The circadian rhythm is a 24-h cyclical change in environmental factors such as light and temperature caused by the rotation of the earth, which has an important impact on the physiology and behavior of organisms (Fustin et al., 2013). The circadian rhythms inside and outside the organism are closely related to the rhythm changes of the external environment. When the rhythm of environmental factors is changed, the nervous system and endocrine system of animals will change accordingly, leading to changes in their behaviors, lifestyles, and physiological conditions (Wu et al., 2002).

The feeding rhythm of animals affected by cyclical change factors (e.g., light and tide) is essential for the establishment of scientific feeding mode (Mistlberger, 1994; Sanchezvazquez, 1995; Wang, 2004; Connor and Gracey, 2011). Many activities such as feeding, oxygen consumption and digestion have been used to study rhythmic behavior in European oyster Ostrea edulis, New Zealand cockle Austravenus stutchburyi, clam Saxidomus purpuratus, etc (Morton, 1971; Williams and Pilditch, 1997; Kim et al., 2003). Furthermore, the absorption of food after feeding is closely related to the process of digestion and metabolism. Meanwhile, digestive enzyme activity is an important indicator of digestion and absorption capacity, which determines the ability to digest and absorb nutrients for food (Bobrowska et al., 2011; Wu et al., 2013). The relationship between the activity of digestive enzymes and the feeding pattern has been studied in cockle Cerastoderma edule (Ibarrola et al., 1998), clam Ruditapes decussatus and Venerupis pullastra (Albentosa and Moyano, 2008), and scallop Patinopecten yessoensis (Li et al., 2010). In addition, some previous studies have found that the activities of digestive enzymes such as pepsin, amylase, cellulase, and lipase are served as indicators that can measure the digestion and absorption of protein and carbohydrates in bivalves (Supannapong et al., 2008; Tizon et al., 2013). For animals, digestion is a complex process that requires signal transduction regulation inside and outside the body. The enzyme-related protein precursor mRNA is regulated to stimulate transcription and synthesis after ingestion, so that the enzyme precursors are released to promote digestion, absorption, and growth (Yúfera et al., 2018). For example, amylase (AMY) gene is proved to be involved in the growth of razor clam Sinonovacula constricta and Pacific oyster Crassostrea gigas, while cellulase (CEL) can improve the digestibility of food and synthesize glucose to provide energy for the body (Meenu et al., 2014; Thongsaiklaing et al., 2014; Rong et al., 2015; Liu et al., 2017). Moreover, lipase (LIP) gene expression is correlated with feeding status, whereas pepsin (PEP) has important digestive functions in both vertebrates and invertebrates (Liang et al., 2003). The presence of various forms of pepsin precursors such as pepsin A and pepsin C may be related to food or feeding habits (Carginale et al., 2004).

S. constricta is an economically and ecologically important benthic marine bivalve, which naturally distributes along the western Pacific coasts of China, Japan, and South Korea (Morton, 2010). It has been widely cultivated in the intertidal zone and estuary waters with more than 400 years of cultivating history in the Zhejiang and Fujian provinces, China (Shen et al., 2013). Due to relatively short production cycle and high productive efficiency, it has been become an important marine aquaculture species in China with 852,925 tons of production in 2018 (FAO, 2020). Food intake is one of the important conditions for increasing the yield of industrial aquaculture (Rønnestad et al., 2013). Good ingestion and efficient digestion of nutrients are necessary to meet the high demand of matter and energy, which are able to support the high-growth rate (Navarro-Guillén et al., 2018). In order to determine the optimal feeding time and increase the growth rate of S. constricta, we continuously measured feeding rate, digestive enzyme activity, and relative expression of digestive enzyme genes at different time points in the day and night. The present results will be beneficial for reducing farming cost and increasing farming benefits.

For most organisms, circadian rhythm is affected by endogenous factors and external environmental factors (Mata-Sotres et al., 2016). Under natural light conditions, the behavior of most animals is divided into two types: diurnal and nocturnal. Understanding and clarifying the circadian rhythm can provide a scientific basis for revealing accurately the laws and inner mechanisms of many life phenomena. Although the feeding rhythm is of great significance for establishing a scientific feeding mode to farmed animals, there are few studies on the feeding rhythm and their mechanisms in aquatic animals. In fish, as reported, the Gilthead sea bream Sparus aurata showed an feeding rhythm during the day under a light/dark condition (Mata-Sotres et al., 2015). In contrast, the Japanese prawn Penaeus japonicus feeds at night under the same conditions (Reymond and Lagardère, 1990). In mollusks, many species showed similar feeding and metabolic rhythm. For example, Manila clam Ruditapes philippinarum exhibited a 24 h circadian rhythm of feeding rates, having a higher feeding rate at night than that in the day (Jiang et al., 2009). A study on circadian feeding activity and digestive physiology in the abalone Haliotis discus hannai found that the percentages of ingesting food were significantly higher at night than in the daytime, demonstrating their nocturnal characteristics (Gao et al., 2021). An obvious circadian rhythm in water filtration rate was also found in the scallop Chlamys farreri, which had a significantly higher water filtration rate at night than that in daytime (Du et al., 2012). In S. constricta, the obvious diurnal changes in food intake rates were proved by the energy budget study (Li et al., 2006). In the present study, we found that the feeding rate of razor clam in dark was significantly higher than that in light, speculating that there may be a circadian rhythm of feeding, which is indirectly regulated by biological clock genes. Additionally, the highest feeding point of mollusks at night may be related to avoiding being preyed by predators. Moreover, some studies in aquatic animals have found that light is the most important external factor influencing the feeding activity and different modes of light conditions will reduce the intake of food (Li et al., 2010). Especially, for the nocturnal animals that forage at night, light inhibits their feeding behavior. However, the long-term evolutionary internal biological clock can regulate the life patterns of animals, which may control the time and amount of consumed food (Annie et al., 1999). These findings may thus help us optimize the feeding time and frequency in clam aquaculture.

