Feed efficiency (FE) is an essential trait in aquacultural animals because feed accounts for 30–70% of production costs in the aquaculture industry (De Verdal et al., 2017). Improved FE of animals will not only reduce feeding costs and increase profitability but also reduce the environmental impact of large-scale farming (Huntington and Hasan, 2009). Residual feed intake (RFI) reflects the difference between the actual feed intake of the animal and the expected feed intake for growth and maintenance of body weight. Therefore, an animal with a low RFI that consumes less feed per unit of body weight than expected is efficient, while an animal with a high RFI that consumes more feed than expected is inefficient (Patience et al., 2015). RFI was first proposed in 1963 as an index for evaluating the animal FE in beef cattle (Koch et al., 1963) and has subsequently been used to assess FE in other species (Dai et al., 2017; De Verdal et al., 2017; Zhang et al., 2017). Experts from the Animal Genetic Resources Committee of FAO also recommend RFI as an indicator for evaluating animal FE traits (Zhang, 2010).

The intestinal microbiota and the digestive enzyme activity in the intestines are regarded as two key factors in animal digestion (Motlagh et al., 2012; Fang et al., 2015). It is hypothesized that these two factors are strongly associated with the RFI of animals. However, there is an important symbiotic relationship between intestinal microbiota and hosts that contribute to energy balance, metabolism, intestinal health, immune activity, and neural development of hosts (Turnbaugh et al., 2007; Cho and Blaser, 2012; Shreiner et al., 2015). Intestinal microbiota are involved in the regulation of host energy-uptake efficiency, which was significantly associated with weight gain (Looft et al., 2014; Kim et al., 2015) and FE (Singh et al., 2012; Mccormack et al., 2017) and plays an important role in maintaining the stability of the intestinal environment (Chung and Kasper, 2010). Changes in intestinal microbiota could inevitably influence intestinal metabolic function and further affect the physiology of organisms (Levy et al., 2017), which can be partially indicated by intestinal metabolites (Yin et al., 2021). Therefore, numerous studies have analyzed intestinal microbiota structure and metabolites to explore the regulatory mechanisms of FE (Metzler-Zebeli et al., 2019; Gardiner et al., 2020).

Digestive enzyme activity also reflects the digestive and absorptive capacity of the intestine, which in turn determines feeding and growth of animals (Baiao et al., 2017; Ydsr et al., 2019). Digestive enzymes are known to influence diet, growth, and many other traits of abalone (Gao et al., 2016, 2021). In addition, intestinal microbiota can influence the production of some digestive enzymes to some extent (Ekhart et al., 2012).

Abalone is an economically important aquaculture mollusk with high-quality protein resources that is widely cultured around the world (Froehlich et al., 2018). Its feed cost has been elevated with the industrialized production mode and rising feed prices, constraining further development of the abalone aquaculture industry in China, whose abalone aquaculture production accounts for 90% of the world. Studies on the FE of abalone from the perspective of intestinal microbiota are limited, although other investigations have shown that intestinal microbiota are irreplaceable for the growth and immunity of abalone (Jing et al., 2012; Choi et al., 2021). In this study, intestinal microbiota, digestive enzyme activity, and metabolome of the Pacific abalone, Haliotis discus hannai, with high FE (low RFI) and low FE (high RFI) were compared, and their interconnections in the regulation of FE of abalone were explored.

In this study, the hypothesis that microbiota structure, digestive enzyme activity, and metabolome in the intestines are associated with FE of animals was tested. For the assessment of growth and resistance traits in abalone, the feeding experiment period was generally 60–90 days (Huang et al., 2018; Wang et al., 2020). In this experiment, a 72-day feeding experiment period was sufficient to obtain growth and feeding efficiency traits for abalone. Low-RFI abalone individuals were selected as the high-FE group (group A) and high-RFI abalone individuals were selected as the low-FE group (group B). The RFI values of group B were significantly higher than those of group A; however, there was no significant difference between the two groups in ADG, FER, and DFI, possibly because of the small number of individuals. It has been reported that FER is correlated to ADG (Case et al., 2012), whereas RFI has a weak correlation to ADG (Dai et al., 2017). Each abalone in the experiment was raised individually, with adequate food and no competition, which may account for the small difference in DFI.

Recently, the development of high-throughput sequencing has facilitated comprehensive profiling of the microbiota co-occurring in the intestines of the animal (Argumedo-Hernández et al., 2010; Zhao et al., 2018). Diet, living environment, and other factors greatly impact the animal intestinal microbiota (Looft et al., 2014; Singh et al., 2014; Kim and Isaacson, 2015). This is one of the first studies to exploit this technology to examine the intestinal microbiota in abalone with varying FEs. The two groups of abalone in this experiment were subjected to the same management, environmental, and nutritional conditions to eliminate additional effects. The intestinal microbiota of all of the abalones were predominated by the phyla Proteobacteria and Tenericutes, which is consistent with previous findings (Wang et al., 2020; Choi et al., 2021). Proteobacteria are carbohydrate-fermenting bacteria and a high percentage in the intestines of abalone enhances the ability to utilize carbohydrates and improve production performance (Elolimy et al., 2019). Abalones with higher FE had a higher percentage of Proteobacteria, which is consistent with the results in the study on the high-feed conversion ratio (FCR) and low-FCR broiler growers (Singh et al., 2012). In addition, changes in the abundance of Firmicutes may affect energy harvesting ability (Turnbaugh, 2006), which may account for the high abundance of Firmicutes in the high-FE group.

