Pacific white shrimp (Litopenaeus vannamei) is highly favored in China and other countries for its abundant protein and low-fat content, making it the most popular shrimp species for consumption (Li et al., 2021). As the breeding industry increased rapidly, epidemic diseases have become one of the major risk factors for shrimp aquaculture in recent years (Yu et al., 2022). Vibrio alginolyticus and other marine-dwelling Vibrio species are conditional pathogens that cause white feces syndrome (WFS), acute hepatopancreatic necrosis disease (AHPND), and other vibrioses in shrimps (Janda et al., 2015; Baker-Austin et al., 2018; Ngo et al., 2020; Foysal et al., 2021; Kumar et al., 2021; Shen et al., 2021; Munkongwongsiri et al., 2022).

When pathogenic Vibrio species or other pathogens infect the host, the immune system will be activated by recognizing several molecules called pathogen-associated molecular patterns (PAMPs). These molecules are crucial for the pathogens’ virulence and survival (Kumar et al., 2011). The host’s pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), and DNA receptors, sense these PAMPs (Medzhitov, 2007; Kumar et al., 2009). After the PAMPs recognized by PRRs, the immune system responds quickly to eliminate the pathogens through several signaling pathways (Li et al., 2009; Fitzgerald and Kagan, 2020).

Being a major PAMP, the bacterial flagellum is also an important virulence factor for its function in motility, chemotaxis, adhesion, biofilm formation, and secretion (Duan et al., 2013; Chaban et al., 2015; Rossez et al., 2015). In mammals and teleost fish, the bacterial flagella recognition and signaling are through TLR5, NLRC4, and NAIP5, and that activation of these receptors mobilizes nuclear factor NF-kappaB and stimulates the production of tumor necrosis factor-alpha (TNF-α) (Feuillet et al., 2006; Zhao and Shao, 2015; Fitzgerald and Kagan, 2020). Some pathogens also develop strategies to avoid the recognition of the flagella and escape the host immune system clearance. During infection, S. typhimurium significantly reduces the expression of genes involved in flagellar machinery and chemotaxis when present inside macrophages (Eriksson et al., 2003). By day 3 after feeding, the motility of E. coli introduced into the mouse gut decreased by 45-50%, and by day 15, it dropped further to 80-90% loss of motility (Gauger et al., 2007). Other methods for evading detection by the host immune system include regularly alternating the expression of flagellin proteins (phase variation), enabling different subsets of the population to express flagella (bistability). Additionally, modifying the structure of flagellin proteins to make them unrecognizable to TLR5 (flagellin modification) and adding post-translational modifications to flagellins to mask target sites (glycosylation) are also effective strategies (Chaban et al., 2015). Yet, there is scant research about how crustacean, including shrimps, recognizes bacteria flagella and actives their immune system. Several TLRs have been found in shrimps and carbs, but none has been confirmed to respond to flagella (Habib and Zhang, 2020).

In this research, we injected the wild-type and mutant strains of V. alginolyticus strain HN08115 with different flagella phenotypes into L. vannamei and analyzed the different gene expression patterns in shrimp hepatopancreas. Our results might help explain how shrimps’ immune systems recognize bacterial flagella and activate the immune system through signaling pathways.

Although there is only one polar flagellum on their rod-shaped cells, Vibrio species are commonly mobile in water, and flagellar motility is important for them to survive and infect the host (Zhu et al., 2017; Khan et al., 2020). V. alginolyticus is one of the pathogenic Vibrio spp. commonly found in diseased marine cultured species. In V. alginolyticus the assembly of the polar flagellum is promoted by FlhF and inhibited by FlhG which is a MinD homolog and an ATPase (Ono et al., 2015; Takekawa et al., 2016). FlhG gene-deficient bacteria will lose the negative regulation ability of flagella and result in the hyperflagellated form (Gulbronson et al., 2016). Since flagella is a major PAMP molecules recognized by the immune system, down-regulation of the flagella is one of the strategies used by pathogens to survive inside the host (Chaban et al., 2015). The role of V. alginolyticus flhG in host invasion remains unclear to us. In this research, we deleted the flhG gene in a V. alginolyticus strain HN08155 which was isolated in our previous research from diseased groupers (Xie et al., 2020). The flhG mutant strains of HN08155 exhibit hyper flagella according to our TEM results (Figure 1B), suggesting that the mutants have lost their ability to negatively regulate the number of flagella and might not escape the immune recognition by inhabiting the flagella.

The transcriptome technology is an efficient method to study the pathways or gene expression patterns in shrimps and other invertebrates. Peng et al. studied the molecular mechanism of L. vannamei’s cold tolerance by hepatopancreas transcriptomic analysis (Zhuo et al., 2021). Zhang et al. compared the different gene expressions before and after L. vannamei was infected by decapod iridescent virus 1 (DIV1) and found that TPI-like genes were crucial in DIV1 infection (Liao et al., 2020). Lin et al. also discovered several unique immune-related genes from V. parahaemolyticus infected L. vannamei by RNA-seq (Qin et al., 2018). Although numerous reports have focused on the gene expression changes of shrimps after being challenged with Vibrio spp., the signaling pathways in which the shrimp immune system recognizes the bacteria flagellum and starts the activation are still unknown to us. In this study, we have injected V. alginolyticus strains with different flagella regulation abilities into L. vannamei and explored the different gene expression responses of the shrimp hepatopancreas by transcriptome. The wild-type strains of V. alginolyticus HN08155 have a full flagella regulation function and may possess one or zero polar flagellum after being infected into the host tissue. On the contrary, the flhG mutant strains will have multiple flagella under all conditions. In this case, the different gene expression profiles of the shrimp response to the two types of strains might help us to understand the molecule mechanisms of flagella recognition and signaling transduction in L. vannamei. The transcriptome analysis identified a total of 104 DEGs, with 52 being up-regulated and 52 being down-regulated. Among the DEGs, several immune-related genes have been identified, which might play important roles in pathogen recognition and immune signaling.

