Berberine hydrochloride is an isoquinoline alkaloid found mainly in the stems and roots of the genus Berberis, and is the main pharmacological component of the Chinese herbal medicines Coptis chinensis and Phellodendron chinensis (Habtemariam, 2016). As a botanical medicine, berberine hydrochloride has been used to treat intestinal infections, especially bacterial diarrhea, for more than 2,000 years (Tang et al., 2009). Coptis coptidis is used as a prescription drug to control bacterial fish diseases (Ya-Nan et al., 2014). Berberine hydrochloride has demonstrated bacteriostatic and bactericidal effects against Vibrio cholerae, Aeromonas hydrophila, Salmonella typhi, Staphylococcus aureus, and Escherichia coli (Zhang et al., 2009). Its main pharmacological activities include antioxidant and anti-inflammatory activities (Xu et al., 2017). Some studies have also shown that berberine hydrochloride improved growth performance, enhanced skin mucolytic enzyme activity, and increased the growth rate and disease resistance of fish in aquaculture. However, the exact mechanisms of berberine hydrochloride are not yet fully understood. In addition, most studies have focused on fish, with little research on crustaceans. We therefore explored the effect of berberine hydrochloride in the crab, Charybdis japonica.

Charybdis japonica is widely distributed in the coastal areas of Southeast Asia, and its rapid growth, high nutritional value (Lou et al., 2018) have made it one of the most economically important species of crabs. C. japonica has been widely cultivated, but its high culture density has led to outbreaks of infections caused by various bacterial and viral pathogens (Johnson, 1983). One of the most harmful pathogens widely present in crabs is A. hydrophila, which is responsible for diseases that cause major economic losses in crab farming worldwide (González-Escalona et al., 2006; Igbinosa et al., 2012). We therefore tested the effects of berberine hydrochloride against this bacterium in the current study.

Transcriptome sequencing can be analyzed in relation to different environmental stresses, viral and bacterial invasion, and gene expression patterns across tissue types (Czesny et al., 2012). Advances in high-throughput RNA-Seq technology now offer the opportunity to explore the transcriptome and gene expression patterns associated with crab immunity (Xie et al., 2014; Jia et al., 2018). This sequencing technology has been used to identify many immune-related genes in various immune-related organs of crabs, including swimming crabs (Liu et al., 2011), Scyla paramamosain (Liu et al., 2017), Eriocheir sinensis (Zhang et al., 2018), and Sinopotamon henanense (Sun et al., 2016). It provides an efficient technique for further gene expression analysis, novel transcript discovery, and differentially expressed gene identification in crab transcriptome sequencing (Garber et al., 2011).

Improving the immunity and disease resistance of crustaceans is one of the basic measures for preventing and controlling diseases. Immune defense in crabs is entirely dependent on an innate, non-adaptive mechanism to resist environmental stress, and pathogen invasion (Kurata et al., 2006). The crab’s innate immune system plays an important role in early pathogen recognition and relies on a limited number of pattern recognition receptors to recognize evolutionarily conserved pathogen-associated molecular patterns (Neves et al., 2009). The gills are an essential organ for regulating osmotic pressure in crustaceans and play an important role in innate immunity. The gills are directly exposed to the external water environment containing a large number of microorganisms (Bao et al., 2019), and are thus the first organ to be exposed to pathogenic bacteria, including members of the Vibrio family. We therefore investigated the gills of C. japonica as the study organ in these experiments.

The application of big amounts of antibiotics has been depended by the treatment of bacterial diseases in aquatic animals, which has resulted in negative effects such as drug resistance, drug residues, and cross-infection of pathogenic bacteria, endangering human health and contaminating soil and water sources when released into the environment (DuPont, 2007; Martínez, 2008; Seal et al., 2013). The aquaculture department is responsible for producing edible crabs for human consumption. Therefore, chemical drugs are generally not preferred in aquaculture for safety reasons. Secondary metabolites isolated from different plants have been shown to be beneficial for both humans and animals (Lazreg Aref et al., 2011). In this study, we examined the use of berberine hydrochloride as an alternative to antibiotics and an immune-stimulant for the prevention and treatment of A. hydrophila-infection in C. japonica. This study aimed to investigate the effects of berberine hydrochloride on the metabolic pathways of C. japonica based on transcriptomic data, to validate the corresponding immune genes in the transcriptomic metabolic pathways in berberine hydrochloride-treated P. aeruginosa-infected C. japonica using the RT-polymerase chain reaction (RT-PCR), and to examine changes in the metabolic pathways and map the complete pathway system.