The degree of digestion is derived from the intestinal transport factor and digestive enzyme activity that reflects the digestion and absorption capacities of intestinal tract (Mata-Sotres et al., 2015; Navarro-Guillén et al., 2015). Generally, there is a close relationship between the digestive enzyme activity and feeding behavior, which the increase of digestive enzyme activity is several hours earlier than the time of peak feeding, suggesting that the early enzyme secretion can optimize the percentages of digestion and absorption, and reduce the risk of predation due to shortened feeding time. For nocturnal animals, even under an unnatural light/dark cycle, the digestive enzyme activity is prone to be higher at night than in the daytime. In the current study, the light/dark cycle and feeding rate had a significant impact on four digestive enzyme activities in the visceral mass of S. constricta, including α-amylase, lipase, cellulase, and pepsin. The vitality of digestive enzymes after the increase of food intake in ZT2 and ZT8 at night is significantly higher than that in low food intake ZT14 and ZT20 in the daytime, demonstrating that food digestion has a circadian rhythm behavior. There is also the same finding that the production of digestive enzymes increased significantly after increasing food intake, like in C. edule (Ibarrola et al., 1998) and clam R. philippinarum (Houki and Tomohiko, 2020). However, relatively speaking, lipase activity is significantly lower than the other three. Numerous studies have shown that carnivorous animals have higher lipase content than omnivorous and herbivorous animals, and those with high lipase content are generally seen in animals with mature intestines, lipid hydrolysis mainly occurs in the pyloric caeca and/or proximal intestine (Caruso et al., 2009; Tengjaroenku et al., 2000). The razor clam, as a filter-feeding shellfish, has an incompletely differentiated intestine, which the ability to hydrolyze lipids is weak.

The expression of digestive enzyme genes is strongly correlated to their enzyme activities, which directly reflect the digestive physiology of aquatic animals. Existing research has shown that the increase of enzyme genes expression at night was the regulation of translation process and may be the anticipation of next food intake (José Antonio et al., 2016). A recent research on the feeding and digestion physiology of abalone H. discus hannai under the light/dark cycle found that digestive enzymes and feeding related genes were significantly higher at night than during the day with rhythmic 24 h oscillations (Gao et al., 2021). In this research, the mRNA levels of four key digestive enzyme genes (AMY, LPS, CEL, and PEP) by qRT-PCR during the light/dark cycles were basically consistent with the production of their enzymes. Furthermore, the genes expression showed a periodic pattern of high at night and low during the day for consecutive 3 days, indicating that circadian rhythm may be the internal mechanism that was regulated by circadian clock genes (Paredes et al., 2014; Qin et al., 2020). Then the circadian clock genes may regulate the expression of AMY, LPS, CEL, and PEP, thereby participating indirectly in the feeding and digestion activities of S. constricta. Although the similar trends of gene expression and activities of key digestive enzymes, and feeding rates in this experiment were discovered, deep understanding of their corresponding relationships still need a lot of in-depth and meticulous research.

The feeding rate, digestive enzyme activities and relative expression of their genes all had a circadian rhythm in S. constricta, which it can be basically determined that ZT0-ZT2 will reach the peak at night and ZT12-ZT14 will reach the lowest value during the day from feeding to digestion. Therefore, it can be speculated that razor clams have higher nighttime activities than during the day, suggesting that the feeding time in the industrial farming can be arranged at night to reduce cultivating costs and increase farming benefits.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

The adult hard clams Sinonovacula constricta at the age of 1 year were collected from the genetic breeding research center of Zhejiang Wanli University, China. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Zhejiang Wanli University, China.

HY and YD conceived and designed the project. TZ collected the samples and contributed reagents. YL performed the experiments and data analysis, and wrote and revised the manuscript. All authors read and approved the final manuscript.

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.