Identification of a new pharyngeal mucosal lymphoid organ in zebrafish and other teleosts: tonsils in fish?

The constant exposure of the fish branchial cavity to aquatic pathogens must have driven local mucosal immune responses to be extremely important for their survival. In this study, we used a universal marker for T lymphocytes/natural killer cells (ZAP70) and advanced imaging techniques to investigate the lymphoid architecture of the zebrafish branchial cavity. We identified a new lymphoid organ, which we tentatively named “Nemausean Lymphoid Organ” (NEMO), situated below the pharynx, and closely associated with gill lymphoid tissues. Besides T/NK cells, NEMO is enriched in plasma/B cells and antigen-presenting cells embedded in a network of reticulated epithelial cells. Presence of activated T cells and lymphocyte proliferation but not V(D)J recombination or hematopoiesis, suggests a function as secondary lymphoid organ. In response to infection, NEMO displays structural changes including the formation of T/NK cells clusters. NEMO and gill lymphoid aggregates form a cohesive unit within a lymphoid network that extends throughout the pharyngo-respiratory area. Collectively, our findings reveal a new mucosal lymphoid organ reminiscent of mammalian tonsils that evolved in fish. Importantly, NEMO could clearly be identified in multiple teleost fish families. One sentence summary A previously unreported lymphoid organ has been identified within the pharyngo-respiratory tract of the zebrafish, and other teleost fish, providing new insights into the immune system of teleost fish and the evolution of vertebrate immunology. ### Competing Interest Statement The authors have declared no competing interest. * Aa : Afferent artery Aaa : Afferent arch artery ALT : Amphibranchial Lymphoid Tissue B : Bone Bc : Branchial cavity Bm : Basement membrane BTK : Bruton Tyrosine Kinase Bv : Blood vessel C : Cartilage Cbe : Cavo-branchial epithelium Ct : Connective tissue Cvs : Central venous sinus d-ILT : distal Interbranchial Lymphoid Tissue Dpi : Day post-infection Ea : Efferent artery Eaa : Efferent arch artery Eav : Endothelial anastomotic vessels Ec : Endothelial cell EM : Electron Microscopy Ft : Fat tissue Ga : Gill arch GALT : Gut-Associated Lymphoid Tissue Gi : Gills GIALT : Gill-Associated Lymphoid Tissue H&E : Hematoxylin and Eosin HK : Head-Kidney ILT : Interbranchial Lymphoid Tissue IHNV : Infectious Hematopoietic Necrosis Virus La : Lamellae Lu : Lumen M : Muscles MALT : Mucosa-Associated Lymphoid Tissue NALT : Nasal-Associated Lymphoid Tissue NEMO : Nemausean lymphoid Organ NK : cell Natural Killer cell Op : Operculum PCNA : Proliferating Cell Nuclear Antigen p-ILT : proximal Interbranchial Lymphoid Tissue Rbc : Red blood cell S : Septum SALT : Skin Associated Lymphoid Tissue Sk : Skin Spi : Sub-pharyngeal isthmus SVCV : Spring Viremia of Carp Virus Tc : Thymus cortex Td : tendon TEM : Transmission Electron Microscopy Tf : Thyroid follicle Tm : Thymus medulla Tu : Tubule Uh : Urohyal bone Um : Urohyal marrow Va : Ventral aorta Wpf : week post-fertilization ZAP70 : Zeta-chain-Associated Protein kinase 70.