Global freshwater pearl production accounting for more than 75% is achieved by the triangle sail mussel Hyriopsis cumingii from China (Tang et al., 2015). On the one hand, water pollution, disease outbreaks, and benthic ecological destruction often occur in the traditional cultivation of H. cumingii because of the use of massive amounts of organic and chemical fertilizers (Hu et al., 2015). The development and exploitation of a new sustainable cultivation mode such as industrialized culture are urgent, and the selection of microalgae with high quality and nutritional value is the key. On the other hand, freshwater bivalves have been considered a biomanipulation tool to control water eutrophication based on ecological functions such as biofiltration, biodeposition, and bioturbation (Gulati et al., 2008; Gu et al., 2020). In small-scale bivalve biomanipulation experiments, H. cumingii can effectively reduce concentrations of total suspended solids and the number of cyanobacteria, increase transparency, and change the community structure of phytoplankton in water (He et al., 2014; Wang et al., 2018). Toxic algae Microcystis aeruginosa is the most common species from cyanobacterial blooms, which causes adverse effects on the physiological responses of H. cumingii (Hu et al., 2016). Therefore, urgent studies about contrasting molecular and biochemical effects of high-quality microalgal diets with the toxic M. aeruginosa on the metabolism and immune physiology of H. cumingii are needed.

As the main food for bivalves, algae is the natural nutritional base and primary source of nutrients. Diatoms such as Navicula sp., Cyclotella sp., and green algae Chlorella vulgaris, which are rich in nutrients including carbohydrate, lipid, and protein, have been applied to bivalve culture. Previous studies have shown that different algae affect the growth, pearl production, fatty acid composition, and energy metabolism of H. cumingii (Chen et al., 2021). Mussels have sensitive physiological responses such as changes in enzyme and gene levels to different algal species or concentrations (Blanco-Rayon et al., 2019; Sun et al., 2021). Metabolic enzymes including glutamic oxaloacetate transaminase (GOT), glutamic pyruvate transaminase (GPT), pyruvate kinase (PK), and hexokinase (HK) have a key role in protein, carbohydrate, and glucose metabolism (Xia et al., 2015). Low-density lipoprotein receptor (LDLR) and carnitine palmitoyltransferase 1 (CPT1) have a crucial function in lipid uptake, transport, and catabolic metabolism (Liu et al., 2018). Hydrolases alkaline phosphatase (AKP) and antioxidase superoxide dismutase (SOD), and catalase (CAT), as non-specific humoral immune factors, participate in immune responses (Hu et al., 2015). Interleukin-17 (HcIL-17) and inhibitor of apoptosis protein (IAP) have crucial functions in the inflammatory reaction, immune responses, and defense of bivalves (Rosani et al., 2015; Zhu et al., 2021). Bivalve gill is the tissue that first comes into contact with foreign particles, and changes in the hemolymph can reflect the process of oxidative damage and the metabolism of pollutants (Pan et al., 2006). Functional features of the bivalve liver include the metabolic reserve site and the helper of transferring these reserves to other organs (Albentosa et al., 2012).

Therefore, detecting the above molecular and biochemical parameters could reflect the health and physiology status of mussels when exposed to three microalgal diets and the toxic M. aeruginosa. This study provides new considerations for technical issues such as the sustainable cultivation of bivalves and the implementation of bivalve biomanipulation in eutrophic water.

The cultivation mode of H. cumingii urgently needs transformation and upgrading, and industrialized culture has become a new direction for the sustainable development of freshwater shellfish in the future because of its characteristics of environmental protection, high yield, and easy operation. The key to success in industrialized culture is the exploration of preferred microalgal diets of H. cumingii. In our study, enzyme and gene expression levels of digestion (showed in Figure S1 in the Supplementary Material ), metabolism and immunity in H. cumingii maintained higher levels in the Cyclotella sp. treatment on day 28, indicating that Cyclotella sp. was suitable algae for H. cumingii, and mussels in this treatment had better metabolic abilities, immune function, and physiological status. In C. vulgaris and N. pelliculosa treatments, enzyme and gene expression levels of metabolism were lower than those in the Cyclotella sp. treatment. This is because the effects of algal species on bivalves are critically related to their abilities to supply essential nutrients to bivalves, in addition to physical properties of algae such as size and shape. The nutritional value of algae is mainly reflected in differences in biochemical components, and compared with proteins and carbohydrates, the lipid composition is more likely to lead to differences (Geng et al., 2016). Because of the low ability to synthesize and biotransform essential fatty acids, bivalves must obtain these nutrients from diets. Similar to our findings, a study proves that Cyclotella sp. is the optimum microalgal diet for H. cumingii from the perspective of the greatest increases in growth and pearl weight, saturated fatty acids, and monounsaturated fatty acids of mussels.