In this study, 5,409 transcripts were significantly up-regulated or down-regulated by berberine hydrochloride, and these genes were distributed in various metabolic pathways. Immersion in berberine altered pathways related to glutathione metabolism, oxidative phosphorylation, fatty acid degradation, retinol metabolism, pyruvate metabolism, HIF-1 signaling, and lysosomes and phagosomes. Glutathione metabolism is one of the main components of the oxidative defense system in C. japonica (Pan and Zhang, 2006) and some genes involved in glutathione metabolism, such as PRDX2-4 (AH < CK < BHAH < BH), were altered by berberine hydrochloride immersion. The oxidative phosphorylation pathway is responsible for the production of ATP in mitochondria (Pagliarini and Rutter, 2013). Upregulation of the lipolytic gene CPT1A was accompanied by an increase in energetic ATP during catabolism (Cheng et al., 2016). In this experiment, berberine hydrochloride regulated the CPT1A gene involved in fatty acid degradation metabolism (AH < CK < BHAH < BH), which may ameliorate the damage caused by Vibrio spp. and A. hydrophila infection in crabs. Vitamin A deficiency impairs retinol metabolism and reduces growth and impairs intestinal immune function in fish (Zhang et al., 2017). Vitamin A deficiency also impairs the physical barrier function associated with impaired antioxidant capacity and exacerbates apoptosis (Jiang et al., 2019). However, berberine hydrochloride can affect retinol metabolism and can up-regulate the RDH12 gene in flavanol metabolism (AH < CK < BHAH < BH). The pyruvate metabolism network, including up-regulation of pyruvate kinase, is involved in the pathogenesis of A. hydrophila in fish, thus enhancing the immunity of zebrafish infected with A. hydrophila (Lin et al., 2019). In the current study, berberine hydrochloride up-regulated genes involved in pyruvate metabolism, including PCCA (CK < AH < BHAH < BH), which may be help to resist infection by A. hydrophila.

Lysosomes are digestive organelles in the endocytic pathway, and increased lysosomal enzyme activity may contribute to resistance to pathogens (Habtemariam, 2016). Lysosomes contain acid hydrolytic enzymes, including LGMN, ATP6, NAGA, LIMP-2, and PSAP. Previous studies found that the LGMN gene played a role in both primary and adaptive immunity, including the generation of major histocompatibility complex II-presented antigens, which have effective enhancement of kinetic energy changes between heat absorption and exotherm during contraction in scleractinian fish (Marra et al., 2017). The oxidative phosphorylation pathway is responsible for the production of intracellular ATP in mitochondria, including ATP6, which can supply energy to other metabolic pathways (Hatefi, 1985; Pagliarini and Rutter, 2013). LIMP-2 belongs to a subfamily of scavenger receptors, known as lysosomal transmembrane glycosylation receptors (Tan et al., 2019), and plays an important role in the immune system in response to bacterial infection in fish. LIMP-2 was significantly up-regulated in all mucosal tissues (skin, gills, and intestine) in Scophthalmus maximus following infection by the Gram-negative bacterium Vibrio anguillarum (Tan et al., 2019). PSAP is the precursor of saposin, which is found in lysosomes and secretory proteins. PSAP-1 and PSAP-2 significantly inhibited the growth of various bacteria and killed Miamiensis avidus in Platichthys stellatus, and significantly inhibited the formation of bacterial biofilms (Ghana et al., 2015). A microbial feed additive in Atlantic salmon has been shown to reduce intestinal inflammation by up-regulating the NAGA gene (Yong et al., 2007). The LGMN gene for antigen processing and presentation (BH < BHAH < CK < AH) was down-regulated, the ATP6 gene for the V-type H + transporter ATPase subunit a (CK < BH < AH < BHAH), the NAGA gene for alpha-N-acetylgalactosaminidase (CK < AH < BHAH < BH), the lysosome membrane protein 2 of LIMP2 (CK < AH < BHAH < BH), and PSAP of saposin. SGP1 (CK < BH < AH < BHAH) were all upregulated. Based on the characteristics of these genes, we deduce that berberine hydrochloride improves the immunity of crabs by increasing the levels of some hydrolytic enzyme genes. Berberine hydrochloride was previously shown to induce the expression of acid hydrolase genes that enhanced the degradation and clearance of methicillin-resistant S. aureus and enterosoluble E. coli from macrophages (Yong et al., 2007). Based on the ability of berberine hydrochloride to induce acid hydrolase gene expression and thus remove bacteria, immersion in berberine hydrochloride could remove A. hydrophila, and thus improve survival of C. japonica.

