Histological differentiation of mucus cell subtypes suggests functional compartmentation in the eel esophagus

We investigated the morphological and histological changes in eel esophagus during the course of freshwater (FW) to seawater (SW) transfer and identified multiple types of mucus cells from tissues that were fixed using Carnoy's solution to retain the mucus structure. The FW esophageal epithelium is stratified and composed of superficial cells, mucus cells, club cells (exocrine cells with a large vacuole), and basal cells. Two types of periodic acid-Schiff (PAS)-positive mucus cells were identified, and they can be further distinguished by the periodic acid-thionin Schiff/KOH/PAS (PAT) method, indicating that C7/9- and C8-sialic acids were produced. Isolectin B4-positive mucus cells were found among the C8-sialic acid-producing cells and located at the tips of the villi at mid-posterior regions of the FW esophagus. The two different muci were immiscible and may form separate layers to protect the tissues from the high osmolality of imbibed SW during early SW acclimation. The densities of club cells and isolectin B4-positive cells decreased after SW acclimation, and cuboidal/columnar epithelial cells subsequently developed for active Na⁺ and Cl^- absorption. Cuboidal/columnar epithelial cells proliferated in scattered array rather than at the bases of the villi, thereby retaining the characteristic of the stratified epithelium. Prominent leukocyte invasion was found at the base of the stratified epithelium at early SW transfer, indicating that the immune system was also activated in response to antigen exposure from imbibed SW. The mucus composition in FW is more complicated than that in SW, fueling further studies for their functions to form unstirred layers as osmoregulatory barriers.