*3.3. NO3-N Uptake Rate*

A significant effect of the culture model on the NO3-N uptake rates of *G. lemaneiformis* was detected (Figure 3A and Table 1). The NO3-N uptake rate at BDR of 2:1 was significantly greater than those of the other culture models. The NO3-N uptake rates of *S. horneri* were significantly different among culture models (Figure 3B and Table 1). The NO3-N uptake rates of the co-culture treatments were significantly greater than that at mono-culture. The NO3-N uptake rates decreased significantly with increasing biomass of *S. horneri*, from BDR of 2:1 to 1:2.

**Figure 3.** The NO3-N uptake rates of *Gracilariopsis lemaneiformis* (**A**) and *Sargassum horneri* (**B**) cultured for twelve days at mono-culture and co-culture with biomass density ratios of 2:1, 1:1, and 1:2. Different lowercase letters represent significant differences (*p* < 0.05) among culture models.

#### *3.4. Chl a*

The Chl *a* contents of *G. lemaneiformis* differed significantly among culture models (Figure 4A and Table 1). The Chl *a* content at BDR of 1:2 was significantly lower than at mono-culture and BDR of 2:1. Similarly, the Chl *a* contents of *S. horneri* differed significantly among culture models (Figure 4B and Table 1). The Chl *a* content at BDR of 2:1 was significantly greater than at mono-culture and at BDR of 1:2.

**Figure 4.** The contents of Chl *a*, soluble protein, and soluble carbohydrate of *Gracilariopsis lemaneiformis* (**A**,**C**,**E**) and *Sargassum horneri* (**B**,**D**,**F**) cultured for twelve days at mono-culture and co-culture with biomass density ratios of 2:1, 1:1, and 1:2. Different lowercase letters represent significant differences (*p* < 0.05) among culture models.

#### *3.5. Soluble Protein and Carbohydrate*

The one-way ANOVA results showed a significant difference among culture models on the soluble protein contents of *G. lemaneiformis* (Figure 4C and Table 1). The soluble protein content at BDR of 2:1 was significantly greater than at BDRs of 1:1 and 1:2. The soluble protein contents of *S. horneri* differed significantly among culture models (Figure 4D and Table 1). The soluble protein contents at BDRs of 2:1 and 1:1 were significantly greater than at mono-culture and BDR of 1:2.

A significant effect of the culture model on the soluble carbohydrate contents of *G. lemaneiformis* was detected (Figure 4E and Table 1). Compared to the mono-culture, the soluble carbohydrate content had significantly higher values at co-culture with different BDRs. Moreover, the soluble carbohydrate content at BDR of 1:2 was significantly greater than at BDRs of 2:1 and 1:1. Similarly, the soluble carbohydrate contents of *S. horneri* varied significantly among culture models (Figure 4F and Table 1). The soluble carbohydrate content was significantly higher at mono-culture than co-culture with different BDRs. The soluble carbohydrate contents increased significantly with increasing biomass of *S. horneri*, from BDR of 2:1 to 1:2.

#### **4. Discussion**

Diverse positive and negative interspecific interactions exist among plants in natural communities [50]. The principal mechanisms of these interactions, such as resource competition or allelopathy, are often species-specific and environment-dependent [51,52]. In this study, the RGR of *G. lemaneiformis* was significantly enhanced under co-culture with BDR of 2:1 compared to under mono-culture, indicating that the presence of *S. horneri* with a relatively lower biomass had a stimulating impact on *G. lemaneiformis*. A similar finding

was documented for *G. lemaneiformis* and epiphytic *U. prolifera*, showing that the RGR of *U. prolifera* was greatly increased under co-culture with BDR of 1:1 [53]. This 'hormesis' effect may have resulted from the division and expansion of cells through the enzymatic or non-enzymatic processes following low-dose phytotoxin stimulation, thereby further increasing the growth of thalli [54,55]. Additionally, this may be due to an overcompensation response following the initial damage caused by low concentrations of phytotoxins [56,57]. Therefore, we speculated that the secretion of allelochemicals played a more critical role in interspecific interactions than resource competition when *G. lemaneiformis* suffered from a low biomass value of *S. horneri*. Moreover, allelopathy presents complex mechanisms that can influence a variety of physiological and biochemical processes [58]. In this study, we observed a significant increase in the net photosynthetic rate, NO3-N uptake rate, and soluble protein content of *G. lemaneiformis* under co-culture with BDR of 2:1. Similarly, Pan et al. [53] showed that, compared to mono-culture, the Chl *a* content and photosynthetic rate of *G. lemaneiformis* were significantly enhanced under co-culture with 0.5 g L−<sup>1</sup> of *U. prolifera*. Further experiments are required to clarify the correlative mechanisms of these findings.

