The barrier function of mucosal epithelia is structurally supported by the epithelial cell lining and in a more dynamic manner by the overlying mucus secretion, which is constantly flowing through renewal and off-sloughing. In fish, all exposed body surfaces, including skin, gills, nostrils, and digestive tract, are covered by mucosal epithelia, which constitute first line of defense against external offenders, friction, and dehydration and also participate in disease resistance, respiration, and ionic and osmotic regulation (1, 2). Thus, the role of mucosal health and mucous modulation should not be underestimated in the context of intensive aquaculture rearing systems in a global warming scenario, in which finfish health is under constant threat of imbalance.

The regulation of the mucus-secreting cells and mucus composition upon disease and infection (3–6), including parasitosis (7, 8), is an appealing study topic in human medicine for its diagnostic and therapeutic value (9). Mucus responses in fish skin, gills, and gut have also gathered research interest (10–16). More specifically, gill parasites often provoked mucus over-secretion, as a host mucosal response intended to expel the parasite invader (17–21), but the fish host may have to deal with several drawbacks such as ion and gas exchange imbalance or microbiota dysbiosis.

The flatworm gill ectoparasite Sparicotyle chrysophrii, formerly classified in the Monogenea class and recently reclassified into the Polyopisthocotyla class (22), is currently considered the most distressing pathogen for gilthead seabream (Sparus aurata) Mediterranean aquaculture (23). The high stocking densities, the lack of fallowing strategies, and the enzootic locations of the off-shore cage farms are favoring parasite outbreaks and transmission. The growing concern in regard to the production losses has directed recent research efforts toward studies on the parasites’ biology (24–28), the host response (29–32), and the search for treatments (33–38). However, a closer look at the modulation of the mucous secretion and the goblet cells in the gills of gilthead seabream during the course of this parasitosis has not been thoroughly taken.

Mucus is mainly produced by goblet cells, secretory cells present in the epithelial cell lining, which synthesize and expel secreted mucins, the main component of the adherent mucus gel. Mucins are high-molecular weight hydrophilic glycoproteins forming a complex matrix, in which water and a cocktail of bioactive molecules are retained, avoiding direct contact between the environment and tissue. By modulating the mucus layer physically (viscosity), biologically (immunoglobulin, lysozyme, antimicrobial peptide contents), and chemically (pH, mucin glycosylation), organisms are able to cope dynamically with the changing external stressors, including their own mucosal microbiota.

The mechanisms that orchestrate goblet cell proliferation, differentiation and distribution patterns, and mucin expression and glycosylation in fish upon parasite challenge are still obscure. This study intends to integrate the study of goblet cell distribution patterns with their transcriptional regulation, together with mucin expression and glycosylation in the gills of gilthead seabream during the course of sparicotylosis using histochemical, lectin-binding, transcriptional, and transcriptomic approaches.

Sparicotyle chrysophrii infections in gilthead seabream farms have become a major issue for aquaculture. In the current study, we unveil some aspects of the local mucosal response triggered by this gill ectoparasite, providing valuable data on mucin and goblet cell distribution and regulation. Studies focusing on mucosal immune responses in fish have traditionally focused on the mucosa-associated lymphoid tissues (MALT), their cell effectors, and their molecular signaling. Thereby, the importance of the strictly mucus-related compartment, i.e., the mucus secretion itself, its mucin components, and the responsible goblet cells and goblet cell glycosidases, have been mostly neglected. Nevertheless, in the current-omics era, recent advances in glycomics granted mucus secretion its position as key mediator between epithelial cells, MALT, and microbiota and their joint interactions with external factors such as pathogens (47–49).

The main clinical sign during sparicotylosis is anemia, which is caused by the hematophagous nature of a parasite (32, 39, 50). Furthermore, the parasite’s attaching mechanism to the gill filament through specialized clamps in its opisthaptor region (51) inflicts evident histopathological lesions such as lamellar synechiae and clubbing, resulting in disruption of the epithelium and marginal blood vessels (50). Gill hemorrhages and an increased mucoid exudate are among the recognized pathogenic effects and host response elicited by gill monogeneans, as reviewed by Ogawa (52). Regarding goblet cells, histological scoring of experimentally infected gilthead seabream showed an overall hyperplasia of neutral goblet cells in gill filaments. At the four gill locations where goblet cells were analyzed, a significant increase in neutral goblet cells was detected in R fish compared to C fish at some points during the parasite challenge (Figures 1, 2). This hyperplasia was especially notable and significant over the course of parasite exposure in the interlamellar pockets and the lamellar epithelium from 32 dpe on. A shift in the position of goblet cell distribution toward interlamellar cavities upon infection with the larval parasitic stage of the freshwater mussel Margaritifera margaritifera in Atlantic salmon was attributed to their role in gill clearance and remodeling (18). Similarly, the present rearrangement of goblet cell distribution is probably helping, by an increased mucus secretion between lamellae, to lubricate the surface of hyperplasic epithelia avoiding lamellar fusion. Thus, goblet cell hyperplasia was not evident at the other locations away from the lamellae, i.e., filament tips and the epithelium covering the proximal cartilage.

