The rapid development of aquaculture has propagated aquaculture in China, and the output has reached 75% of the world (Pauly and Zeller, 2017). As fish production increases, fish diseases (such as Nocardia, Aeromonas, and Edwardsiella) have become a serious problem in aquaculture, often causing major economic losses (Bondad-Reantaso et al., 2005; Lee et al., 2014; Watts et al., 2017). Previously, chemical drugs such as antibiotics have been the common choice for preventing fish diseases, but the high use of antibiotics has caused problems of drug residues, multi-drug resistance, and environmental pollution (Watts et al., 2017), as well as damage to the immune system of fish and adversely affecting the digestive tract flora of the fish (Lee et al., 2014; Capkin et al., 2017). The intestinal environment of fish is a critical protective barrier against the occurrence of disease, and the dynamic interaction between the intestinal mucosa and pathogenic microorganisms induces inflammatory responses and alters the barrier function of the intestine (Honda and Littman, 2012). Some studies have shown that probiotics prevent the colonization of pathogenic bacteria by regulating the intestinal mucosal barrier function of fish, competitively inhibiting the growth of fish intestinal pathogenic bacteria, stimulating the specific or non-specific immune function, promoting animal growth, improving the feed conversion rate, enhancing the animal immune function, and improving ecological environment in vivo (Zhang et al., 2019a). Therefore, probiotics, as one of the safe alternatives to antibiotics, have been widely used in aquaculture in the form of feed additives in recent years (Selim and Reda, 2015; Mukherjee and Ghosh, 2016; Yi et al., 2018). Compared to Lactobacillus, Bacillus can form endospores that protect it from environmental influence, which is more advantageous in the prevention of fish diseases.

Lysinibacillus sphaericus, discovered in 1965, was first proposed as a new genus associated with Bacillus (formerly known as Bacillus sphaericus) and is gaining importance due to the commercial use of some members for the biological control of vector-borne diseases (Baumann et al., 1991). Subsequently, L. sphaericus was widely used as a reference for the study of larvicidal activity of Lactobacillus coccidiosis (Lozano and Dussán, 2013), metal tolerance (Davidson et al., 1977; Lozano and Dussán, 2013; Peña-Montenegro et al., 2015), surface protein structures such as the s layer (Brown et al., 2004), and biosurfactant production comparison points (Lacey, 2007). However, at present, no studies have reported that L. sphaericus microorganisms promote growth and prevent bacterial diseases in fish. In addition, only a few studies have yet reported the whole genome analysis related to L. sphaericus.

Whole genome sequencing can detect the entire genome sequence for a species and can decipher the encompassed genetic mechanism information by analyzing the base sequence of the species comprehensively and accurately; it has the advantages of high sequencing coverage and strong accuracy (Bartram et al., 2011). Third-generation sequencing technology refers to Oxford Nanopore Technologies, nanopore technologies nanopore, single-molecule sequencing, and SMRT technology developed by PacBio, which does not require polymerase chain reaction (PCR) amplification for the sequencing process and has the characteristics of sequencing read length and high sequencing efficiency (Clarke et al., 2009). With the rapid development of technology, third-generation whole genome sequencing has become an efficient detection method, widely used in medicine, biology, and other fields, and plays a critical role in the study of the biological characteristics of environmental microorganisms and intestinal microflora (Peña-Montenegro et al., 2015; Finton et al., 2020; Jose et al., 2020).

This study, for the first time, a potential probiotic strain AEB18 with significant inhibition of N. seriolae was isolated from healthy fish intestines, and the whole genome of this strain was sequenced on the Nanopore platform. Subsequently, the antibacterial gene cluster of strain AEB18 was analyzed by gene function annotation and antiSMASH online tool to further explore its antibacterial mechanism. In addition, after feeding with AEB18 supplemented diets, the effects of promoting growth and preventing nocardiosis of hybrid snakehead were determined.

The isolation of strains from the breeding environment including that of the host is the most effective way to screen probiotics. The majority of aquatic probiotics are screened according to the effect of inhibiting specific pathogenic bacteria. This antagonistic screening method has become a specific mode to identify probiotic strains. Several studies are focused on wild fish from an Indian maior carp, Catla catla (Hamilton) (Mukherjee and Ghosh, 2016), Atlantic cod (Gadus morhua) (Gildberg and Mikkelsen, 1998), turbot (Scophthalmus maximus) (Chen et al., 2016), goldfish (Carassius auratus) (Ghosh, 2008), gray mullet (Mugil cephalus L.) (Lin et al., 2013), and carp (Cyprinus carpio L.) (Feng et al., 2019) isolated from the intestines of probiotics that have antagonistic effects on common aquatic pathogens. In this study, we isolated microorganisms from healthy hybrid snakehead intestines and screened strains that have antagonistic effects on N. seriolae, of which Bacillus exerted a significant inhibitory effect, seeming to be a promising probiotic. Meanwhile, the 16S rRNA identification method was used to identify the strain of Bacillus AEB18 in depth. The results showed that the genetic correlation between AEB18 and L. sphaericus DSM 28 exhibited a 99.36% homology, indicating that AEB18 belongs to L. sphaericus. Studies have shown that L. sphaericus is widely used in larvicide activity and metal tolerance (Lozano and Dussán, 2013), however, its role in the prevention and control of aquaculture diseases has not been reported. Therefore, we screened and identified L. sphaericus AEB18 with potential probiotic effects for follow-up research.

