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The skin of vertebrates is the outermost organ of the body and serves as the first line of defense against external aggressions. In contrast to mammalian skin, that of teleost fish lacks keratinization and has evolved to operate as a mucosal surface containing a skin-associated lymphoid tissue (SALT). Thus far, IgT representing the prevalent Ig in SALT have only been reported upon infection with a parasite. However, very little is known about the types of B cells and Igs responding to bacterial infection in the teleost skin mucosa, as well as the inductive or effector role of the SALT in such responses. To address these questions, in this study, we analyzed the immune response of trout skin upon infection with one of the most widespread fish skin bacterial pathogens, Flavobacterium columnare. This pathogen induced strong skin innate immune and inflammatory responses at the initial phases of infection. More critically, we found that the skin mucus of fish having survived the infection contained significant IgT- but not IgM- or IgD-specific titers against the bacteria. Moreover, we demonstrate the local proliferation and production of IgT+ B cells and specific IgT titers, respectively, within the SALT upon bacterial infection. Thus, our findings represent the first demonstration that IgT is the main Ig isotype induced by the skin mucosa upon bacterial infection and that, because of the large surface of the skin, its SALT probably represents a prominent IgT-inductive site in fish.

The crosstalk between the immune system and microbiota drives an amazingly complex mutualistic symbiosis. In mammals, the upper respiratory tract acts as a gateway for pathogen invasion, and the dynamic interaction between microbiota and mucosal immunity on its surface can effectively prevent disease development. However, the relationship between virus-mediated mucosal immune responses and microbes in lower vertebrates remains uncharacterized. In this study, we successfully constructed an infection model by intraperitoneally injecting common carp (Cyprinus carpio) with spring viremia of carp virus (SVCV). In addition to the detection of the SVCV in the nose and pharynx of common carp, we also identified obvious histopathological changes following viral infection. Moreover, numerous immune-related genes were significantly upregulated in the nose and pharynx at the peak of SVCV infection, after which the expression levels decreased to levels similar to those of the control group. Transcriptome sequencing results revealed that pathways associated with bacterial infection in the Toll-like receptor pathway and the Nod-like receptor pathway were activated in addition to the virus-related Rig-I-like receptor pathway after SVCV infection, suggesting that viral infection may be followed by opportunistic bacterial infection in these mucosal tissues. Using 16S rRNA gene sequencing, we further identified an upward trend in pathogenic bacteria on the mucosal surface of the nose and pharynx 4 days after SVCV infection, after which these tissues eventually reached new homeostasis. Taken together, our results suggest that the dynamic interaction between mucosal immunity and microbiota promotes the host to a new ecological state.

The mucosa of vertebrates is a particularly complex but dynamic environment in which the host constantly interacts with trillions of commensal microorganisms and pathogens. Although the internal and external mucosal microbiomes with immune defense of mammals have been well investigated, the relationship between mucosal microbes and their host’s immune responses has not been systematically understood in the early vertebrates. In this study, we compared the composition and distribution of mucosal microbiota in common carp (Cyprinus carpio), and found that there were significant differences of microbiota between in the internal (gut) and external mucosal (buccal mucosa, gills and skin) tissues. Next, we successfully constructed an infection model with spring viremia of carp virus (SVCV). Specifically, following viral infection, the immune and antiviral related genes showed different up-regulation in all selected mucosal tissues while significant morphological changes were only found in external tissues including buccal mucosa, gills and skin. Using 16S rRNA gene sequence, we revealed that the abundance of Proteobacteria in mucosal tissues including buccal mucosa, gills and gut showed increased trend after viral infection, whereas the abundance of Fusobacteria significantly decreased in gut. In addition, the loss of dominant commensal microorganisms and increased colonization of opportunistic bacteria were discovered in the mucosal surfaces indicating that a secondary bacterial infection might occur in these mucosal tissues after viral infection. Overall, our results firstly point out the distribution of internal and external mucosal microbiota and analyze the changes of mucosal microbiota in common carp after SVCV infection, which may indicated that the potential role of mucosal microbiota in the antiviral process in early vertebrates.