In addition to physical and chemical methods, different nature-based biomanipulation methods are used in a large number of practical cases to control algal blooms (Triest et al., 2016). With the emergence of shortcomings in fish manipulation (an inadequate reduction in the standing stock of planktivorous fish and the inability of piscivorous fish to control small fish) (Gulati et al., 2008), utilizing interactions of bivalves and algae makes bivalves a potential biomanipulation tool to effectively control eutrophication of water bodies. In small-scale bivalve biomanipulation experiments for mostly less than 30 days, freshwater bivalves can effectively reduce concentrations of total suspended solids, chlorophyll content, and concentrations of total phosphorus and increase transparency and concentrations of soluble reactive phosphorus in water (Wu et al., 2018; Gu et al., 2020). A 14-day exposure experiment of M. aeruginosa (1.7 × 10⁷ cells ml⁻¹) found that short-term ingestion of toxic M. aeruginosa is not fatal to H. cumingii and that mussels also can recover to health (Liu et al., 2020). In our study, M. aeruginosa stimulates immune responses of H. cumingii in a short-term exposure (7 days), and there was a significant reduction in the metabolism and immune abilities of mussels on the 28th day of this treatment. On the one hand, there were no dead mussels, and the concentration of M. aeruginosa used in the experiment (4.5 × 10⁶ cells ml⁻¹) was higher than that in natural environment (about 2 × 10⁵ cells ml⁻¹ in China Lake Taihu and Dianchi), indicating that H. cumingii has a high tolerance to M. aeruginosa and the ability to control eutrophication in the natural environment. On the other hand, it should deserve attention that the toxic M. aeruginosa exhibits negative effects on the physiology such as the clearance rate and absorption efficiency of bivalves (Hu et al., 2016). The same as the results of our study, exposure to M. aeruginosa still influences the metabolism and the immunological mechanism of bivalves (Hu et al., 2015). Therefore, more studies are needed to use H. cumingii for bivalve biomanipulation. The purpose of this study was to help screen suitable algal diets for H. cumingii in industrialized cultures and provide supporting theories for bivalve biomanipulation by contrasting the effects of high-quality microalgal diets and the toxic M. aeruginosa on the physiological state of H. cumingii. The following is a specific analysis of how these microalgae affected the metabolism, immune enzyme, and gene expression levels of H. cumingii.

Transaminases have a key role in mobilizing amino acids for gluconeogenesis and function in carbohydrate and protein metabolism (Ramaswamy et al., 1999). Levels of GOT and GPT can reflect the turnover efficiency of amino acids, and they are usually used as biochemical markers to determine tissue damage in the liver when aquatic animals face changes in physiology and pathology induced by environmental conditions (Hong et al., 2015). The ability of aquatic animals to use carbohydrates depends on their ability of glucose oxidation, glycogen biosynthesis, and lipogenesis (Guo et al., 2006). HK and PK are two key glycolytic regulatory enzymes, and they can reflect the capacity to metabolize glucose in aquatic animals (Xia et al., 2015). A study about effects on aminotransferases activities of sea cucumber Apostichopus japonicus fed with different diets such as brown algae, red algae, and green algae and addition of benthic matter (composed of benthic diatoms, dinoflagellates, detritus, and fine clay particles) for 70 days shows that the mixture of red algae and benthic matter significantly increases GOT and GPT activities, transamination, and protein metabolism, and A. japonicus has a higher specific growth rate (Wen et al., 2016). To assess the influence of food type on biomarkers, the PK activity of mussels Mytilus galloprovincialis fed with commercial food is much lower than those fed with live microalgae (Isochrysis galbana and Tetraselmis chuii), suggesting decreased aerobic scope, increased gluconeogenesis, and lower nutritional status of mussels (Blanco-Rayon et al., 2019). In our study, GOT, GPT, PK, and HK activities of mussels in the Cyclotella sp. treatment remained stable and were significantly higher than those in C. vulgaris and N. pelliculosa treatments after 7 days. The 28-day toxic M. aeruginosa treatment significantly reduced these metabolic enzyme activities in the liver of H. cumingii. Results suggested that mussels in the Cyclotella sp. treatment had a higher capacity to metabolize protein and glucose, and decreased metabolic enzyme activities in mussels from the M. aeruginosa treatment might be caused by liver tissue damage.

