Competitive effects of the macroalga Caulerpa taxifolia on key physiological processes in the scleractinian coral Turbinaria peltata under thermal stress

Sample collection

T. peltata (with a skeleton size of length:width:height = 20:18:12 cm, one colony) and C. taxifolia (5 kg) were collected from the Xuwen Coral Reef National Nature Reserve (109° 55′ E, 20° 16′ N) at a depth of approximately 4 m. The samples were transported to the laboratory and cultured in two 200-L tanks at a temperature of 26.5 °C, pH of 8.0, salinity of 33, and 200 µmol photons m⁻²s⁻¹ with a 12: 12 h light/dark cycle for 3 months. Blue lighting was provided by a photosynthetically active radiation (PAR) lamp (Aqua Knight M029). An illuminometer (UNI-T UT383) was used for light measurement. After 3 months of acclimation, the corals from the same colony were cut into 54 pieces, approximately 4 cm in diameter, and were fixed on a ceramic base with aqua rubber (Aron Alpha GEL-10). The samples were then placed in another 200-L tank for 1 week until the anthopolyp had stretched naturally.

Experimental design

After acclimation, 54 coral nubbins were randomly allocated into 10 L experimental tanks. To mimic the coral-macroalgal interaction in coral reefs, the same amount of macroalgae was co-cultured with the coral samples in the following ways: (1) no algae were added to the tank, i.e., the control group (Fig. 1A). (2) Algae (25 g) were cultured in external algae boxes with no direct contact between the algae and the coral samples. The allelopathic substances produced by algae could enter the tank with the corals by water flow to determine whether the algae had a water-mediated interaction in the co-occurrence group (Fig. 1B). (3) The algae and corals were co-cultured in the same tank with direct contact, and a panel made of polymethyl methacrylate (PMMA) was used to fix the height of the algae parallel to the coral samples; this was referred to as the direct contact group (Fig. 1C). The experimental tanks were subjected to ambient conditions (27 °C) and the shared socioeconomic paths (SSPs) scenario SSP2-4.5 (30 °C) (Zhongming, Linong & Xiaona, 2021), to mimic the typical temperature range that the corals experienced at Xuwen Coral Reef Nature Reserve in order to explore the coral-macroalgae contacts at different temperatures. Each treatment contained three replicate tanks, within which three coral nubbins were placed per tank.

The temperature in each experimental group was increased to the set temperature by 1 °C per day. The first 3 days were the temperature adjustment period. Some algae tips decomposed due to metabolism or blue light intolerance. Therefore, the algae were checked and replaced each day to ensure that the experimental group had 25 g (0.0025 g cm⁻³) of fresh algae, which is the amount of algae with the density of 0.0022 g per cubic meter of water surveyed from the inshore reef of Xuwen Coral Reef Nature Reserve. Fifty percent of the seawater was replaced every 3 days in each tank. Organisms were kept under treatment conditions for a period of 28 days and physiological measurements were subsequently performed.

Endosymbiont density and Chl a content

At the end of the experiment, coral tissue were removed from the nubbins using a waterpick (0.45 µm filtered seawater), and the slurry was homogenized. Six 15 ml samples were taken from the slurry, centrifuged (4,000 rpm min⁻¹, 4 °C, 10 min), and the supernatant was removed. Part of the pellet was suspended in 5 mL formaldehyde to count the endosymbiont density under an inverted microscope (DMI 6001B, magnification eyepiece × magnification objective: 10 × 20) with a blood counting plate (CKSLAB CB30). Another portion was resuspended in 8 mL methanol. The pigments were extracted at 4 °C for 24 h. The extract was centrifuged (4,000 rpm min⁻¹, 4 °C, 10 min), and Chl a was determined according to the method described by Ritchie (2006). Data were normalized to skeletal surface with the aluminium foil technique (Marsh, 1970).