Currently, the nanopore sequencing technology (third-generation sequencing technology) has been widely used for whole-genome sequencing on bacteria isolated from varied environmental media. Sher et al. (2020) performed whole-genome sequencing of one strain of bacteria carrying polymetallic genes isolated from industrial wastewater on the Illumina and Nanopore sequencing platforms, then, a complete sequence was assembled and identified as Micrococcus luteus, which was named AS2 based on 16S rRNA sequence, with a genome size of 2.86 Mbp and GC content of 72.8%. Jose et al. (2020) used nanopore sequencing technology to sequence the whole genome of one water-based strain of Vibrio cholerae, MinION sequencer produced 17,901 reads and 151,833,237 base pairs, with an average length of 8,551 bp and the longest sequence length of 86,745 bp, and the assembled sequence GC content was 47.72%, the N50 value was 2,984 kbp. The full-length genome of V. cholerae was successfully obtained by nanopore sequencing. Finton et al. (2020) performed whole-genome sequencing of antibiotic-resistant bacteria isolated from campus pond water samples through the second-generation sequencing platform Illumina MiSeq and the third-generation sequencing platform Oxford Nanopore MinION and identified Escherichia coli, Klebsiella pneumoniae, and Holderia parapot. Whole-genome sequencing data analysis showed that Enterobacter carried β-lactamase antibiotic-resistance genes, including blaCTX-M, blaTEM, blaCMY, blaSHV, blaKPC-like, and other genes (Finton et al., 2020). Sorokin et al. isolated a new halophilic strain AB-CW3 from the high-saline lake of Kulunda Steppe, assembled the complete genome of AB-CW3 by combining Nanopore and Illumina sequencing technologies, and found that it expressed an antimicrobial peptide antibiotic that had not yet been produced by Gram-positive bacteria (Sorokin et al., 2020). In our study, the whole genome of L. sphaericus AEB18 was first sequenced on the Nanopore sequencing platform, and the total length of the L. sphaericus AEB18 gene sequence was 4,653,716 bp, the length of N50 (bp) was 23,336, the length of N90 (bp) was 6,536, the GC content was 37.4%, and it contained 108 tRNAs, 37 rRNAs, and 95 ncRNAs, assembled into a complete genome of AEB18.

Secondary metabolites are a series of macromolecular non-essential substances synthesized by microorganisms in a specific growth period with primary metabolites as precursors, such as antibiotics, toxins, hormones, and pigments (Harwood et al., 2018). In this study, the antiSMASH online tool was used to predict the secondary metabolite gene clusters of strain AEB18, and a total of 9 lipopeptide and polyketone antibiotic gene clusters were obtained, involving polyketones, non-ribosomal synthetic peptides, β-lactones, terpenes, and ribosomal synthetic peptides. Among them, 6 antibiotic synthetic gene clusters (bacillibactin, kijanimicin, molybdenum cofactor, petrobactin, bacillopaline, and fengycin) were less similar to the currently known antibiotic gene clusters, of which one antibiotic gene cluster (butirosin) was only 7% similar to the currently known antibiotic gene cluster; it also included three gene clusters with unknown functions. The study has shown that bacillibactin is considered virulent against animal pathogens (Miethke et al., 2008), but its role in microbial competition remains to be elucidated. In addition to antimicrobial activity, kijanimicin also has antitumor activity (Bradner et al., 1983). Petrobactin exerts a protective effect against oxidative stress and improves spore-producing efficiency in Bacillus anthracis (Hagan et al., 2018). Fengycin is a bacteriostatic substance synthesized by Bacillus subtilis that targets the immune response to fungal infections and disrupts the cell membranes of the target cells (Vanittanakom et al., 1986). Based on the above information, it is speculated that the AEB18 can achieve biological control by producing a variety of bacteriostatic substances and CEs, and has great potential for aquatic applications.