LDLR has a crucial function in lipid uptake and transport into liver cells, then lipids are targeted to catabolic and anabolic pathways, and CPT1 has a key role in lipid catabolic metabolism (Liu et al., 2018). Similar to the results of the above metabolize enzymes, mussels in the Cyclotella sp. treatment had higher expression levels of LDLR and CPT1 than mussels in C. vulgaris and N. pelliculosa treatments, suggesting that these mussels had higher lipid uptake and metabolic efficiency. The 28-day toxic M. aeruginosa treatment led to metabolism dysfunction and affected the expression of related genes in mussels.

How to boost the immunity of mussels through nutritional methods should be a concern. Bivalves rely on innate immunity to provide defense, and this immunity involves both cell-mediated and humoral systems. The phosphomonoesterase AKP has a crucial function in the digestion and degradation of foreign proteins, carbohydrates, and lipids in the lysosome, participating in non-specific immune actions of aquatic animals (Liang et al., 2014). Under oxidative stress, when the production of free radicals exceeds the level of natural antioxidant defense regulation, it triggers the oxidation of DNA, protein, and lipids, resulting in multiple disease conditions (Ighodaro and Akinloye, 2018). Antioxidant enzymes such as SOD and CAT are the first and most important line of defense in the antioxidant system, respectively, dismutase superoxide radicals into H₂O₂ and water and breakdown of H₂O₂ into O₂ and water. Hydrolases AKP and antioxidase SOD and CAT, as non-specific humoral immune factors, participate in immune responses, and they are often used as reliable indicators in assessing the immune status of bivalves (Hu et al., 2015). Our results showed that AKP, SOD, and CAT activities of H. cumingii in the Cyclotella sp. treatment remained stable, and mussels in this treatment had higher immunity. The fluctuation of these immune enzyme activities in C. vulgaris and N. pelliculosa treatments may be related to changes in immune responses and physiological instability of H. cumingii caused by these algae. Similar to our study, after 45 days, feeding diets such as Chlorella sp. powder and Spirulina platensis powder significantly increased AKP, SOD, and CAT activities and absolute growth rates of pearl oyster Pinctada fucata martensii as compared with the corn gluten diet, suggesting that immunity of P. f. martensii can be improved by these algal diets. However, most AKP, SOD, and CAT activities in the M. aeruginosa treatment significantly increased at first and then decreased. It suggested that toxic algae stimulated immune responses of mussels in a short time (Hu et al., 2015) and then caused immune damage and enzyme inactivation.

HcIL-17 is a key inducer of proinflammatory cytokine secretion, IAP has a crucial function in regulating apoptosis and immune defense, and these genes have been verified to be involved in the bivalve immune responses (Rosani et al., 2015; Zhu et al., 2021). HcIL-17 and IAP expression levels of H. cumingii in the M. aeruginosa treatment increased on day 1 and then decreased after 14 days. Toxic algae created an inflammatory milieu in liver tissues and stimulated the immune response of mussels (Dou et al., 2020), and long-term stress of toxic algae caused immune disorders and metabolic imbalance and reduced the expression of related immune genes at last.

Results of PCA ( Figure 4 ) visually supported that the suitable algal diet played a significant role in metabolic abilities, immune function, and physiological status, shown by the strong clustering of the side with higher enzyme activities in the Cyclotella sp. treatment during the experimental period. When accompanied with the 28-day toxic M. aeruginosa treatment, the clustering effect weakened, suggesting that toxic algae caused metabolic disorders, immune injury, and poor physiological status.

Metabolism, immune enzyme, and gene expression levels in H. cumingii had no significant changes and remained at a high level in the Cyclotella sp. treatment on day 28, suggesting that Cyclotella sp. was the suitable algae for H. cumingii, followed by C. vulgaris and N. pelliculosa. The toxic M. aeruginosa treatment resulted in metabolic disorders and immune injury, though no mussels died. These findings provide basic information on metabolism and immune mechanisms in H. cumingii exposed to different algae. How the suitable algae can apply to practices of the long-term industrialized culture of H. cumingii by considering mussel growth and the pearl quality should be studied in the future. How to apply H. cumingii to bivalve biomanipulation in a large-scale or whole ecosystem needs more studies about the tolerance of mussels to M. aeruginosa, specific stocking methods, and adjusting measures to local conditions.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed by the authors.

This work was supported by the earmarked fund for CARS (Grant No. CARS-49), the National Natural Science Foundation of China (Grant No. 31770965), and the Chongqing Natural Science Foundation Project (Grant No. cstc2020jcyj-msxmX0971).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.