The olfactory organ of vertebrates receives chemical cues present in the air or water and, at the same time, they are exposed to invading pathogens. Nasal-associated lymphoid tissue (NALT), which serves as a mucosal inductive site for humoral immune responses against antigen stimulation in mammals, is present also in teleosts. IgT in teleosts is responsible for similar functions to those carried out by IgA in mammals. Moreover, teleost NALT is known to contain B-cells and teleost nasal mucus contains immunoglobulins (Igs). Yet, whether nasal B cells and Igs respond to infection remains unknown. We hypothesized that water-borne parasites can invade the nasal cavity of fish and elicit local specific immune responses. To address this hypothesis, we developed a model of bath infection with the Ichthyophthirius multifiliis (Ich) parasite in rainbow trout, Oncorhynchus mykiss, an ancient bony fish, and investigated the nasal adaptive immune response against this parasite. Critically, we found that Ich parasites in water could reach the nasal cavity and successfully invade the nasal mucosa. Moreover, strong parasite-specific IgT responses were detected in the nasal mucus, and the accumulation of IgT⁺ B-cells was noted in the nasal epidermis after Ich infection. Strikingly, local IgT⁺ B-cell proliferation and parasite-specific IgT generation were found in the trout olfactory organ, providing new evidence that nasal-specific immune responses were induced locally by a parasitic challenge. Overall, our findings suggest that nasal mucosal adaptive immune responses are similar to those reported in other fish mucosal sites and that an antibody system with a dedicated mucosal Ig performs evolutionary conserved functions across vertebrate mucosal surfaces. The olfactory organ is a vitally important chemosensory organ in vertebrates but it is also continuously stimulated by pathogenic microorganisms in the external environment. In mammals and birds, nasopharynx-associated lymphoid tissue (NALT) is considered one of the first lines of immune defense against inhaled antigens and in bony fish, protecting against water-borne infections. However, although B-cells and immunoglobulins (Igs) have been found in teleost NALT, the defensive mechanisms of parasite-specific immune responses after pathogen challenge in the olfactory organ of teleost fish remain poorly understood. Considering that the NALT of all vertebrates has been subjected to similar evolutionary forces, we hypothesize that mucosal Igs play a critical role in the defense of olfactory systems against parasites. To confirm this hypothesis, we show the local proliferation of IgT⁺ B-cells and production of pathogen-specific IgT within the nasal mucosa upon parasite infection, indicating that parasite-specific IgT is the main Ig isotype specialized for nasal-adaptive immune responses. From an evolutionary perspective, our findings contribute to expanding our view of nasal immune systems and determining the fate of the host–pathogen interaction.

The pharyngeal organ is located at the crossroad of the respiratory and digestive tracts in vertebrate, and it is continuously challenged by varying Ags during breathing and feeding. In mammals, the pharyngeal mucosa (PM) is a critical first line of defense. However, the evolutionary origins and ancient roles of immune defense and microbiota homeostasis of PM are still unknown. In this study, to our knowledge, we are the first to find that diffuse MALT is present in PM of rainbow trout, an early vertebrate. Importantly, following parasitic infection, we detect that strong parasite-specific mucosal IgT and dominant proliferation of IgT⁺ B cell immune responses occurs in trout PM, providing, to our knowledge, the first demonstration of local mucosal Ig responses against pathogens in pharyngeal organ of a nonmammal species. Moreover, we show that the trout PM microbiota is prevalently coated with secretory IgT and, to a much lesser degree, by IgM and IgD, suggesting the key role of mucosal Igs in the immune exclusion of teleost pharyngeal bacteria. Overall, to our knowledge, our findings provide the first evidence that pharyngeal mucosal immunity appear earlier than tetrapods. Key Points A mucosally associated lymphoid tissue is identified in teleost pharyngeal cavity. Trout pharyngeal bacteria are mainly coated with secretory IgT. Teleost IgT plays a vital role in protecting the PC from invading pathogens.

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. Key Points Strong IgT responds to bacterial infection in fish skin mucosal immunity. The skin represents a mucosal Ig-inductive site in fish.

Background: The visual organ plays a crucial role in sensing environmental information. However, its mucosal surfaces are constantly exposed to selective pressures from aquatic or airborne pathogens and microbial communities. Although few studies have characterized the conjunctival-associated lymphoid tissue (CALT) in the ocular mucosa (OM) of birds and mammals, little is known regarding the evolutionary origins and functions of immune defense and microbiota homeostasis of the OM in the early vertebrates. Results: Our study characterized the structure of the OM microbial ecosystem in rainbow trout (Oncorhynchus mykiss) and confirmed for the first time the presence of a diffuse mucosal-associated lymphoid tissue (MALT) in fish OM. Moreover, the microbial communities residing on the ocular mucosal surface contribute to shaping its immune environment. Interestingly, following IHNV infection, we observed robust immune responses, significant tissue damage, and microbial dysbiosis in the trout OM, particularly in the fornix conjunctiva (FC), which is characterized by the increase of pathobionts and a reduction of beneficial taxa in the relative abundance in OM. Critically, we identified a significant correlation between viral-induced immune responses and microbiome homeostasis in the OM, underscoring its key role in mucosal immunity and microbiota homeostasis. Conclusions: Our findings suggest that immune defense and microbiota homeostasis in OM occurred concurrently in early vertebrate species, shedding light on the coevolution between microbiota and mucosal immunity.