C-type lectins (CTLs) are PRRs specifically recognize the sugar residues or motifs present in the glycans of pathogens (Mnich et al., 2020). In shrimp and other invertebrates’ immunity, CTLs play much more important roles than in mammals since the invertebrates lack adaptive immunity and largely depend on innate immunity to detect and combat pathogen invasion. Studies on shrimp C-type lectin also showed that they have multiple antibacterial activities, such as promoting phagocytosis, inhibiting bacterial attachment, regulating downstream immune effectors, and directing antimicrobial activity (Wang et al., 2020). In this research, among the up-regulated DEGs, two lectin-related genes, galactose-specific lectin nattectin-like (LOC113811918) and macrophage mannose receptor 1-like (LOC113819956), were found to increase their mRNA level in the △flhG infected shrimp hepatopancreas. In Thalassophryne nattereri fish, nattectin is involved in the Th1 responses and macrophage differentiation as a signal molecular (Lopes-Ferreira et al., 2011; Saraiva et al., 2011). The agglutinating activity against bacteria of nattectin was found in Misgurnus anguillicaudatus and Carassius auratus (Wang et al., 2017; Zhang et al., 2020). Lv et al. isolated a nattectin from Larimichthys crocea and investigated its role as a PRR in innate immunity (Lv et al., 2016). Another common C-type lectin, mannose receptor (CD206), also can bind to microbial surface glycan structures with terminal fucose, mannose, and N-Acetylglucosamine (GlcNAc) and active various immune cells (Van Der Zande et al., 2021). The mannose receptors of blunt snout bream (Megalobrama amblycephala) can recognize and mediate chitooligosaccharide internalization into macrophages (Ouyang et al., 2021). Xin et al. cloned and analyzed a mannose receptor from red swamp crayfish (Procambarus clarkii), investigating its role in bacteria binding and agglutination (Man et al., 2018). In our results, these two CTL genes’ up-regulated expression might relate to the presence of more flagella compared to the WT type group, suggesting that they have involved in bacteria flagella recognition and pathogen clearance.

The TNF receptor-associated factor (TRAF) 6-like in hepatopancreas also up-regulated significantly after being infected with the hyper flagella strain. TRAF proteins directly bind to the cytoplasmic tail of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) and there are seven members of the TRAF (TRAF1–7) (Ishida et al., 1996; Yamamoto et al., 2021). TRAF2, 5, and 6 activate nuclear factor-κB (NF-κB) and are involved in the canonical NF-κB pathway (Hayden and Ghosh, 2014). Multiple studies have elucidated TRAF6’s function as a molecular bridge that connects upstream TLRs, MyD88, and IRAKs to the downstream NF-kappa B and MAPK-signaling pathways (Kim and Rikihisa, 2002). In Penaeus monodon, PmTRAF6 up-regulated constantly after being challenged by the white spot syndrome virus (WSSV) but remained unchanged after poly I:C stimulation (Deepika et al., 2014). Zhao et al. reported that MST4 which phosphorylates TRAF6 responded to V. alginolyticus infection and activated the TLR-TRAF6 signaling pathway to increase respiratory burst (RB) activity and decrease the total hemocyte count (THC) in L. vannamei (Zhao et al., 2017). However, according to the studies of Wang et al., the mRNA level of LvTRAF6 in the L. vannamei’s hepatopancreas was at a lower level compared to other tissues and increased by the WSSV infection but unchanged after V. alginolyticus challenge (Wang et al., 2011). These findings imply that V. alginolyticus possesses a mechanism to evade the activation of the TRAF6 pathway. In this study, the transcriptome data show that the flhG mutant strains increased the gene expression of TRAF6 (LOC113810339) significantly than the WT strains of V. alginolyticus HN08155, suggesting that the TRAF6 signaling pathway is vital for the bacteria immune defense in shrimp and can be activated by the flagella.

The hepatopancreas of L. vannamei displayed distinct gene expression patterns after infection with a V. alginolyticus mutant strain that possessed constant hyper flagella, achieved through the deletion of the flhG gene, as compared to the wild type strain. Among the differentially expressed genes (DEGs), galactose-specific lectin nattectin and macrophage mannose receptor 1, both belonging to the CTL family, were found to potentially participate in flagella recognition. Additionally, the up-regulation of the TNF receptor-associated factor (TRAF) 6 gene in the hepatopancreas suggested the involvement of the TRAF6 pathway in immune activation.

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 manuscript presents research on animals that do not require ethical approval for their study.

JZ: Writing – original draft. KL: Data curation, Writing – review & editing. XG: Writing – original draft. NZ: Writing – original draft. YZ: Writing – review & editing. WR: Writing – review & editing. AH: Writing – review & editing. HL: Supervision, Writing – review & editing. ZX: Supervision, Writing – review & editing.