Immunoglobulins (Igs) are crucial components of the immune response in vertebrates including teleost fish. In the present study, we firstly cloned the full-length cDNA of IgT and IgD heavy chain genes in largemouth bass (Micropterus salmoides), one of the important farmed fish species in China. Importantly, IgH locus covering τ, μ and δ genes of largemouth bass was also illustrated. The genomic organization of the IgH locus presented as V26-D3-J5-Cτ-D6-J6-Cμ-Cδ, which was similar with some other teleost fish. Furthermore, The IgH locus of largemouth bass contained 26 VH segments and they belonged to three subgroups, whose members showed a greater than 80% amino acid identity. This founding would allow us to gain further insight into the evolution of adaptive immune system. To further understanding the immune response of immunoglobulins, we successfully constructed the infection model with bacteria (GFP–Flavobacterium columnare G4). Our results showed the expression of IgM was up-regulated in head kidney, and the expression of IgT was significantly induced mainly in skin and gill, suggesting their potential roles in systemic and mucosal immune response to bacterial infection, respectively.

The skin of vertebrates is the outermost organ of the body and serves as the first line of defense against external aggressions. In contrast to mammals, the skin of early vertebrates like fish, lacks keratinization and has evolved to operate as a mucosal surface. As such, it has been shown to contain a skin-associated lymphoid tissue (SALT) in the epidermis. Prior to the discovery of IgT as a key mucosal immunoglobulin (Ig) in fish, several studies reported IgM-specific titers in the skin mucus upon pathogenic infection or vaccination. In contrast, a recent report showed IgT being the main Ig responding in the skin upon parasitic challenge. Whether IgT is the main Ig isotype elicited in the skin against bacterial pathogens, and whether such responses are locally induced within the SALT are important questions that remain unanswered. Here we analyzed the immune response of trout skin upon infection with one of the most widespread fish skin bacterial pathogens, Flavobacterium columnare (F. columnare). To gain insight into the pathogenesis and progression of the infection in skin, a recombinant F. columnare expressing green fluorescent protein (GFP) was constructed and showed to successfully invade the skin mucosa. This pathogen induced strong skin innate immune and inflammatory responses at the initial phases of infection. More critically, we found that the skin mucus of fish having survived the infection contained significant IgT- but not IgM- or IgD-specific titers against the bacteria. Moreover, we demonstrate the local proliferation and production of IgT+ B-cells and pathogen-specific IgT titers respectively within the SALT upon bacterial infection. Thus, our findings represent the first demonstration that IgT is the main Ig isotype induced by the skin mucosa upon bacterial infection, and that because of the large surface of the skin, its SALT probably represents a prominent IgT inductive site in fish. Author summary In contrast to mammalian skin, that of teleost fish is the largest mucosal surface containing a skin-associated lymphoid tissue (SALT). Thus far, IgT representing the prevalent immunoglobulin (Ig) in SALT have only been reported upon infection with a parasite. However, very little is known about the types of B cells and Igs responding to bacterial infection in the teleost skin mucosa, as well as the inductive or effector role of the SALT in such responses. To address these questions, we infected rainbow trout (Oncorhynchus mykiss) with F. columnare an important skin pathogen that causes significant losses both in wild and farmed fish populations. Remarkably, strong pathogen-specific IgT responses and accumulation of IgT+ B cells were induced in the cutaneous mucus and skin epidermis respectively upon infection. Evidence for the skin being an IgT inductive site was shown by the finding of local IgT+ B cells proliferation and pathogen-specific IgT tites in cultured trout skin explants. Thus, our findings demonstrate that IgT is the prevailing Ig isotype induced by the skin mucosa upon bacterial infection, and that the skin represents an IgT inductive site in fish.

The buccal mucosa (BM) of vertebrates is a critical mucosal barrier constantly exposed to rich and diverse pathogens from air, water, and food. While mammals are known to contain a mucosal associated lymphoid tissue (MALT) in the buccal cavity which induces B-cells and immunoglobulins (Igs) responses against bacterial pathogens, however, very little is known about the evolutionary roles of buccal MALT in immune defense. Here we developed a bath infection model that rainbow trout experimentally exposed to Flavobacterium columnare (F. columnare), which is well known as a mucosal pathogen. Using this model, we provided the first evidence for the process of bacterial invasion in the fish BM. Moreover, strong pathogen-specific IgT responses and accumulation of IgT+ B-cells were induced in the buccal mucus and BM of infected trout with F. columnare. In contrast, specific IgM responses were for the most part detected in the fish serum. More specifically, we showed that the local proliferation of IgT+ B-cells and production of pathogen-specific IgT within the BM upon bacterial infection. Overall, our findings represent the first demonstration that IgT is the main Ig isotype specialized for buccal immune responses against bacterial infection in a non-tetrapod species.