Meaningfully, goblet cell hyperplasia occurred after the first month of parasite challenge once infective oncomiracidia, post-larvae, and juveniles of S. chrysophrii turned into the more pathogenic adult worm stages, which was in agreement with the previous observations (39). At 32 dpe, the highest adult stage infection intensity reached 112.8 ± 43.62, with each adult worm bearing 2 rows of up to 72 clamps (personal observation). These, pinch on the lamellae, eroding the mucosal surface and disrupting the gill tissue, probably triggering the increased mucus secretion to protect the nude or damaged gill epithelium. Mucosal and waterborne bacteria infecting such wounds also contribute to an inflammatory response in the parasitized gills (53). In fact, the current S. chrysophii-infected fish had developed epitheliocystis at the later sampling point. Sparicotyle chrysophrii and epitheliocystis co-infection in gilthead seabream are common under farm and experimental conditions (39, 50, 54), and its specific bacterial etiology was a matter of discussion until recently (43, 55, 56).

In the context of intestinal helminth infections, hyperplasia and hypertrophy of the mucous cells were described at the site of parasite attachment, especially for the ones with acid glycoconjugates (57). Hypertrophy of mucus cells with an increase in carboxylated glycoproteins containing sialic acid was observed in gills of gilthead seabream fed an essential oil-supplemented diet and exposed to S. chrysophrii, but no change occurred in cell numbers (34). In the present study, gilthead seabream responded to parasite infection with an increase in goblet cells with neutral glycoconjugates. In healthy gills, the abundant secretion of neutral mucus characterized by glycoproteins with oxidizable vicinal diols is associated with pH regulation for acidity buffering of acidic mucins, with the maintenance of homeostasis and lubrication (58–60). The shift toward mucin acidification upon infection, related to viscosity increase in the mucus secretion and trapping of offending microorganisms due to higher amount of O-sulfate esters (60, 61), was not detected by histochemistry in the gills of S. chrysophrii-infected gilthead seabream. Conversely, the predominantly neutral mucin production found from 32 dpe on involves a less viscous mucus layer which facilitates laminar water flow and gas exchange in the gill lamellae (61, 62). Lethargy, as a sign of respiratory distress, is common upon sparicotylosis (38), and even a mild S. chrysophrii infection elicited the downregulation of hypoxia-related oxygen homeostasis genes not only locally but also in the spleen of gilthead seabream (31). Thus, the reduced oxygen availability induced by the direct parasitic damage on the gills and blood consumption resulting in anemia provokes severe hypometabolic effects at systemic level during mild infections. Our current results point to a local mucosal response by the fish aiming to counterbalance parasite-induced hypoxia by secreting less viscous mucus on the lamellar surface that facilitates gas exchange.

Goblet cells bearing acidic, carboxylated mucins were found only in the epithelium covering the proximal cartilage of the arches, away from the gill respiratory epithelium, both in C and R fish (Figure 2; Supplementary Figure S1). Rakers and gill arches lubricated with highly viscous and sticky mucus, predominantly containing glycoproteins with O-sulfate esters and sialic acids, were reported from diverse teleost species in order to protect the epithelium of the pharyngeal cavity against mechanical injuries during food ingestion and transport food particles (62). Moreover, glycoproteins in such secretions cross-link sulfate and sialic acid groups resulting in a more resistant barrier against bacterial enzymatic degradation and thus, more difficult to breach by bacteria and more efficient in containing microbial infections (61).