At the genus level, Mycoplasma is the most abundant bacteria and has been reported several times in abalone (Cicala et al., 2018; Nam et al., 2018). Pseudoalteromonas and Cobetia accounted for a higher proportion of the intestinal microbiota in group A than in group B, and both groups could produce alginate lyase (Chaki et al., 2008; Gong et al., 2016). Alginate lyase, a digestive enzyme catalyzing the degradation of alginate, has been isolated from various kinds of organisms with different substrate specificities and is extremely important in food digestion and nutrition absorption in abalone (Zhu and Yin, 2015; Bansemer et al., 2016). The higher proportion of Pseudoalteromonas and Cobetia in the high-FE group may have increased the activity of alginate lyase in abalone, which promoted the digestion and absorption of food and thus improved FE. The presumed possibility was initially established by measuring intestinal enzyme activity, although the gene for alginate lyase is present in abalone (Sim et al., 2012). Combined with the results of other digestive enzyme activities and protein enzyme activities, we hypothesized that alginate lyase and cellulase enzyme activities may play important roles in inducing changes in RFI in abalone. However, whether the increase in cellulase enzyme activity was influenced by the intestinal microbiota needs to be further investigated.

Microbial culture analysis of healthy and diseased abalone intestines revealed a decrease in microbiota diversity in diseased abalone (Huang et al., 2010) and we theorize that microbiota diversity may represent the health condition of the abalone intestine. The reduction in intestinal microbiota diversity in group B may be an indication of abnormal intestinal health conditions, which in turn contributes to the low-FE trait in abalone. Another study showed that the addition of probiotics to feed promoted growth, immunity, and intestinal microbial richness in the small abalone Hediste diversicolor (Zhao et al., 2018), again suggesting that high microbiota diversity is associated with positive aspects of the trait. Abalone individuals with high FE had high microbiota diversity and richness, which is consistent with the results in most animal studies (Bergamaschi et al., 2020).

By establishing associations between microbiota and the FE of abalone, a number of microbiota biomarkers were found in the high- and low-FE groups, which could provide a reference for screening abalone with high FE and improve the FE traits of abalone through targeted regulation of intestinal microbiota. The biomarker Saprospira between groups A and B is a member of Bacteroidetes, and members of this bacteria are closely associated with intestinal microbiota in pigs with low RFI (Mccormack et al., 2017), indicating the strong ability of such bacteria to ferment complex carbohydrates (UmuJeremy et al., 2015). Rhodanobacteraceae are considered to be potentially beneficial bacteria because of their ability to produce beneficial chemicals (Saudo-Wilhelmy et al., 2013). Rhodobacteraceae has the role of catalytic denitrification, degradation of various organic pollutants, and inhibition of the growth of harmful pathogens (Liu et al., 2012; Wang et al., 2018). Therefore, we hypothesized that the immunity of abalone in group A that was enriched with Rhodobacteraceae was higher than that of group B, which in turn influenced FE, and this hypothesis coincided with the results of upregulation of the immune pathway in group A. Actinobacteria has been recognized as an important source of biologically active natural products with a variety of biological properties such as antibacterial, anticancer, and antiviral (Manivasagan et al., 2014). The biomarkers Llumatobacteraceae and Gaiellales in group A belong to Actinobacteria. Taken together, we hypothesized that Saprospira, Rhodanobacteraceae, Llumatobacteraceae, and Gaiellales in the intestines may potentially be utilized as biomarkers for distinguishing high-FE abalone from low-FE abalone. Improvement of intestinal microbiota through the transplantation of specific bacteria or the use of dietary supplements has progressed in other species (Vos, 2013; Wlodarska et al., 2015). Therefore, it is possible to improve the FE of abalone by regulating the intestinal microbiota composition.

Metabolite identification is the main metabolomic bottleneck, especially in marine non-model organisms (Reverter et al., 2020). In this study, the intestinal metabolome of abalone in the high- and low-FE groups was analyzed, and a total of 344 metabolites were identified. The number of metabolites identified was similar to previous studies on abalone (Shen et al., 2021), and major metabolites were identified, including taurine, citrate, succinate, lactate, glutamate, glutamine, and L-carnitine, thus confirming the reliability of our results. Arginine is involved in RNA synthesis and protein glycosylation, and it is an essential amino acid for cellular function (Li et al., 2018). The growth and FE of red drum (Sciaenops ocellatus) have a standard requirement for arginine (Barziza et al., 2000). In this study, the arginine synthesis pathway was upregulated in the low-RFI group, and we hypothesized that arginine plays a regulatory function in abalone FE. Furthermore, differences in amino acid and fatty acid metabolic pathways indicate the importance of intestinal metabolic capacity on FE traits in abalone. This study was also the first to elucidate the mechanisms influencing feed efficiency traits in abalone through metabolome profiles.