Autophagy is a lysosomal-dependent degradation pathway in eukaryotic cells (Levine et al., 2011). The process of autophagy can be divided into four steps: formation of autophagosomes, fusion of autophagosomes and lysosomes, degradation of autophagosome substrate, and regeneration of autophagosomes. Autophagy can also be induced by conditions such as hypoxia, oxidative stress, and radiation (Radomski et al., 2018). The autophagosome fuses with the lysosome to form the autophagosome and acidic hydrolases within the lysosome lumen degrade the autophagic inner membrane and internal macromolecules to produce small molecules such as amino acids and monosaccharides. In the current study, berberine hydrochloride upregulated some autophagosome genes, including NOS1 and NOS2, which may be related to the microbial-resistance effects of berberine hydrochloride. Nitric oxide is produced by NOS with L-arginine as a substrate, and acts as an antimicrobial host defense effector, which is produced by many immune-associated cells during infection (MacMicking et al., 1997; Bogdan, 1998). The expression and induction of NOS1 has been detected on the mucosal surface of the gills and intestines after challenge, and significant up-regulation of NOS1 was detected in the intestinal tract of turbot following V. anguillarum infection (Pederzoli et al., 2007; Yao et al., 2014). Vibrio has been shown to induce fluid secretion in the intestine, while nitric oxide regulates blood flow and peristalsis and has cytotoxic and antimicrobial activities in the intestine. Vibrio-infected intestinal epithelial cells showed significantly increased NOS activity (Janoff et al., 1997). In addition, the NOS gene also showed high expression in the gut in invertebrates, with the highest expression of the NOS1 gene in the gills (CK < BH < AH < BHAH) following attack by Vibrio parahaemolyticus (Li et al., 2012). This was in accord with the current results in C. japonica infected with A. hydrophila. CTSL is a lysosomal cysteine protease found in rock bream (Whang et al., 2011) and orange spotted groupers (Liang et al., 2012), which acts as a barrier against invading pathogens and is involved in vertebrate immune responses. CTSL acts as a chemical barrier against microbial invasion in the innate immune system (Ellis, 2001). The solubility of CTSL is the main factor leading to the infection of E. sinensis (Li et al., 2012) and Chinese white shrimp (Ren et al., 2010), suggesting its potential role in the immune defense system. In the present study, berberine hydrochloride up-regulated the CTSL (CK < BH < AH < BHAH) in C. japonica to resist A. hydrophila infection.

The HIF-1 signaling pathway plays a central role in the rapid adaptation to an inadequate oxygen supply to prevent cell damage, and the HIF maintenance pathway thus affects many diseases and disease processes (Kim et al., 2017). HIF-1 signaling reduced GAPDH in anaerobic respiration and had a protective effect against Edwardsiella tarda infection (Liu et al., 2005). Fusing GAPDH from pathogenic A. hydrophila, as a putative protective antigen, with four different delivery systems produced a candidate vaccine that induced effective host protection against V. anguillarum, A. hydrophila, and Aspergillus hydrophila (Zhou et al., 2010; Yan et al., 2011). In the current study, berberine hydrochloride also reduced GAPDH gene expression (BH < BHAH < AH < CK), which may have reduced the toxicity of A. hydrophila. Berberine hydrochloride reduced the activity of the eno gene (BH < BHAH < CK < AH), which may have reduced the toxicity of A. hydrophila. The virulence gene eno in Vibrio alginolyticus and A. hydrophila strains has a good toxicological potency on combination with other virulence genes and against the corresponding eno binding of the host (Zuo et al., 2019).

This study revealed the molecular mechanisms following infection of C. japonica by A. hydrophila and immersion in berberine hydrochloride. Exposure to berberine hydrochloride altered the expression of genes involved in glutathione metabolism, oxidative phosphorylation, fatty acid degradation, retinol metabolism, pyruvate metabolism, HIF-1 signaling, and other pathways. Berberine hydrochloride may thus have an adjuvant therapeutic effect against A. hydrophila infection. Transcriptome analysis showed that berberine-soaked C. japonica adapted to A. hydrophila invasion by increasing the expression levels of genes involved in the acid hydrolase and endophagosome, and anaerobic respiration-related genes. These transcriptomic data for the effects of berberine hydrochloride on C. japonica provided strong support for its further development as a therapeutic anti-bacterial agent. The results of this study provided a new scientific basis for the potential role of berberine hydrochloride in protecting crustaceans against bacterial infections in aquaculture. It also provided an additional option for the use of secondary metabolites to replace antibiotics in the treatment of bacterial diseases in aquatic animals.

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

Ethical review and approval was not required for the animal study because crabs are crustaceans or arthropods, which do not belong to vertebrates and cephalopods.

MH was responsible for experimental design, experimental operation, and article writing. YL was responsible for data analysis. TZ and DL were responsible for instrument operation. ZZ and QJ were responsible for overall thinking. All authors contributed to the article and approved the submitted version.

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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