At the transcriptional level, goblet cell hyperplasia was supported by the overexpression of mucin genes and regulatory factors involved in goblet cell differentiation (Figure 4). Apomucin, the linear polypeptide backbone of the mucin, is encoded by specific Muc genes in goblet cells. Assembly, automatic annotation, and in silico identification of mucins in non-mammalian species are challenging mainly due to their very long repetitive sequences and the poorly conserved sequence of mucin domains (63). Only a few studies have been conducted on teleost mucin gene expression with mucin transcription data that are barely available for Atlantic salmon (64, 65), zebrafish (66–68), and common carp (69, 70), and one previous record exists for gilthead seabream (15). In the later study, partial sequences and structure of some mucins were described for gilthead seabream from transcriptomic data. In the current study, we integrated this previously defined information with the newly available genome data from two different genome assemblies for this species [fSpaAur1.1 in ensembl.org, NCBI and (40)] and identified nine mucin sequences. Our analysis of the automatic annotation of the sequences showed a coherent complete mucin structure for the membrane-bound mucins (muc4, muc13, and muc18), except for imuc, whose C terminus part was the only part identified. However, upstream of the imuc sequence in chromosome 6, several large repetitive PST domains can be identified that probably encode a part of the N terminus sequence of this protein, or belong to a different mucin sequence encoded in tandem, as typically found in gel-forming mucin gene organization (71). Long-read sequence analyses should be conducted to unravel the complete sequence of this mucin. Similarly, although muc2 and muc5 sequences appear almost complete from N to C terminus, the predicted coding sequences in the middle repeat part are not very consistent, with atypical intron–exon boundary sequences (Supplementary Table S1). In addition, the similarities between muc2 and muc5 sequences, and the poor conservation of mucin domains, are challenging for mucin classification and nomenclature in teleosts and other non-mammalian species. In this line, zebrafish muc5 sequences have been termed muc5.1–3 instead of using the mammalian nomenclature MUC5AC/MUC5B (67). Therefore, the nomenclature used in the current study is somewhat arbitrary, and a more accurate annotation of fish mucins should be performed integrating mammalian and non-mammalian sequences, since up to 133 Muc2-type and 263 Muc5-type proteins have been identified from vertebrate genomes (68). Muc2, Muc19, and Muc5 are very large, secreted, gel-forming mucins, whereas I-Muc, Muc4, Muc13, and Muc18 are membrane-bound mucins (15, 72, 73). Pérez-Sánchez et al. (15) found that Muc18, the most abundant gill mucin, and I-Muc constitutively expressed in the gills of gilthead seabream. These authors annotated the imuc sequence, which had no clear orthologous genes in mammals and was found highly expressed in the posterior intestine, where it revealed itself as a biomarker of intestinal health changing its expression upon parasite infections. Additionally, expression of muc2, muc13, and muc19 was found restricted to the gastrointestinal tract, in agreement with our current results which found no detectable expression in gills. Our results further reveal that I-Muc has an additional role in gill protection and mucosal response (15).

Expression and a robust upregulation of muc2 genes, muc5, imuc, muc4, and muc18 were found in the parasitized gills of gilthead seabream at 32 dpe, recovering their initial levels at 61 dpe for all mucin genes except for muc5a/c, which remained upregulated along the course of the entire experiment. Apparently, the secreted muc2 sequences and the membrane-bound imuc, muc4, and muc18 responded acutely once the highly pathogenic adult worms were established, with fold changes up to >20. The highest fold changes among them corresponded to the secreted muc2a and muc2b, while upregulation of the membrane-bound mucins imuc, muc4, and muc18 was not as high. Mucin upregulation in response to a gill parasite was described in Atlantic salmon, displaying clinical amoebic gill disease at 21 dpe, when a 30-fold muc5 upregulation was found (64). In addition, in gills, those authors found downregulation of the membrane-bound muc18 and very low and variable expression of a muc2 sequence, which they considered to have a major role in the intestine of salmon. Sveen et al. (65) found three different muc5a/c genes, two muc2 genes, and one muc5b gene expressed in the gills of Atlantic salmon, from which two muc5a/c and two muc2 genes were significantly upregulated only 3 h after handling stress. In common carp, different pathogenic viruses provoked severe mucosal distress in gills by downregulating muc2-like and muc18 gene expression at the time that clinical signs of the disease appeared and susceptibility to secondary infections increased (69). In contrast, the expression of three different muc5 genes was upregulated in the whole body of zebrafish larvae in response to bacterial challenges upon pectin administration, which was considered an innate antimicrobial immune response (66). Therefore, it seems evident that mucin expression is involved in protective responses upon exposure to different pathogens and stressors, but there are large differences in mucin expression patterns among different fish species.

