Single-nucleus RNA sequencing illuminates a functional analog of lymphoid organ in crustacean

The evolution of lymphoid organs is a complex topic in the animal kingdom. A presumptive invertebrate lymphoid organ (Oka) was previously reported in shrimp based on its morphological and histological observations. However, whether it truly functions as a lymphoid organ akin to those in vertebrates remains uncertain.

This study aims to characterize the cell composition of Oka at single-cell resolution and its function similarity with vertebrate lymphoid organs.

Single-nucleus RNA-seq and cross-species analysis were conducted to identify the major cell types in Oka of the whiteleg shrimp L. vannamei. The spatial expressions of the key genes were detected by in-situ hybridization and immunohistochemical analyses. Cytotoxic activity against heterologous cells was examined using fluorescence microscopy and flow cytometry.

Oka comprised diverse cell types including ECM-producing cells, macrophage-like cells, lymphocyte precursor-like cells, capsular cells, hemocytes, SLC-rich cells, epithelial cells and progenitors. Notably, typical vertebrate macrophage markers (NLRP3, LAMP2 and LGMN) and ZAP-70, a marker of T lymphocytes/natural killer (NK) cells were expressed in the macrophage-like cells. The lymphocyte precursor-like cells, characterized by the expression of YBX3 and SOX4, were further distinguished by the up-regulated expression of CD39, CD49d, and CD133 homologues following WSSV infection. These two cell types were observed to migrate into the lymphoid organ spheroid during structural remodeling of the Oka following WSSV infection. Functionally, Oka cells of shrimp also exhibited cytotoxic activity against heterologous cells.

The presence and WSSV infection-induced aggregation of ZAP-70 positive cells in Oka of shrimp and its cytotoxic activity against heterologous cells suggest that shrimp Oka might perform similar function as the lymphoid organ in vertebrates. The present results will not only provide evidence to confirm the function of Oka in shrimp, but also greatly widen the knowledge about the origin and evolution of lymphoid organ in the animal kingdom.