Feeding rate

A total of 1 ml of Artemia solution was added to 1 L of water and a plankton counter was used to measure the mixed solution and to determine the individuals of the Artemia solution per milliliter. Nubbins were moved into the feeding tanks (1 L) with an Artemia concentration of ~ 2 ind mL⁻¹, while one tank served as a control (without coral). After an incubation period of 1 h, the coral nubbins were rinsed with seawater and returned to their respective positions in the experimental tanks. The feeding rate was calculated as the decline in Artemia concentration in the feeding tanks and normalized per polyp. The measurement was performed once a week between 11:00–12:00 a.m. The number of polyps in each nubbins were visually counted before the experiment.

Growth rate

The coral nubbins were weighed on a balance (UW 2200H, accuracy = 0.01 g) using the buoyant weight technique (Davies, 1989). Before each measurement, the surface of the coral ceramic base was lightly brushed with a toothbrush to remove algae. A glass beaker was filled with 1 L of filtered seawater (27 °C, salinity 32). Then the coral nubbins were placed on the bottom of the beaker and the weight (minus the weight of the beaker without samples) was measured. The growth rate (g d⁻¹) was calculated as $\left(M_{ti}-M_{t0}/T_i\right)$, where M_t0 represents the nubbin weight at the beginning of experiment, M_t1 the weight at the measureing time point and T_i represents the duration in days. The measurement was repeated every 7 d. Data were normalized to skeletal surface area determined with the aluminium foil technique (Marsh, 1970).

SOD and CAT

The homogenized coral tissue slurry that was used to measure the endosymbiont density and Chl a content was centrifuged (4,000 rpm min⁻¹, 10 min, 4 °C), and the supernatant was collected to measure the SOD and CAT activities. These measurements were determined in the dilution using kits (A001-1-1, A007-1-1; Nanjing Jicheng, Nanjing, China). A BCA (Bicinchoninic Acid Assay) kit was used to determine the protein concentration (A045-3-1; Nanjing Jicheng, Nanjing, China). The enzyme activities were normalized to total protein content as U mg prot⁻¹.

Data analysis

The results are presented as the means ± standard deviations. Data were tested for homogeneity of variance (visual inspection of residuals vs. fitted values), and the normality of the residuals was tested using the Shapiro‒Wilk normality test. All response data of corals were tested using a two-factor analysis of variance (ANOVA) with “temperature (27 °C, 30 °C)” and “algae (control, direct contact, indirect interaction)” as fixed factors, including the interaction term. When significant effects of factors occurred, ANOVAs were followed by Tukey’s multiple comparisons test for identifying differences between algal treatments at the same temperature and Sidak’s multiple comparisons test for identifying differences between temperature treatments. The data was analysed and graphed using GraphPad Prism 8.0 and p < 0.05 was considered a significant difference (the statistical results of p-value supported by the Supplemental Material).

Seawater chemistry monitoring

The temperature, pH, and salinity values were measured continuously during the experiment (Fig. 2).

Endosymbiont density and Chl a content

Figures 3A and 3B show that the density and pigment content of the endosymbiont were significantly influenced by temperature. Without algae, elevated temperature resulted in an average 44.6% (from 1.3 ± 0.1 to 0.72 ± 0.07 × 10⁶ cells cm⁻²) decrease in endosymbiont density (p < 0.01) and a average 58% (from 17.1 ± 1.5 to 7.2 ± 2.6 µg cm⁻²) decrease in Chl a content (p < 0.01). Compared with the control group, the co-occurrence group (0.95 ± 0.1 × 10⁶ cells cm⁻², p = 0.03) and direct contact group (0.89 ± 0.2 × 10⁶ cells cm⁻², p = 0.01) in 27 °C temperature treatments showed lower mean values of endosymbiont density. However, there were not significantly differences between the control, co-occurrence and direct contact groups at 30 °C. The Chl a concentration only differed significantly between the control and the direct contact group at ambient temperature (p = 0.03).

Feeding rate

The results of the feeding rate are displayed in Fig. 4A. Primarily, elevated temperature had a significantly detrimental effect on the feeding rate among the treatments. There was significant difference between the the co-occurrence group (p = 0.02) and the direct contact group (p = 0.03) compared with control group at 27 °C. At 30°C, direct contact with algae caused the feeding rate to fall to a minimum of 12.8 ± 1.1 ind polyp⁻¹ h⁻¹ compared with control group (p < 0.01). The interaction of temperature and algae influenced the feeding rate significantly (F = 4.7, p = 0.04, Table 1).