Regulatory mediators are responsible for the tuning of the goblet cell differentiation, leading to their hyperplasia and consequent mucus hypersecretion (74, 75). Accordingly, elf3 and agr2, both involved in goblet cell differentiation, were synchronically upregulated with the observed mucin upregulation in R gills. In the intestines of Elf3-deficient mice, poor differentiation of goblet cells occurred (76). In addition, gene homologs of agr2 in humans and mice are strongly expressed in mucus-secreting cells of the digestive and respiratory epithelia, and in zebrafish, agr2 gene expression was found in mucus cells from all mucosal tissues including gills (77). Such regulatory mediators are, however, also responsible for avoiding a chronic inflammatory/repair response, which would worsen respiratory distress and hypoxia (74), and consequently, their expression levels were restored after the second month of parasite exposure. The transcriptional response of hes1b was slightly different, presenting a minor downregulation in the gills of R fish along the entire parasite challenge. Nevertheless, in fish as in mammals, the Notch-Hes1 pathway drives intestinal differentiation, and its inhibition results in goblet cell differentiation (78, 79), which is in line with our results. Accordingly, downregulation of hes1 was described before as an intestinal helminth defense in order to increase mucin secretion and prevent worm attachment or promote detachment (80).

Regarding terminal glycosylation of the mucins contained in gill goblet cells, we found moderate changes including an overall increase in Gal, GalNac, and Fuc in R fish (Table 4). In addition, GlcNac/sialic acid terminal residues also increased slightly in the goblet cells of the interlamellar pockets of R fish. Glycosylation of the apomucin is carried out post-transcriptionally by glycotransferases and glycosidases in the Golgi apparatus during the secretory pathway. The resulting glycan oligomerization and branching and defining of the terminal sugar moieties produce the structural diversity of the glycome. Terminal sugar residues determine the charge and antigens of the mucins and are the molecular basis for interspecies recognition being capable of activating immune responses. In fact, chemical modifications of glycans have been proposed as diagnostic and therapeutic strategies against diseases (81). Firmino et al. (34) suggested that diet supplementation with essential oils during sparicotylosis enhanced the mucosal defense mechanism, in part by an increase in sialic acid-containing mucins, which helped parasite trapping and shedding and led to a significant reduction in parasite abundance and prevalence. Here, in the absence of any particular dietary treatment, WGA binding to almost all sialylated glycans and GlcNAc showed a moderate increase in the goblet cells of the interlamellar pockets and the interlamellar mucus secretion of R fish at 32 dpe. In contrast, SNA, binding only to sialic acid attached to terminal α2, 6Gal, gave the weakest of all lectin labels, being almost negligible. Thus, overall sialylated and Glc-N-acetylated glycans seem to be increasingly secreted in the gills of gilthead seabream as a response to the establishment of adult S. chrysophrii stages. The increase in sialic acid and GlcNAc together with goblet cell hyperplasia is recognized as part of the expulsion response against helminth infections, since sialic acids serve for pathogen binding and dumping (8). In agreement, six N-acetylglucosaminyltransferases were upregulated in the RNAseq dataset of gilthead seabream gills with severe sparicotylosis (Figure 5). The addition of GlcNAc to the oligosaccharide chain can generate core 2 and 4 structures, allowing branching and providing the substrate for the further addition of sugar by other glycosyltransferases. This is a regulatory turning point of glycosylation, since many biologically important oligosaccharide structures involved in recognition and adhesion are constructed on this branch, and improper N-acetylglucosaminyltransferase expression in mammals is related to pathological conditions (82). In addition, the expression profile of sialyltransferases was clearly downregulated in the RNAseq dataset of fish with low infection intensity, whereas in highly infected fish, only one sialyltransferase of the five we identified was significantly downregulated, and sialidases presented opposite expression profiles, depending on the infection intensity of the fish.

While sialic acids confer a negative charge to the glycoconjugate, terminal fucose residues confer hydrophobicity, but both are associated with mucosal protection and their alteration may result in disease (72, 75). Thus, human fucosyltransferases and their expression have been studied for their importance in inflammation, mucosal colonization, and host immune response modulation (83, 84), and more recently, their importance for the maintenance of healthy gut microbiota profiles has been stressed out (85, 86). In R gills, a moderate increase in Fuc terminal residues was observed in the lamellar goblet cells and those of the epithelium covering the proximal cartilage, and fucosyltransferase expression appeared upregulated in fish with high infection intensity (Figures 2, 5). Such results may also point to a protective host response through modulation of mucin glycosylation. Conversely, the only previous study including glycosylation of gilthead seabream gills mucus found scarce Fuc residues after feeding with an essential oil-supplemented diet and challenging fish with the polyopisthocotylidan (34). However, those fish had a mean infection intensity of 2.7 parasites/fish, much lower than the current one of 112.8 ± 43.62, and very similar to the 2.73 parasites/fish of the low-intensity RNAseq dataset (31), in which fucosyltransferases were downregulated. Thus, some protective mechanisms of mucosal modulation in the gills of gilthead seabream may only be triggered upon severe polyopisthocotylidan infection. Through a glycomic approach, the presence of complex fucosylated mucin structures was found in Atlantic salmon gills, which would lead to increased structure diversity of glycan epitopes to diversify its repertoire as a possible defensive immune strategy (87). Similarly, the skin mucin structure of Atlantic salmon subjected to chronic stress carried increased Fuc, sialic acid, and core 1 glycans (47).

