate cysts of harmful species (e.g., G. catenatum and O. cf. ovata) from the southern Mediterranean Sea.

Additionally, our macroalgal methanol extracts were about 50–200 times more efficient against the germling viability of all cyst species than the aqueous extract. These results supported the fact that alcoholic solvents such as ethanol are superior to water in extracting bioactive compounds from plants (Sultana et al., 2009). This study also revealed that although there was no statistical difference in the susceptibility of germling cells to macroalgal extracts between G. catenatum and O. cf. ovata cysts (p > 0.05), O. cf. ovata germlings were more vulnerable than G. catenatum germling. These findings are thus consistent with previous studies demonstrating that the inhibitory effects of macroalgal extracts on microalgal growth differed depending on the target species of microalgae (Mohamed et al., 2022; Zhang et al., 2021).

Such inhibitory effects of macroalgal extracts could be attributed to the active substances found in macroalgae, which may inhibit metabolic processes that govern the division and growth of germling cells formed after cyst excystment (Gémin et al., 2020; Mohamed et al., 2022). In this study, the GC-MS analysis of the extracts of tested macroalgal species revealed the dominance of long chain fatty acid ester, unsaturated fatty acids, alcohols, saturated fatty acids, diisooctyl phthalate ester, and unsaturated aldehydes (Tables S1–S7 and Figures S1-S7 in Supplementary Material). These allelochemicals were previously isolated from higher plants and macroalgae, and most of them, especially unsaturated fatty acids (e.g., linoleic acid), diisooctyl ester, and polyunsaturated aldehyde, have been confirmed to inhibit the growth of many harmful microalgae such as Karenia mikimotoi, Alexandrium tamarensem Prorocentrum micans, and H. akashiwo (Lenzo et al., 2022; Patil et al., 2024; Pichierri et al., 2016; Wang et al., 2018). Most likely, these allelochemicals might have affected the division and growth of germling cells of G. catenatum and O. cf. ovata cysts. Regarding the algicidal action, it has been stated that macroalgal allelochemicals primarily disrupt photosystem II (PSII) components leading to reduction in photosynthetic pigments, and ultimately inhibit the proliferation of the algal cells (Cheng et al., 2024; Zhang et al., 2019). Simultaneously, allelochemicals cause oxidative stress and formation of excessive reactive oxygen species (ROS), triggering the death of microalgal cells (i.e., cell apoptosis) through the action of caspase-3 and -9 (Wang et al., 2023). However, more research is needed to separate and characterize such active substances produced by these macroalgae in order to potentially use them to control and mitigate HABs in the natural environment.

This study provided evidence that macroalgae from the Libyan Mediterranean coast, particularly Cystoseira species, inhibited the germination success of G. catenatum and O. cf. ovata cysts through suppressing the germling viability after cyst germination. This indicates that these macroalgae could retard the recurrence of blooms of these dinoflagellate species in the water column. The study also showed that methanol extracts of these macroalgae have stronger inhibitory effects on the germination success with lower IC₅₀ values (0.05–1.5 mg extract g⁻¹ sediment) than aqueous extracts (IC₅₀ = 3.9–590 mg extract g⁻¹ sediment). Given that cyst germlings are more sensitive toward active substances than mature individuals (Bond et al., 1999; Mohamed et al., 2022), this study suggests that the growth of harmful microalgae should be controlled at the early stages (i.e., germlings) rather than treatment of algal blooms, which may cause algal cell lysis and release massive amounts of algal toxins into the aquatic environment. Since macroalgae or their extracts have been previously reported as ecofriendly materials (Jeong et al., 2000; Wang et al., 2007), the extracts of our macroalgae could be applied to a confined coastal area (i.e., bloom area) to inhibit the growth and division of germling cells after cyst germination, and thus restrict recurrence of HABs in these regions. However, further in situ study or mesocosm experiments are needed to determine ecological safety and efficacy under natural conditions. The potential impacts of these macroalgal extracts on other beneficial microalgae and microbial communities should also be examined in future studies.