Protein content

The results of protein content are shown in Fig. 4B. At 27 °C, algae caused a loss of 37.5% of protein content in coral tissue in the co-occurrence group (p = 0.01) and 49.0% protein content in the direct contact group (p < 0.01). Although contact with algae further decreased the mean of protein content, the difference was not significant with the co-occurrence group. At 30 °C, the direct contact with algae resulted in the lowest protein content of 1.2 ± 0.3 mg cm⁻² (p < 0.01, p < 0.01) compared with the control and co-occurrence groups. No significant difference was observed between the temperature treatments in the presence or absence of algae (p = 0.11, Table 1). There was no significant interaction between algae and the temperature treatment (F = 2.86, p = 0.12, Table 1).

Growth rate

As indicated by the change in buoyant weight, the growth rate was affected by algae at 27 °C and the effect of elevated temperature was only seen in the control group (Fig. 4C). At 27 °C, the growth rate of corals in the control group was highest, with a mean value of 4.1 ± 1.4 mg cm⁻² d⁻¹. Compared with control group, the coculture with macroalgae decreased the growth rate of coral by 57.7% in the co-occurrence group (p < 0.01) and 65.5% in the direct contact group (p < 0.01). But no significant difference between co-occurrence and direct contact group. Elevated temperature had an inhibitory effect on the growth rate in the coral culture system without algae. The growth rate in this group was 83.4% lower than the control group (p < 0.01). The elevated temperature combined with direct contact with algae resulted in the lowest coral growth rate, with a value of 0.57 ± 0.45 mg cm⁻² d⁻¹. However, the differences among algae treatments at an elevated temperature were not significant. The interaction of temperature and algae influenced the growth rate significantly (F = 28, p < 0.01, Table 1).

SOD and CAT

As shown in Fig. 5A, macroalgae treatments increased the SOD antioxidant capacity of corals under both temperature conditions. At 27 °C, co-occurrence with algae increased the SOD activity of coral 1.85-fold compared with control group (p = 0.03). Moreover, the SOD activity was higher for the direct contact group (288.1 ± 16.6 U mgprot⁻¹) than control groups (p < 0.01) and co-occurrence group (p = 0.03). At 30 °C, the mean SOD activity of coral without the presence of algae increased by 1.77-fold compared with its counterpart at ambient temperature (p = 0.03). In the direct contact group at 30 °C, SOD activity increased to the highest level of 354.3 ± 59.56 U mg prot⁻¹ compared with control group (p < 0.01). However, in the co-occurrence and direct contact system, there was no significant difference caused by elevated temperature, indicating that both factors did not interact (F = 2.37, p = 0.16, Table 1).

The CAT activity also increased after algae interaction and elevated temperature, as shown in Fig. 5B. At 27 °C, co-occurrence with algae caused the CAT activity in coral tissue to rise 5.3-fold (p = 0.046), which is comparable to the level of CAT activity in the direct contact group (p = 0.04). At 30 °C, the CAT activity in the control also increased by 7.1-fold (p < 0.01). Moreover, when cultured in contact with the algae, the CAT activity further doubled compared with the control group (p = 0.03). The combined effect of temperature and macroalgae was significant (F = 5.13, p = 0.04, Table 1).

Effects of C. taxifolia on endosymbiont of T. peltata

Algae was found to influence the average endosymbiont density and chl a content of T. peltata, however, no bleaching occurred. A number of studies have reported that coral’s photosynthetic efficiency decreased (Fv/Fm), or bleaching occurred, when there was direct or indirect contact with macroalgae. However, not all coral species are equally susceptible to algae and not all algae will have deleterious effects on corals (Smith et al., 2006; Rasher & Hay, 2010; Fong et al., 2020). Rasher & Hay (2010) suggested that Padina perindusiata and Sargassum sp. did not inhibit photosynthetic efficiency or induce bleaching of Porites porites, which might be explained by the fact that the 20-day interaction period was too short to impact the coral health. Additionally, T. peltata is a massive coral that could resist environmental pressure by increasing its basic metabolism (Loya et al., 2001). This may explain why there was no significant bleaching effects of macroalgae on endosymbiont density. There was a stronger effect for direct contact compared to co-occurrence in chl a concentration at 27 °C, suggesting that physical mechanisms are still the main way in which macroalgae affect corals.