In addition, GalNAc, which is incorporated by N-acetylgalactosaminyltransferases to the apomucin initiating the oligosaccharide sidechains, seemed to increase in the goblet cells of the R fish lamellar epithelium and the mucus secretion. The detection of this sugar moiety at the terminal position in secreted mucins was considered a sign of immature mucin secretion in gilthead seabream upon intestinal parasite infection (88) and also in vertebrates, in general (89). Secretion of not fully mature mucins seems to contradict the previous observations of mucins with increased GlcNAc, sialic acid, and Fuc, indicators of complex glycans. Future glycomic work will be conducted to validate or reject if the mucosal gill secretion of gilthead seabream contains immature, not fully glycosylated mucins upon sparicotylosis.

Phosphomannomutase isozymes, as known from mammals, are required for the process of N-glycosylation. They provide Man-1-P, the substrate needed by mannosyltransferases, to incorporate Man into glycoconjugates (90). Identified mannosyltransferase sequences participate in this early N-linked glycosylation, from which derived glycan structures are key for functions such as cell recognition, host-defense, and protein secretion in many organisms (91). The downregulation of mannosyltransferase observed in the gills of fish with low infection intensity was not detected in fish with high intensity, as also happened for one phosphomannomutase and most mannosidases identified (Figure 5). Mannosidases and sialidases are both relevant glycosidases for the release of mucin glycans and would play their role in sloughing mucus with the retained parasites off, as previously suggested for an endopeptidase by Firmino et al. (34). Overall, enzyme gene expression from the RNAseq analyses performed herein pointed to a hyporegulative profile in the gills of the fish with low infection intensity, which was mostly reverted or even inverted for many glycosyltransferases and glycosidases in fish with high infection intensity. This would correspond to a defense response in gills involving higher mucin biosynthesis and release, which is in line with the observed goblet cell hyperplasia and mucus hypersecretion upon severe S. chrysophrii infection, once the adult parasite stages are established.

Many aspects beyond our current scope, such as glycosyltransferase competition and their intracellular location, epigenetics, health status, or microbiome/pathobiome signaling, can influence the final O-glycosylation profile, which compromises mucin secretion, conformation, involvement in adhesion and recognition events, and microbiome niches. Furthermore, each glycosyltransferase accounts for the proper arrangement of the individual monosaccharides in each unique oligosaccharide structure, and their gene expression serves as fine tuning of the glycosylation process. Future transcriptomic and glycomic approaches will help us understand the scope of the detected mucin modulation in this host–microbiome–parasite interaction by integrating the transcriptional, glycosylation, and microbiome viewpoints. We are still far from understanding the entire interplay occurring between host mediators and effectors, microbiota and microbiota-derived factors, and parasites and their excretory–secretory products. However, this study helps to understand how the gill mucosal microhabitat responds to the S. chrysophrii offender and consequent microbiota shift (43) by increased goblet cell differentiation leading to neutral goblet cell hyperplasia on gill lamellae, acutely increased mucin expression, and a probable increase in more complex glycoconjugates with sialylated, fucosylated, and branched structures.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

The animal study was approved by the Ethics and Animal Welfare Committee of the Institute of Aquaculture Torre de la Sal (IATS-CSIC, Castellón, Spain) CSIC and “Generalitat Valenciana” (permit number 2018/VSC/PEA/0240). The study was conducted in accordance with the local legislation and institutional requirements.

ER-F: Formal analysis, Investigation, Methodology, Writing – original draft. RP: Investigation, Writing – original draft. UM-B: Investigation, Writing – review & editing. OP: Funding acquisition, Supervision, Writing – review & editing. AS-B: Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – review & editing. IE: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Visualization, Writing – original draft, Writing – review & editing. MP: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Visualization, Writing – review & editing.