Several studies have shown improved growth performance and survival rate of fish, which is a direct embodiment of the effect of probiotics. For example, Bjornsdottir et al. (2010) found that artemia was fortified with fish-derived probiotics (Vibrio sp., V. splendidus, and Pseudoalteromonas elyakovii) (10⁷ CFU/g) that was then fed to open-stage Atlantic halibut (Hippoglossus hippoglossus L.), after 57 days, the survival rate and body weight of juvenile fish were significantly higher than the control group. Avella et al. (2010) reported that feeding three strains of Bacillus (B. subtilis, B. licheniformis, and B. pumilus) strengthened rotifer (Brachionus plicatilis) in the breeding of Sparus aurata, and the length and weight of larvae in the probiotic group increased significantly. Mohapatra et al. compared the probiotic compound feeding methods and found that feeding three active probiotics B. subtilis, Saccharomyces cerevisiae and Lactococcus lactis significantly improved the immunity of Labeo rohita species and the survival rate after Aeromonas hydrophila infection (Mohapatra et al., 2014). In this study, the results showed that compared to the control group, the L. sphaericus AEB18 (1 × 10⁹ CFU/g) significantly (P< 0.05) promoted the growth of hybrid snakehead and increased the activity of intestinal digestive enzymes after 21 days applications, indicating an improved food utilization efficiency of hybrid snakeheads. This application effect was similar to that reported previously (Avella et al., 2010; Bjornsdottir et al., 2010; Mohapatra et al., 2014).

Accumulating evidences suggested that adding probiotics (e.g. Bacillus and Lactobacillus) to aquatic feed can inhibit the occurrence of bacterial diseases in fish (Bahi et al., 2017; Srisapoome and Areechon, 2017; Zhang et al., 2019b). The bacteriostatic activity of probiotics is manifested in the active substances, which exert antibacterial effects on a variety of fish pathogenic microorganisms. Additionally, probiotics improve the host’s disease resistance through direct antagonism to pathogenic bacteria. Antagonism is indispensable in maintaining the balance between potential probiotics and pathogenic bacteria in fish (Pandiyan et al., 2013). The growth inhibition effect of probiotics on pathogenic bacteria may be achieved by producing bacteriocins, lysozyme, proteases, siderophores, and other antimicrobial metabolites alone or in combination with other probiotics or prebiotics or by altering the pH in the intestine or competing with pathogenic bacteria for essential nutrients (Verschuere et al., 2000). On the other hand, probiotics regulate the balance of fish intestinal microflora, elevate the dominant flora and bacteria content in the intestine, compete with pathogenic bacteria for nutrient space, increase the number of beneficial bacteria, reduce the number of harmful bacteria, and enhance the function of the fish immune system, thereby inhibiting the pathogenicity of pathogenic microorganisms to fish (Kuebutornye et al., 2020). Similarly, in the present study, L. sphaericus AEB18 had a specific effect on the prevention and control of hybrid snakehead nocardiosis. The results showed that compared to the positive control group, the L. sphaericus AEB18 + N. seriolae group significantly reduced the mortality rate of hybrid snakeheads (P< 0.05). Thus, we speculated that the inhibitory effect of L. sphaericus AEB18 on pathogenic bacteria may be achieved by producing antibacterial metabolites, such as bacillibactin, kijanimicin, petrobactin, fengycin, and esterase, but its specific inhibitory mechanism needs to be investigated in depth.

In summary, the strain L. sphaericus AEB18, derived from the healthy intestine of hybrid snakeheads, demonstrates a notable inhibitory effect on N. seriolae in laboratory conditions. The genome of L. sphaericus AEB18 consists of a circular DNA with 4,653,716 base pairs (bp) and a GC content of 37.4%. Through the prediction of secondary metabolites, nine gene clusters associated with various bacteriostatic secondary metabolites, such as polyketones, non-ribosomal synthetic peptides, β-lactones, terpenes, and ribosomal synthetic peptides, were identified in L. sphaericus AEB18. Moreover, in vivo studies have shown that L. sphaericus AEB18 significantly enhances the growth of hybrid snakeheads and increases the activity of intestinal digestive enzymes. Additionally, it has a positive impact on the survival rate of fish infected with N. seriolae. These findings suggest that L. sphaericus AEB18 holds significant potential as a biocontrol agent and could be utilized to enhance disease management in freshwater aquaculture.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

The animal study was approved by [please insert the full name and affiliation of the ethics committee/institutional review board: the Animal Ethics Committee of South China Agricultural University, Guangzhou, Guangdong, China (approval ID: SYXK-2019-0136). The study was conducted in accordance with the local legislation and institutional requirements.

TZ: Funding acquisition, Writing – original draft, Writing – review & editing, Project administration. PC: Data curation, Writing – original draft. JL: Investigation, Writing – original draft. ZL: Writing – review & editing. XD: Resources, Validation, Writing – review & editing. XH: Resources, Validation, Writing – review & editing. XZ: Resources, Visualization, Writing – review & editing.