Differential mucins secretion by intestinal mucous cells of Chelon ramada in response to an enteric helminth Neoechinorhynchus agilis (Acanthocephala)

Highlights

• Intestinal mucous cells have a role in the innate immune response.

• Mucus secretion increases in the intestine of Chelon ramada infected with Neoechinorhynchus agilis.

• The high protective carboxylated and sulphated mucins are the most increasing mucus components in parasitized intestine.

• A high number of intestinal mucous cells containing oligosaccharide residues is involved in the protection against pathogens.

Abstract

Intestinal mucous cells produce and secrete mucins which hydrate, lubricate and protect the intestinal epithelium from mechanical injuries due to the transition of digesta or action of pathogens. Intestinal mucous cells are considered elements of the innate immune system as they secrete lectins, toxins, immunoglobulins, and anti-microbial peptides. Acid mucins can surround and eliminate many pathogenic microorganisms. We performed a quantitative analysis of the density and mucus composition of different intestinal mucous cell types from mullet (Chelon ramada) that were infected solely with Neoechinorhynchus agilis. Most N. agilis were encountered in the middle region of the intestine. Mucous cell types were identified with Alcian Blue (pH2.5) and Periodic acid-Schiff (PAS) histochemistry, and by staining with a panel of seven lectins. Mucus enriched for high viscosity acid mucins was accumulated near points of worm attachment. Parasites were surrounded by an adherent mucus layer or blanket. Ultrastructural examination showed intestinal mucous cells typically possessed an elongated, basally positioned nucleus and numerous electron dense and lucent vesicles in the cytoplasm. The results show both an increase in mucus production and changes in mucin composition in infected mullet in comparison with uninfected conspecifics.

Introduction

Mucous cells of fish intestine continuously produce and secrete mucins that form a seamless layer at the epithelial surface. Mucins perform multiple functions including lubrication, protection of the tunica mucosa against mechanical damages, constitution of a hypertonic medium in the stomach and intestine, acting as a diffusion barrier for various ions, and involvement in epithelial ion transport (Fiertak and Kilarski, 2002; Gupta, 1989; Loretz, 1995). Fish live in an ideal medium for the growth and proliferation of several organisms which results in the constant presence of bacteria, parasites, toxins and viruses in the luminal content (Neuhaus et al., 2007a, 2007b). The mucus barrier represents a site of colonization for many beneficial microorganism but can also be compromised by pathogens (Soffientino et al., 2006).

Mucous cells and the mucus layer are the main mediators of the luminal colonizing organisms and are involved in their interaction with the host (Malin et al., 2014). As a consequence, intestinal parasites might induce a heavy mucus production and its discharge into the lumen (Bergstrom et al., 2010; Grootjans et al., 2016), as are described in several papers on fish-helminth systems (Bosi and Dezfuli, 2015; Bosi et al., 2005, 2015, 2017; Chambers et al., 2001; Dezfuli et al., 2010, 2016). Most studies are focused on the conventional histochemical use of alcian blue pH2.5 (AB) followed with the periodic acid Schiff (PAS). The AB/PAS method can distinguish among acidic mucins (stain blue, AB positive), neutral mucins with periodate reactive vicinal diols (stain magenta, PAS positive), and mucins containing a mixture of both mucins (stain violet, AB/PAS positive).

Lectins are macromolecules, each with a specific binding affinity for a particular sugar residue. Lectins can be employed as probes for the localization of specific sugar residues in mucus glycoconjugates (Danguy et al., 1994; Ihida et al., 1991) to identify mucous cell populations and to characterize changes in mucus composition (Danguy et al., 1994; Spicer and Schulte, 1992). In fish mucins, five main monosaccharides are present, namely fucose, mannose, N-acetyl-α-galactosamine, N-acetyl-β-galactosamine, and neuraminic acid (= sialic acid = N-acetyl-Neuraminic Acid) (Alexander and Ingram, 1992; Schroers et al., 2009; Shephard, 1994). Fish mucins are structurally similar to their mammalian counterparts (Shephard, 1994). The AB/PAS method has a limited sensitivity but can be combined with lectin histochemistry to yield more detailed information.

The Mugillidae, known as grey mullets are one of the most ubiquitous teleost families in coastal waters of the world (Crosetti, 2016). Frequent migration of the mugilids from freshwater to sea water are believed to increase their vulnerability to infection by different taxa of parasites (Paperna and Overstreet, 1981; Sayyaf Dezfuli et al., 2018). Thinlip mullet Chelon ramada is the most abundant mullet species which lives in the Scardovari lagoons. We compared different intestinal subpopulations of mucous cells in C. ramada parasitized with the acanthocephalan Neoechinorhynchus agilis with uninfected mullets. Changes in production and secretion of mucins sugar residues in parasitized vs. uninfected mullet were also analyzed using lectin histochemistry.