Effects of C. taxifolia and thermal stress on the physiology of T. peltata

The feeding rate was affected by elevated temperature. Johannes & Tepley (1974) also found that the feeding rate of coral decreased in heat stress because of the polyp contraction or a loss of nematocyst function. Our results suggest that a decrease in chl a content and endosymbiont density was the reason why the feeding rate was impacted in elevated temperature. Endosymbionts provide photosynthate to host cells (Van Oppen & Blackall, 2019). A decrease in the endosymbiont density at high temperatures may result in reduced energy expenditure to maintain normal physiological functions and reduce resistance to predation. This study showed that contact with C. taxifolia resulted in the greatest reduction in the feeding rate at 30 °C. In summary, thermal stress might a crucial factor affecting the feeding ratio of T. peltata, which became more severe when in contact with macroalgae.

Macroalgae can induce reduced protein content in corals. Damage to coral tissue by contact with macroalgae has been documented in many studies. Bender, Diaz-Pulido & Dove (2012) asserted that Acropora sp. lost tissue and decreased its growth rate due to allelopathy mechanisms after coming into contact with Chlorodesmis fastigiata. In fact, macroalgae may transfer many allelopathic substances to corals, altering the structure of the microbial community and impacting the physiological processes of corals (Fong et al., 2020). This damage may ultimately reduce the protein content. Under stress, massive corals with thicker tissues may overcome the effects of endosymbiont loss through catabolism (DeCarlo & Harrison, 2019). Macroalgae may affect coral tissue by creating anoxic zones. Barott et al. (2009) demonstrated that after the interactions between corals (Pocillopora verrucosa, Montipora sp.) and some species of macroalgae (e.g., Gracilaria sp., Bryopsis sp., and various turf algae), the characteristic patterning of coral pigments and polyps was altered and the tissue appeared damaged.

The growth rate of coral was altered by the macroalgae and temperature, the results were consistent with previous studies (Tanner, 1995; Rölfer et al., 2021; Vega Thurber et al., 2012; Vermeij et al., 2009). At 27 °C, the growth rate of the direct contact group was lower than that of the co-occurrence group. Therefore, the effects of seaweed on coral growth may require direct contact at ambient temperatures (Clements et al., 2020). Brown et al. (2019) also demonstrated that coral growth was reduced or even negative at 30 °C when in contact with algae. Longo & Hay (2015) determined that corals showed signs of stress at 30 °C, and contact with Halimeda heteromorpha further contributed to a decreased growth rate and increased mortality rate. These results may be due to the simultaneous decline in the autotrophic and heterotrophic activities of corals under the impacts of thermal stress or macroalgae, resulting in a drop of protein content and ultimately affecting the growth rate.

Effects of C. taxifolia and thermal stress on oxidative stress of T. peltata

Corals under thermal stress may produce ROS (Blanckaert et al., 2021). Downs et al. (2002) documented that when exploring the varied oxidative stress response of coral under seasonal change, the SOD in summer was three times higher than that in winter. This study determined that SOD in corals was higher when macroalgae were present (the effect was stronger for direct contact compared to co-occurrence group), the increased temperature, or there was the synergistic effect of both. Thus, weakened corals were found to be more vulnerable to competition from algae, which was also supported by the results of Diaz-Pulido et al. (2010). The level of both antioxidant enzyme activities was similar to thermal stress alone when C. taxifolia indirectly contacted T. peltata. These results indicate that the stress triggered by macroalgal allelochemicals on coral was equivalent to that induced by increased temperature. The temperature effect was stronger in CAT activity compared with SOD activity under direct contact, which may be related to the reduced protein content in coral tissues caused by elevated temperature under the direct contact treatment. Due to the evident decrease in the protein content of coral tissues in the direct contact group, the amount of antioxidant enzymes produced by coral is not enough to resist the damage of ROS.