Changes in intestinal microbiota may depend on or in turn influence metabolite production in the host (Levy et al., 2017). Therefore, changes in metabolites may be closely related to the intestinal microbiota. Proteobacteria in the intestines has been reported to play a crucial role in amino acid utilization (Neis et al., 2015). The three intestinal bacteria (Uncultured beta proteobacterium, BD1_7_clade, and Lautropia) that were significantly different in the high-/low-FE groups all belonged to Proteobacteria. DL-methionine sulfoxide is involved in the methionine metabolic pathway, which promotes protein synthesis in animal growth (Hu et al., 2016) and production performance (Fagundes et al., 2016). In addition, methionine is required for the biosynthesis of spermidine and spermine, two compounds that help to reduce oxidative stress (Minois et al., 2011). Indoleacetic acid, a metabolite of tryptophan, is a ligand of the aromatic hydrocarbon receptor (AHR) (Roager and Licht, 2018), a transcription factor that is widely expressed in cells of the immune system. Several studies have shown that AHR activation alters innate and adaptive immune responses in a ligand-specific manner. In addition, another important physiological function of tryptophan is protein synthesis (Gargaro et al., 2016). L-pyroglutamic acid is a metabolite in the glutathione cycle, which can be converted to glutamate by the 5-oxoprolinase enzyme (Mello et al., 1995). The addition of glutamate in feed can increase the FE of animals (Cantalapiedra-Hijar et al., 2016). The tyrosine metabolic pathway is upregulated in pigs with low RFI (Mccormack et al., 2017), in which tyrosine and its subsequent metabolites can reduce the production of reactive oxygen species (ROS), and the upregulation of tyrosine metabolism may play a positive role in reducing oxidative damages in the intestines (Beloborodova et al., 2012). This provides evidence that the intestines or microbiota of abalones that are more feed efficient could potentially be “healthier.” Tyramine (TA), as a neurotransmitter, neuromodulator, and neurohormone, controls or regulates many behavioral and physiological processes in animals (Orchard, 1982). The nerve tissues of invertebrates, such as insects, crustaceans, and mollusks, contain large amounts of TA (Robertson and Juorio, 1976). Tyrosine is also required for the synthesis of 5-hydroxytryptamine and dopamine, both of which are important neurotransmitters (Bergwerff et al., 2016). The upregulation of tyrosine and tryptophan in the HFE group may be an adaptation, which promotes metabolism in organisms. DL-methionine, indoleacetic acid, and L-pyroglutamic acid were positively correlated with Proteobacteria (Uncultured beta proteobacterium, BD1_7_clade, and Lautropia) (Figure 5), and it was hypothesized that the above three intestinal bacteria contributed to the FE of abalone through the following two reasons. Firstly, Proteobacteria promote the utilization of amino acids in abalone, which in turn provides more nutrients to maintain the synthesis of amino acids required for abalone growth, ultimately improving the feed efficiency traits. Of course, the increased protease activity of the HFE group also promoted amino acid hydrolysis and protein absorption (Kumlu and Jones, 2010). Second, Proteobacteria improves the immunity of the abalone and reduces oxidative damage of the intestine to ensure that the intestine performs its digestive and absorption functions better, which in turn improves the feed efficiency of the abalone. In summary, we hypothesized that the microbiota, metabolism, and enzyme activity in the abalone intestines interact to alter amino acid metabolism, immune responses, and signaling pathways, which in turn influence FE.

This study reveals for the first time differences in intestinal microbiota structures and intestinal metabolism in shellfish with different feed efficiencies. Abalone with high FE have higher microbiota diversity and a higher proportion of Proteobacteria and Firmicutes. Four microbiota, namely, Saprospira, Rhodanobacteraceae, Llumatobacteraceae, and Gaiellales, may be potentially utilized as biomarkers for distinguishing between high- and low-FE abalone. The intestinal digestive enzyme activity also contributed to differences in FE in abalone, and Pseudoalteromonas and Cobetia may influence the activity of alginate lyase in the intestine. The interaction between microbiota and intestines influences amino acid metabolism, immune response, and the signal transduction pathway in abalone, in turn causing differences in FE. The differences in amino acid metabolism may also be influenced by intestinal trypsin activity. This study not only analyzed the mechanisms of variations in abalone FE in terms of intestinal microbiota, intestinal metabolism, and intestinal enzyme activity, but it also provided important basic knowledge for improving abalone FE by modulating intestinal microbiota.

The data presented in the study are deposited in the GenBank Sequence Read Archive (SRA) repository, accession number PRJNA805375.

The animal study was reviewed and approved by the ethics committee of Xiamen University.

CK, WwY, and WcY provided the experimental ideas and design of this study. WcY, MZ, JL, FY, ZH, and WZ did the experiments. YL and SG helped to analyze the data. WcY and YL wrote the manuscript. WwY, YS, and XL revised the manuscript. All authors 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.

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