Compared to the stimulating effect of *S. horneri* on *G. lemaneiformis* with a relatively lower biomass, the RGR at BDR of 1:2 was significantly lower than at mono-culture. Similarly, Xie et al. [59] demonstrated that the initial population density rate greatly influences the interaction of *Heterosigma akashiwo* (H) and *Prorocentrum donghaiense* (P). The growth of *H. akashiwo* was restrained by *P. donghaiense* at the inoculation proportion of 1H:4P, whereas *H. akashiwo* possessed a higher growth rate at the inoculation proportion of 4H:1P. Both nutrient competition and allelopathy have been demonstrated to play a critical role in interspecific interaction between these two microalgal species. In addition, the net photosynthetic rate, Chl *a* content, and soluble protein content of *G. lemaneiformis* significantly decrease at BDR of 1:2. A similar result was presented in [24], where the growth, photosynthetic activity, and biochemical metabolism of *N. yezoensis* were significantly inhibited by epiphytic *U. prolifera*, mainly as a result of nutrition and light competition. On the other hand, high concentrations of allelochemicals may lead to cell expansion and rupture, while cells can maintain osmotic pressure and intracellular homeostasis by accumulating soluble carbohydrates [60–62]. In the current study, the soluble carbohydrate content of *G. lemaneiformis* presented a significant increase at BDR of 1:2, indicating that *S. horneri* may have exerted a strong allelopathic effect on *G. lemaneiformis* under this condition. Therefore, the inhibitory effect of *S. horneri* with a relatively higher biomass on *G. lemaneiformis* may be associated with a combination of resource competition and allelopathy.

Compared to the mono-culture, the RGR, net photosynthetic rate, NO3-N uptake rate, Chl *a* content, and soluble protein content of *S. horneri* were enhanced under co-culture conditions with *G. lemaneiformis* at different BDRs. Macroalgal species with a stronger growth capacity usually have advantages in interspecific competition scenarios [51,63]. Our results showed that *S. horneri* (7.23%) has a better growth rate than *G. lemaneiformis* (3.53%) under mono-culture conditions. The outstanding growth performance of *S. horneri* may be correlated with its adaptability to frequent marine environment changes during the long-distance floating processes [64]. Additionally, *U. lactuca* showed greater growth and maximal photosynthetic ability than *G. lemaneiformis* under competitive conditions, benefiting from its larger frond surface area and higher nutrient uptake rate [65]. All these results indicate that *S. horneri* has a dominant role in its competition with *G. lemaneiformis* because of its physiological and morphological advantages. In addition, we observed that all physiological and biochemical parameters of *S. horneri* increased significantly with increasing biomass of *G. lemaneiformis* under co-culture conditions. Wang et al. [66] reported that co-culture with a small quantity of *G. lemaneiformis* thalli was significantly beneficial to the growth of the red tide species *H. akashiwo*. Similarly, *G. lemaneiformis* can stimulate the growth of *S. costatum* by secreting various types of allelochemicals [67]. Therefore, we suggest that certain allelochemicals produced by *G. lemaneiformis* may partially contribute to the increased growth rate of *S. horneri*.

The golden tide caused by *Sargassum* species has become a major ecological threat to the coastal environment and aquaculture production. According to the present data, although the occurrence of small quantities of *S. horneri* can stimulate the growth of *G. lemaneiformis,* its outbreak has a greatly harmful impact on the growth and biochemical metabolism of *G. lemaneiformis*, resulting in reduced yield and quality. Inversely, the growth of *S. horneri* is enhanced by the occurrence of *G. lemaneiformis*, regardless of BDR. This observation suggests that *G. lemaneiformis* cultivation has no inhibitory effect on golden tide, rather favoring its further expansion. Hence, *S. horneri* should be removed or intercepted in time to minimize economic and ecological losses. Due to the limited availability of data in this study, more studies involving related physiological and molecular mechanisms are required to evaluate the coping strategies of *G. lemaneiformis* against golden tide outbreaks.

**Author Contributions:** Investigation, data curation, visualization, writing—original draft, H.S.; conceptualization, methodology, project administration, funding acquisition, writing—review and editing, Y.L. and X.G.; formal analysis, resources, J.L.; validation, supervision, Q.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by the National Key R&D Program of China (No. 2020YFD0900201) and the Fundamental Research Funds for the Central Universities of China (No. 842212015).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data in this study are available from the corresponding author upon reasonable request.

**Acknowledgments:** We are grateful to QiaohanWang of Ocean University of China for experimental assistance.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

